diff --git a/.DS_Store b/.DS_Store
index fa766860224d1f4f1281130b04166967aaa41319..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 100644
Binary files a/.DS_Store and b/.DS_Store differ
diff --git a/src/App/App.css b/src/App/App.css
index c4dac1cc9d41d908ecaa0e1eee346b6c427762f3..0026faea443fad5d7f28e03dc50df70c4ab52855 100644
--- a/src/App/App.css
+++ b/src/App/App.css
@@ -301,7 +301,7 @@ color: var(--text-primary);
}
.navbar{
backdrop-filter: blur(5px);
- background-color: rgb(255,246,242, 0.4);
+ background-color: rgb(255,246,242, 0.8);
transition: visibility 0s, 0.6s, opacity 0.6s linear, transform 1s;
}
nav.navbar {
@@ -844,14 +844,24 @@ img .middle{
max-height: 70% !important;
}
+
.img-round{
border-radius: 50%;
width: 120px;
height: 120px;
- max-height: 13vh;
+ max-height: 13vh;
max-width: 13vh;
object-fit: cover !important;
}
+
+.img-round-panel{
+ border-radius: 50%;
+ width: 100px;
+ height: 100px;
+ max-height: 10vh;
+ max-width: 10vh;
+ object-fit: cover !important;
+}
.img-cube{
max-width: 80%;
}
@@ -1123,12 +1133,13 @@ svg{
max-width: 1100px !important;
}
.one-pdf-line{ /* @media fertig */
- height: 1450px !important;
+ max-height: 650px !important;
+ height: 100vh !important;
+ max-width: 40% !important;
}
.two-pdf-line{ /* @media fertig */
- max-height: 650px !important;
- height: 35vh !important;
- max-width: 50%;
+ min-height: 650px !important;
+ height: 100vh !important;
}
/* SHAPES */
@@ -4092,7 +4103,7 @@ figure img{
#exhead, #ibhead, #jughead, #mmhead, #nothead, #parhead, #reshead, #teamhead
, #sphead, #suphead
{
- background-size: 1650px auto;
+ background-size: 100vw auto;
background-repeat: no-repeat;
}
@@ -4136,4 +4147,16 @@ figure img{
height: auto; /* Maintain aspect ratio of the image */
max-height: 150px; /* Set a maximum height to ensure consistency */
object-fit: cover; /* Ensure the image covers the available space */
+}
+
+.timelinetab{
+ border-radius: 20px;
+ padding: 20px;
+}
+
+
+.iframe-container {
+ display: flex;
+ justify-content: center;
+ align-items: center;
}
\ No newline at end of file
diff --git a/src/App/Timelines.css b/src/App/Timelines.css
index ba3f6cf3270d20ad62269684eedb0d723dd2d03b..c14380430da3e9d637f5566f5e898fbc7c863bb3 100644
--- a/src/App/Timelines.css
+++ b/src/App/Timelines.css
@@ -223,7 +223,7 @@ button.tabbutton:nth-child(1), button.tabbutton:nth-child(6){
.timeline-item-content {
box-shadow: 0 0 5px rgba(0, 0, 0, 0.3);
border-radius: 5px;
- background-color: var(--ourbeige);
+ background-color: white; /* Soll white bleiben! */
display: flex;
flex-direction: column;
align-items: flex-end;
@@ -275,7 +275,7 @@ button.tabbutton:nth-child(1), button.tabbutton:nth-child(6){
.timeline-end{
box-shadow: 0 0 5px rgba(0, 0, 0, 0.3);
border-radius: 5px;
-background-color: var(--ourbeige);
+background-color: white; /* soll white bleiben */
padding: 15px;
position: relative;
text-align: center;
@@ -285,7 +285,7 @@ margin-top: 8vw;
.timeline-begin{
box-shadow: 0 0 5px rgba(0, 0, 0, 0.3);
border-radius: 5px;
-background-color: var(--ourbeige);
+background-color: white; /* soll white bleiben */
padding: 15px;
position: relative;
text-align: center;
@@ -362,4 +362,16 @@ display: flex;
.timeline-item figcaption{
background-color: var(--ourbeige) !important;
+}
+
+
+.timeline-item figcaption, .timeline-item figure, .timeline-item figcaption h3 {
+ background-color: white !important;
+}
+
+
+
+.timeline-item .img-cube{
+ height: 120px;
+ max-height: 13vh;
}
\ No newline at end of file
diff --git a/src/components/BFH-Timeline.tsx b/src/components/BFH-Timeline.tsx
index f6cbbffa06221b6a0f3cfb807c81c7901aeafe46..0a9e027c2ce8ea1b97202411130922d26099f4fd 100644
--- a/src/components/BFH-Timeline.tsx
+++ b/src/components/BFH-Timeline.tsx
@@ -191,9 +191,14 @@ export function BFHTimeline () {
<h5>Team project presentations</h5>
<div className="col bfh-slider">
<SimpleSlider>
- <img className="" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
- <img className="" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
- <img className="" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7428-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7430-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7485-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7498-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7506-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7524-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7532-enhanced-nr.webp"/>
+ <img className="" src="https://static.igem.wiki/teams/5247/photos/meetup/team-presentations/anyconv-com-dscf7548-enhanced-nr.webp"/>
</SimpleSlider>
</div>
<span>
diff --git a/src/components/Pdfs.tsx b/src/components/Pdfs.tsx
index c324859aa9e25635e1371720898c091bec8eaf66..f6340892926d88d50004e0f0239ff88a82b64c89 100644
--- a/src/components/Pdfs.tsx
+++ b/src/components/Pdfs.tsx
@@ -3,13 +3,20 @@ import { ButtonOne, DownloadLink } from "./Buttons"
export function PDF({link, name}: {link: string, name:string}){
return(
- <div className='row align-items-center'>
- <iframe src={link} width="100%" title="title" className='one-pdf-line small-i'>
- </iframe>
- <div className='row download-col'>
- <DownloadLink url={link} fileName={name} ></DownloadLink>
- </div>
- </div>
+
+ <div className="col">
+ <div className='align-items-center'>
+ <div className="iframe-container">
+ <iframe src={link} title="title" className='one-pdf-line small-i'>
+ </iframe>
+ </div>
+ </div>
+ <div className='row download-col'>
+ <DownloadLink url={link} fileName={name} ></DownloadLink>
+ </div>
+ </div>
+
+
)
}
export function TwoLinePDF({link, name}: {link: string, name:string}){
diff --git a/src/components/Tabs.tsx b/src/components/Tabs.tsx
index 37395250ecabc7c68eea9686ba27367755b93e8e..0e1123d97abe6766e59d348666069eabbf85d328 100644
--- a/src/components/Tabs.tsx
+++ b/src/components/Tabs.tsx
@@ -73,7 +73,46 @@ import { stringToSlug } from "../utils";
}
/* textparts */
- if(data[i].aimofcontact === ""){
+ let aiM: JSX.Element = <></>
+ if(data[i].aimofcontact){
+ aiM = <>
+ <h4>{goalheading}</h4>
+ <div className="flexbox">
+ <p className="hp-timeline-p">{data[i].aimofcontact}</p>
+
+ {aim_img}
+ </div>
+ </>
+ }
+
+ let insighT: JSX.Element = <></>
+ if(data[i].insights){
+ insighT = <>
+ <h4>Insights</h4>
+ <div className="flexbox">
+ {inv_img}
+
+ <p className="hp-timeline-p">{data[i].insights}</p>
+ </div>
+ </>
+ }
+
+ let imP: JSX.Element = <></>
+ if(data[i].implementation){
+ imP = <>
+ <h4>Implementation</h4>
+ <div className="flexbox">
+ <p className="hp-timeline-p">{data[i].implementation}</p>
+
+ {imp_img}
+ </div>
+
+ </>
+ }
+
+
+
+ /* if(data[i].aimofcontact === ""){
problem = true;
problem_desc.push(" " + goalheading + " missing");
}
@@ -84,7 +123,7 @@ import { stringToSlug } from "../utils";
if(data[i].implementation === ""){
problem = true;
problem_desc.push(" implementation missing");
- }
+ } */
/* Clarification */
var clari: React.ReactNode = <></>
if(data[i].clarification){
@@ -152,6 +191,34 @@ import { stringToSlug } from "../utils";
</Collapsible>
}
+ let morepics: JSX.Element[] = [];
+ let oldpics: string[] = [];
+ /* if(data[i].more_pictures){ */
+ if (!data[i].aimofcontact && data[i].pictureurl_aim) {
+ console.info("!data[i].aimofcontact && data[i].pictureurl_aim")
+ oldpics.push(data[i].pictureurl_aim!)
+ }
+ if (!data[i].insights && data[i].pictureurl_interview) {
+ oldpics.push(data[i].pictureurl_interview!)
+ }
+ if (!data[i].implementation && data[i].pictureurl_implementation) {
+ oldpics.push(data[i].pictureurl_implementation!)
+ }
+ let newlist = oldpics.concat(data[i].more_pictures!);
+ for (let ind = 0; ind < (newlist.length - 1); ind += 2) {
+ morepics.push(
+ <div className="row">
+ <div className="col">
+ <img src={newlist[ind]}/>
+ </div>
+ <div className="col">
+ <img src={newlist[ind+1]}/>
+ </div>
+ </div>
+ )
+ }
+
+ /* } */
let node = <>
<br/>
@@ -160,7 +227,7 @@ import { stringToSlug } from "../utils";
<div className="row">
<div className="col-6">
<div className={"t-tag " + data[i].tag}>
- {data[i].job} {data[i].affiliation}
+ {data[i].job} - {data[i].affiliation}
</div>
</div>
<div className="col" style={{padding: "5px"}}>{expert}</div>
@@ -190,32 +257,20 @@ import { stringToSlug } from "../utils";
</div>
</div> */}
- <h4>{goalheading}</h4>
- <div className="flexbox">
- <p className="hp-timeline-p">{data[i].aimofcontact}</p>
-
- {aim_img}
- </div>
+ {aiM}
+ {insighT}
- <h4>Insights</h4>
- <div className="flexbox">
- {inv_img}
-
- <p className="hp-timeline-p">{data[i].insights}</p>
- </div>
-
{clari}
- <h4>Implementation</h4>
- <div className="flexbox">
- <p className="hp-timeline-p">{data[i].implementation}</p>
-
- {imp_img}
- </div>
+ {imP}
+
<div className="col">
- <BlockQuoteB text={data[i].quote} cite={quoted}></BlockQuoteB>
+ <BlockQuoteB text={data[i].quote!} cite={quoted}></BlockQuoteB>
</div>
+
+ {data[i].text}
+ {morepics}
{int}
{refs}
</>
@@ -231,14 +286,14 @@ import { stringToSlug } from "../utils";
else{
if (data[i].interviewtabid === "timeline"){
rows.push(
- <div id={data[i].interviewtabid} className={cla} style={{display: "block"}}>
+ <div id={data[i].interviewtabid} className={`${cla} timelinetab bg-db`} style={{display: "block"}}>
{node}
</div>
)
}
else{
rows.push(
- <div id={data[i].interviewtabid} className={cla}>
+ <div id={data[i].interviewtabid} className={`${cla} timelinetab bg-db`}>
{node}
</div>
)
@@ -248,7 +303,7 @@ import { stringToSlug } from "../utils";
}
return rows;
}
-
+
/* Interview */
export function OLDBFHStyleTabs({data, cla}: {data: Array<NodeDatensatz>, cla: string}) {
diff --git a/src/components/VerticalTimeline.tsx b/src/components/VerticalTimeline.tsx
index 7288796d43c60b56fdee3e4facb697f618d9a7e1..31d411f3e6b206e68966163d0541dc826ea853df 100644
--- a/src/components/VerticalTimeline.tsx
+++ b/src/components/VerticalTimeline.tsx
@@ -146,23 +146,23 @@ export const PanelTimelineItem = () => (
<time>Panel Discussion</time>
<div className="row align-items-center panel-talk-gallery">
<div className="col">
- <img className="img-round" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/traci.jpg" alt="pr-sample1" />
+ <img className="img-round-panel" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/traci.jpg" alt="pr-sample1" />
<div className="small middle">Taci <br/> Haddock</div>
</div>
<div className="col">
- <img className="img-round" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/anita-sifuantes.png" alt="pr-sample1" />
+ <img className="img-round-panel" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/anita-sifuantes.png" alt="pr-sample1" />
<div className="small middle">Ana <br/> Sifuentes</div>
</div>
<div className="col">
- <img className="img-round" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/olivia.png" alt="pr-sample1" />
+ <img className="img-round-panel" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/olivia.png" alt="pr-sample1" />
<div className="small middle">Olivia <br/> Mozolewska</div>
</div>
<div className="col">
- <img className="img-round" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/middendorf.jpeg" alt="pr-sample1" />
+ <img className="img-round-panel" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/middendorf.jpeg" alt="pr-sample1" />
<div className="small middle">Lasse <br/> Middendorf</div>
</div>
<div className="col">
- <img className="img-round" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/nemanja.jpg" alt="pr-sample1" />
+ <img className="img-round-panel" src="https://static.igem.wiki/teams/5247/photos/meetup/speakers/nemanja.jpg" alt="pr-sample1" />
<div className="small middle">Nemanja <br/> Stijepovic</div>
</div>
</div>
diff --git a/src/contents/Contribution/contribution.tsx b/src/contents/Contribution/contribution.tsx
index f7b7913377be08bb7e4b28a9f37dd55e8c762c45..21f1a0d3bae3be27933195ba5ef1b23260115df9 100644
--- a/src/contents/Contribution/contribution.tsx
+++ b/src/contents/Contribution/contribution.tsx
@@ -1,6 +1,7 @@
import { useTabNavigation } from "../../utils/TabNavigation";
import { MeetUP } from "./BFH/bfh";
import { CollabContribution } from "./collabs";
+import { PatientContribution } from "./patient-contribution";
import { SafetyContribution } from "./safety-contribution";
import { Wiki } from "./Wiki/wiki";
@@ -11,6 +12,7 @@ export function Contribution() {
<MeetUP/>
<SafetyContribution/>
<CollabContribution/>
+ <PatientContribution/>
<Wiki/>
</>
);
diff --git a/src/contents/Contribution/patient-contribution.tsx b/src/contents/Contribution/patient-contribution.tsx
new file mode 100644
index 0000000000000000000000000000000000000000..d323bf8da27d7ef3e4210c8cdc1cea01835dda5c
--- /dev/null
+++ b/src/contents/Contribution/patient-contribution.tsx
@@ -0,0 +1,26 @@
+
+import { TwoLinePDF } from "../../components/Pdfs";
+import { Section } from "../../components/sections";
+
+
+export function PatientContribution(){
+
+ return(
+ <>
+ <Section title="Patient Matters" id="Patient Matters" >
+ <div className='row align-items-center'>
+ <div className='col '>
+ <TwoLinePDF link="https://static.igem.wiki/teams/5247/pdfs/patienteneinwilligung-mustervorlage-igem.pdf" name="patienteneinwilligung-mustervorlage-igem.pdf"/>
+ </div>
+ <div className='seperator-2 col-2'>
+ </div>
+ <div className='col '>
+ <TwoLinePDF link="https://static.igem.wiki/teams/5247/pdfs/augearbeitetes-hygienekonzept.pdf" name="augearbeitetes-hygienekonzept.pdf"/>
+ </div>
+ </div>
+ </Section>
+
+ </>
+
+ )
+}
\ No newline at end of file
diff --git a/src/contents/Contribution/safety-contribution.tsx b/src/contents/Contribution/safety-contribution.tsx
index 8b9fd7115db47413986c438aa79c97aa3b494e25..cb5a85c1066bdbc278a33a1e77d0e4f75d71634c 100644
--- a/src/contents/Contribution/safety-contribution.tsx
+++ b/src/contents/Contribution/safety-contribution.tsx
@@ -6,12 +6,9 @@ export function SafetyContribution(){
return(
<>
- <Section title="Patient Consent Form" id="Patient Consent Form" >
- <PDF link="https://static.igem.wiki/teams/5247/pdfs/patienteneinwilligung-mustervorlage-igem.pdf" name="patienteneinwilligung-mustervorlage-igem.pdf"/>
+ <Section title="Primary Culture Safety Guideline" id="Safety Guideline" >
+ <PDF link="https://static.igem.wiki/teams/5247/pdfs/primary-culture-guideline.pdf" name="primary-culture-guideline.pdf"/>
</Section>
- <Section title="Primary Culture Safety Guidelines" id="Safety Guidelines">
- <PDF link="https://static.igem.wiki/teams/5247/pdfs/primary-culture-guideline.pdf" name="primary-culture-guideline.pdf"/>
- </Section>
</>
)
diff --git a/src/contents/Human Practices/Conclisuin.tsx b/src/contents/Human Practices/Conclisuin.tsx
index aa6e739fc4db3770e612b09e35a039e0ab872ffd..27335a16746e99106239bc632a524c2aa31b4793 100644
--- a/src/contents/Human Practices/Conclisuin.tsx
+++ b/src/contents/Human Practices/Conclisuin.tsx
@@ -4,6 +4,7 @@ import { useNavigation } from "../../utils";
export function HPconclusion(){
const {goToPagesAndOpenTab} = useNavigation();
+ const {goToPageAndScroll} = useNavigation();
return(
<>
<p>Our project has evolved through a deeply collaborative and human-centered approach, integrating diverse feedback from patients, clinicians, researchers, and industry experts. These insights shaped not only the technical aspects of our gene therapy for cystic fibrosis (CF) but also our commitment to addressing real-world patient needs, ethical considerations, and the disparities in CF treatment worldwide. From<a onClick={() => goToPagesAndOpenTab('maxfirst', '')}>Max Beckmann’s</a> patient perspective to expert guidance on technical and ethical issues, each stakeholder contributed to refining our solution, ensuring it is both innovative and empathetic. Our focus on gene therapy targeting CF’s complex mutations, integrating physiotherapy, and ensuring global accessibility demonstrates our holistic and inclusive vision for this project. Importantly, the collaboration with researchers in nanoparticle stability and gene therapy, along with the development of bilingual surveys and outreach materials, highlights our efforts to make science more accessible and transparent, bridging gaps in knowledge and care. </p>
@@ -14,7 +15,7 @@ export function HPconclusion(){
<H5 text="Ethical, Legal, and Regulatory Considerations "/>
<p>Collaboration with regulatory experts, such as <a onClick={() => goToPagesAndOpenTab('berens', '')}> Dr. Eva-Maria Berens </a> , ensured our work adhered to ethical and legal standards, particularly in patient consent and biosafety protocols. Through feedback from legal and bioethics committees, we refined our consent processes and improved our management of personal data, ensuring our project complied with both German and international regulations. </p>
<H5 text="Technical and Scientific Adaptations "/>
- <p>We continuously integrated expert technical feedback into the project. Contributions from <a onClick={() => goToPagesAndOpenTab('rnhale', '')}>Dr. Benjamin Winkeljann</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a> helped us improve the stability and scalability of our spray-dried lipid nanoparticles (LNPs), which are essential for RNA delivery. This practical focus on scalable solutions allowed us to address global challenges, such as the need for transportable therapies. Collaborating with yeast cultivation experts like <a onClick={() => goToPagesAndOpenTab('nberelsmann', '')}>Nils Berelsmann</a>, we also optimized RNA delivery systems[Link] to ensure effective lung penetration. </p>
+ <p>We continuously integrated expert technical feedback into the project. Contributions from <a onClick={() => goToPagesAndOpenTab('rnhale', '')}>Dr. Benjamin Winkeljann</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a> helped us improve the stability and scalability of our spray-dried lipid nanoparticles (LNPs), which are essential for RNA delivery. This practical focus on scalable solutions allowed us to address global challenges, such as the need for transportable therapies. Collaborating with yeast cultivation experts like <a onClick={() => goToPagesAndOpenTab('nberelsmann', '')}>Nils Berelsmann</a>, we also optimized <a onClick={() => goToPageAndScroll ('lnps', '/materials-methods')}> RNA delivery Systems </a> to ensure effective lung penetration. </p>
<H5 text="Global Impact and Inclusivity "/>
<p>Recognizing the disparities in CF care across different regions, particularly in underrepresented areas like Asia, we adjusted our approach to create a more inclusive therapy. With feedback from stakeholders like <a onClick={() => goToPagesAndOpenTab('joshua', '')}>Joshua Bauder</a> from CF Vest International and <a onClick={() => goToPagesAndOpenTab('sriram', '/human-practices')}>Dr. Sriram Vaidyanathan</a>, we ensured our therapy addressed a wider range of CF mutations. This global focus led to bilingual surveys and expanded outreach efforts to raise awareness about CF and gene therapy. </p>
<H5 text="Feedback Loops and Project Evolution "/>
@@ -325,10 +326,11 @@ function AnalyseWischmeyer(){
)
}
function AnalyseMattijs(){
+ const {goToPagesAndOpenTab} = useNavigation();
return(
<Collapsible title="Mattjis Bulcaen – University of Leuven, CF and Prime Editing Expert" id="mattijsanalyseC">
<p>Mattijs Bulcaen, being a researcher working on a topic very close to ours, provided invaluable guidance in the early stages of our project. He gave us an insight into the current advances in the field that we that we were able to make use of later on. He reviewed with us our own ideas and considerations, such as the use of the PEAR reporter system. Following our interview with Mattijs we integrated the structural motif trevopreQ1 into the pegRNA, which enhanced the prime editing efficiency - a critical improvement we successfully tested and demonstrated in our results. </p>
- <p>Our decision of first testing prime editing in HEK293 cells instead of other cell lines or primary cells was also based on his statement that HEK cells are by far the easiest to archieve editing in. Mattijs additionally recommended the use of HEK293T cell lines overexpressing CFTR and CFTR F508del[link]. However, after conducting patch clamp measurements, we found that the HEK cells were unsuitable for our needs, requiring us to adjust our testing system. As a result, we switched to Ignatova[Link] cells from Hamburg for further experiments. </p>
+ <p>Our decision of first testing prime editing in HEK293 cells instead of other cell lines or primary cells was also based on his statement that HEK cells are by far the easiest to archieve editing in. Mattijs additionally recommended the use of HEK293T cell lines overexpressing CFTR and CFTR F508del[link]. However, after conducting patch clamp measurements, we found that the HEK cells were unsuitable for our needs, requiring us to adjust our testing system. As a result, we switched to <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Ignatova's</a> cells from Hamburg for further experiments. </p>
<figure>
<div className="row">
<div className="col">
@@ -410,6 +412,11 @@ function AnalyseBerens(){
const {goToPagesAndOpenTab} = useNavigation();
return(
<Collapsible title="Dr. Eva-Maria Berens – Head of ethics committee at University of Bielefeld" id="berensanalyseC">
+ <p>In our pursuit of generating primary cultures from human nasal epithelial cells for testing, we sought advice from the Biosafety and Security Committee of iGEM, as well as consult with Dr. Eva-Maria Berens, our ethics officer. Through in-depth discussions with Dr. Berens, we carefully assessed the risks and legal obligations associated with our project. We learned that, given the minimal invasiveness of the procedure, no formal bioethics application was necessary. However, we recognized the importance of developing a legally sound informed consent form for participants, ensuring that we adhered to the relevant legal frameworks. </p>
+ <p>In collaboration with various legal institutions, we drafted a comprehensive informed consent form that complies with both national regulations in Germany and the specific policies of Bielefeld University. As the first iGEM team to tackle the complex cultivation of human primary nasal epithelial cells, we were committed to paving the way for future teams. To support this, we created a guideline documenting the proper handling of human biomaterial obtained through these types of sample collection. </p>
+ <p>Additionally, we worked with Mr. Timm Weber[Link], who is working for the local biobank OWL, to establish protocols for the management and storage of sensitive, personally identifiable data. While no special procedures were required for our specific project, we nonetheless made it a priority to implement anonymized methods to protect participant privacy. </p>
+ <p>The input and feedback from Dr. Berens and Mr. Weber formed the basis of our ethical consideration of our project. With their help, we were able to improve our technology on primary cultures with a higher level of safety and an adapted guidline. In addition, our biosafety contributions were significantly improved as we were able to ensure compliance with all legal and ethical standards.. </p>
+ <p>Having established the necessary ethical and legal groundwork, we turned our focus to the practical aspects of handling and cultivating patient samples. This required specialized expertise, which led us to seek assistance from <a onClick={() => goToPagesAndOpenTab('michaela', '/human-practices')}>Stemcell Technologies</a> ensure the successful cultivation and maintenance of the nasal epithelial cells. Their support was essential in enabling us to push forward with our testing and bring our project closer to real-world applications. </p>
<figure>
<div className="row">
<div className="col">
diff --git a/src/contents/Human Practices/Feedback.tsx b/src/contents/Human Practices/Feedback.tsx
index 7a2fef3290683df55170498d1c2ab83a77304b85..680f7fabd9746e1f3a8c2b20bec354c875b9113a 100644
--- a/src/contents/Human Practices/Feedback.tsx
+++ b/src/contents/Human Practices/Feedback.tsx
@@ -4,9 +4,10 @@ import { H4, H5 } from '../../components/Headings';
import { Collapsible } from "../../components/Collapsible";
import PreCyse from '../../components/precyse';
import { HPconTabs } from './Conclisuin';
+import { useNavigation } from '../../utils';
export function HPFeedback(){
-
+ const {goToPageWithTabAndScroll} = useNavigation();
return(
<div>
<p>Through our project, the insights and feedback from various stakeholders and experts played a crucial role in shaping and refining our approach. We actively integrated their input into the design, execution, and public engagement aspects of our work, ensuring a human-centered, scientifically sound solution. Below, we highlight key contributors and how their feedback impacted the project's development across multiple phases. </p>
@@ -14,7 +15,7 @@ export function HPFeedback(){
<H4 text="Our surveys on cystic fibrosis and gene therapy"></H4>
<p>From our outreach efforts, we learned that many people lack knowledge about cystic fibrosis and desire more education on the subject. The same applies to gene therapy, with most individuals expressing openness to treatment options, which reinforces our commitment to pursuing this approach. </p>
<p>However, we recognize the importance of handling the public's trust and lack of knowledge responsibly. We aim to educate the community about safety and ethical considerations surrounding gene therapy. </p>
- <p>In response, we have decided to implement feedback by creating informative materials such as flyers and utilizing platforms like Muko Move [Link] [PO_MUKOmove.docx], Teuto ruft [Link] [HP_TeutoRuft.docx], SchülerInnenakademie [Link] [PO_Schülerakademie.docx], and social media [Link] [https://www.instagram.com/igem.bielefeld/?hl=de] to raise awareness and provide education. </p>
+ <p>In response, we have decided to implement feedback by creating informative materials such as flyers and utilizing platforms like <a onClick={() => goToPageWithTabAndScroll({tabId: 'mukomove', scrollToId: "cf-month" , path: '/human-practices'})}>mukoMOVE</a>, <a onClick={() => goToPageWithTabAndScroll({tabId: 'teutoruft', scrollToId: "teuroruft-heading" , path: '/human-practices'})}>â€Teuto ruft!â€</a>, <a onClick={() => goToPageWithTabAndScroll({tabId: 'akademie', scrollToId: "student-academy-heading" , path: '/human-practices'})}>SchülerInnenakademie</a>, and <a href='https://www.instagram.com/igem.bielefeld/?hl=de'>social media</a> to raise awareness and provide education. </p>
<figure>
<div className="row">
<div className="col">
diff --git a/src/contents/Human Practices/Introduction.tsx b/src/contents/Human Practices/Introduction.tsx
index 8d839dd125af5749005c4033cf730de44371a20d..a77cf756e346348dabc509b7c960e64a71a437e0 100644
--- a/src/contents/Human Practices/Introduction.tsx
+++ b/src/contents/Human Practices/Introduction.tsx
@@ -50,7 +50,7 @@ export function HPIntroduction(){
while developing an awareness of the responsibilities that come with it.
</p>
<p>
- As part of our <PreCyse/> project, we performed intensive brainstorming sessions and expert consultations. We conducted comprehensive
+ As part of our <PreCyse/> project, we performed intensive brainstorming sessions and expert consultations. We conducted comprehensive
<a onClick={() => goToPageAndScroll("our-surveys-on-cystic-fibrosis-and-gene-therapy", "/human-practices")}>surveys</a> among the public and people with cystic fibrosis and their relatives. We focused on critical aspects such as the <b>needs of our
target groups, safety, ethics, design, implementation, and business</b> — each guided by the core values of our team. Based on these interactions
and the recommendations of the Human Practice committee, we have developed an optimal strategy for our project, ensuring that our work is not
diff --git a/src/contents/description.tsx b/src/contents/description.tsx
index bf0e0cb5a4eab8778c6b010e7b874742d0a8c5f7..9973655ded76d524f5a8fc78081ac3947925cf6b 100644
--- a/src/contents/description.tsx
+++ b/src/contents/description.tsx
@@ -2,9 +2,7 @@ import { InfoBox } from "../components/Boxes";
import { TabButtonRow } from "../components/Buttons";
import Collapsible from "../components/Collapsible";
import { SupScrollLink } from "../components/ScrollLink";
-import { H2, H4} from "../components/Headings";
-import { LoremMedium } from "../components/Loremipsum";
-import { Circle } from "../components/Shapes";
+import { H4} from "../components/Headings";
import { ButtonRowTabs } from "../components/Tabs";
import PieChart from "../components/Graph";
import PreCyse from "../components/precyse";
@@ -14,7 +12,6 @@ import { Section, Subesction } from "../components/sections";
import { symptomdata, SymptomDatensatz } from "../data/symptom-data";
import { drugdata, DrugDatensatz } from "../data/drug-data";
import { useTabNavigation } from "../utils/TabNavigation";
-import { QuizQuestion } from "../components/Quiz";
import PrimeEditingComplex from "../components/Complex-svg";
import { useNavigation } from "../utils";
@@ -26,105 +23,131 @@ export function Description() {
<div className="row mt-4">
<div className="col">
<Section title="Abstract" id="Abstract">
- <p id="obenindescription" >We are proud to introduce our next-generation prime editing technology <PreCyse/> . We aim to develop an innovative gene therapy against cystic fibrosis, tackling the most common mutation F508del of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. We optimize lipid nanoparticles (LNPs) for the efficient and cell-specific delivery of our therapeutic mRNA. Current treatment strategies are limited in terms of speed, precision and effectiveness, often failing to achieve long-lasting improvements. In addition, high costs and limited accessibility of pharmaceuticals contribute to adverse prognosis of many patients. We want to develop a monthly applied which represents a cure that is more advanced and user-friendly compared to other medications due to its longer lasting time, lowering the frequency of use. </p>
+ <p id="obenindescription" >We are proud to introduce <PreCyse/>, our next-generation Prime Editing technology aimed at developing an innovative gene therapy for cystic fibrosis, specifically targeting the most common mutation, <b>F508del</b> of the CFTR gene. PreCyse is designed to address key limitations in current treatments, including limited size, speed, precision, and long-term effectiveness. Our approach integrates <b>PrimeGuide</b>, a highly optimized Prime Editing system, with <b>AirBuddy</b>, a novel lipid nanoparticle (LNP) delivery platform. The <b>SORT LNPs</b> used in AirBuddy are optimized for pulmonary delivery, offering precise organ targeting and structural stability throughout the inhalation process. As a Postdoc at the Broad Institute of MIT and Havard—where Prime Editing was first pioneered—observed, “<i>Everyone is looking for smaller prime editors</i>†and PrimeGuide embodies this vision by utilizing a smaller, more efficient editing complex. Unlike current treatments, which often require frequent administration and provide only temporary relief, PreCyse is developed as a monthly applied therapy with the potential for longer-lasting results, reducing the frequency of use and improving patient outcomes. By lowering costs and improving accessibility, PreCyse aims to offer a more advanced and user-friendly cure for cystic fibrosis. </p>
</Section>
<Section title="Our Motivation" id="Our Motivation">
- <p>We chose to focus on CF and specifically the F508del mutation due to its prevalence and the severe impact it has on patients' lives. Additionally, our team includes members who have close friends affected by this condition, giving us a personal connection and a strong motivation to find a solution. By targeting the F508del mutation, we aim to develop a therapy that could potentially, not only benefit many CF patients and make a significant improvement in their lives, but also can serve as a template, which research groups can use to target other genetic diseases. </p>
<div className="row align-items-center">
<div className="col" >
- </div>
+ <p>Our project started with a personal story. Rather than being driven purely by academic curiosity, our motivation came from someone close to one of our team members — Max Beckmann, a friend who has lived with Cystic Fibrosis (CF) since his birth. Specifically, he carries the F508del mutation, the most common genetic cause of the disease. Seeing the impact of CF on his daily life—frequent treatments and physical strain—made us realize how much more can be done to improve the lives of those affected, which inspired us to pursue this project. </p>
+ <p>As we explored Cystic Fibrosis further, we were struck by how widespread it is, being the most common genetic disorder in Germany. Approximately 70% of those with CF are specifically affected by the F508del mutation [1]. This mutation is the most prevalent and well-studied of the thousands of genetic variations that cause CF, making it an important focus of research and intervention. In fact, about 90% of Europeans and individuals of European descent with CF have at least one F508del allele [2, 3]. This widespread prevalence highlighted the significance of our project—not just for our friend, but for the thousands of others affected by this mutation across Europe and beyond. </p>
+ </div>
<div className="col" >
- <img className="img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
+ <img className="img" src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
</div>
- </div>
- <p>Max</p>
+ </div>
+ <p>By focusing on the F508del mutation, we also hope to contribute valuable insights to the global cystic fibrosis community. Although this mutation is most common in European populations, it is also found in other regions around the world [4, 5]. Our research could thus help inform treatment strategies and health policies on an international scale. </p>
+ <p>With several team members focusing their studies on biomedical fields, we began by examining the current landscape of CF treatments. It quickly became clear that, despite recent progress, there is still no cure. Most therapies, such as CFTR modulators, focus on managing symptoms and improving lung function rather than addressing the underlying cause of the disease [6]. This realization led us to explore gene-editing technologies, thus leading us to Prime Editing—a next generation gene editing method—captured our attention. </p>
+ <p>While Prime Editing holds great promise, we found that its application for cystic fibrosis, particularly the F508del mutation, had not been fully explored. Recognizing this gap in the research inspired us to take on the challenge of optimizing Prime Editing for this specific mutation. Our mission became clear: we want to contribute to the development of a potential therapeutic approach for cystic fibrosis, specifically targeting the F508del mutation with prime editing, and bring us closer to a long-term solution for patients. </p>
</Section>
<Section title="Cystic Fibrosis" id="Cystic Fibrosis">
<Subesction title="Overview" id="Cystic Fibrosis1">
<div className="row align-items-center">
<div className="col">
- <p data-aos="zoom-y-out" >Cystic fibrosis (CF) is the most common life-limiting genetic disorder in the Caucasian population. In Europe, CF affecting about 1 in 3,000 newborns
- <SupScrollLink label="1"/>.</p>
- <p> It is caused by mutations in the CFTR gene, which controls ions and water movement in cells. This leads to thick mucus, clogging airways, and frequent infections. The defective CFTR protein impacts the respiratory and digestive systems, causing chronic lung infections, breathing difficulties, and malnutrition. CF's severity varies, but it reduces life quality and expectancy. There are over 1,700 CFTR mutations; the F508del mutation is most common, present in 70% of cases. It prevents proper protein folding, affecting its function. </p>
- <Collapsible id="fanzorcas-collapsible" title="Cas vs. Fanzor">
- <p>The mutations can be divided into six classes <SupScrollLink label="9"/>:</p>
- <p>Class I mutations prevent the synthesis of CFTR proteins altogether, meaning no channels are produced.</p>
- <p>Class II mutations, which include the common F508del mutation (responsible for about 85% of cases <SupScrollLink label="10"/>), disrupt the maturation process of the protein. As a result, the defective channels are quickly degraded by the cell.</p>
- <p>Class III mutations, known as “gating†mutations, reduce the likelihood that the CFTR channel will open correctly, impairing its function.</p>
- <p>Class IV, V, and VI mutations are rare. These mutations result in the production of unstable or inefficient CFTR proteins, which do not function adequately and are produced in insufficient numbers.</p>
+ <p data-aos="zoom-y-out" >Cystic Fibrosis (CF) is a common life-limiting genetic disorder, particularly affecting the Caucasian population, with approximately 162,400 people worldwide living with the condition [1]. Statistically, about one in every 3,300 white newborns is born with CF [2]. And according to the German Cystic Fibrosis Registry, the average life expectancy for children born with CF in 2021 was around 57 years [3], highlighting the severe and life-shortening nature of the disease. </p>
+ <p>The modern understanding of CF dates back to 1932 when Dr. Dorothy Andersen, a pediatric specialist, first described the disease and coined the term "Cystic Fibrosis" [4]. In Germany, it is commonly known as "Mukoviszidose," derived from the Latin words meaning "mucus" and "viscous" [4], emphasizing the characteristic thick, sticky mucus that defines the condition [5, 6]. </p>
+ <p>Genetic research has identified over 1,700 mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, with the ∆F508 mutation being the most common, affecting about 70% of CF patients. This mutation prevents the proper folding of the CFTR protein, significantly impairing its function [7]. </p>
+ <p>The CFTR protein regulates the flow of chloride ions across the membranes of cells in the lungs, digestive system, and other organs. This ion flow is essential for drawing water into surrounding tissues, which helps maintain the proper hydration and consistency of mucus. In patients with CF, the disruption of this process prevents sufficient water from entering the mucus, making it abnormally thick and sticky. The accumulation of this mucus leads to an obstruction of airways and digestive ducts, resulting in chronic lung infections, inflammation, impaired digestion, and malnutrition[8]. </p>
+ <div className="casettecontainer" >
+ <figure>
+ <img className="img" src="https://static.igem.wiki/teams/5247/project-description/lung-ephitel-biorender.png"/>
+ <figcaption>
+ <b>Figure 1: </b>
+ Lung ephitelium of human with correct CFTR expression (left) and Cystic Fibrosis (right)
+ </figcaption>
+ </figure>
+ </div>
+ <Collapsible id="classes-mutations-collapsible" title="Different classes of mutations">
+ <p>The mutations can be divided into <u>six classes</u> <SupScrollLink label="9"/>:</p>
+ <p><b>Class I</b> mutations prevent the synthesis of CFTR proteins altogether, meaning no channels are produced.</p>
+ <p><b>Class I</b> mutations, which include the common F508del mutation (responsible for about 85% of cases <SupScrollLink label="10"/>), disrupt the maturation process of the protein. As a result, the defective channels are quickly degraded by the cell.</p>
+ <p><b>Class I</b> mutations, known as “gating†mutations, reduce the likelihood that the CFTR channel will open correctly, impairing its function.</p>
+ <p><b>Class IV, V</b> and <b>VI</b> mutations are rare. These mutations result in the production of unstable or inefficient CFTR proteins, which do not function adequately and are produced in insufficient numbers.</p>
</Collapsible>
- <LoremMedium/>
- </div>
- <div className="row-if-small col-2 ">
- <Circle text="1:3000 newborns worldwide"/>
- <Circle text="x:y newborns in Germany"/>
- <Circle text="kosten"/>
- </div>
- {/* <Linear
- xAxis={[{ data: [1, 2, 3, 5, 8, 10] }]}
- series={[
- {
- data: [2, 5.5, 2, 8.5, 1.5, 5],
- },
- ]}
- width={500}
- height={300}
- /> */}
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"></img>
- </div>
-
+ <p>The prevalence of CF varies globally, with higher concentrations of cases in Europe, North America, and parts of Oceania. This geographic variation underscores the need for regionally tailored healthcare solutions. </p>
+ </div>
+ <div className="casettecontainer">
+ <img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"></img>
+ </div>
+ </div>
+ <p>CF is often diagnosed early through newborn screening programs, which detect elevated levels of immunoreactive trypsinogen (IRT). A positive result typically leads to a sweat test, the gold standard for diagnosing CF, which measures the concentration of chloride in sweat. </p>
+ <p>Although there is currently no cure for CF, patients must manage the disease throughout their lives, relying on treatments that alleviate symptoms but do not address the root cause. This lifelong management imposes significant financial burdens on affected families and healthcare systems, particularly in regions with a high prevalence of CF [9]. In recent years, <b>CFTR modulators</b>, which target the underlying genetic defect, have offered new hope for many patients. </p>
</Subesction>
<Subesction title="The CFTR Protein" id="Cystic Fibrosis2">
- <div className="row align-items-center">
- <figure>
- <div className="row">
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
- </div>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
- </div>
- </div>
- <figcaption><b>Figure x.</b> </figcaption>
- </figure>
-
- <div className="col">
- <p>Text about CFTR </p> <LoremMedium/>
- <div className="figure-wrapper">
- <figure>
- <div className="col gif-wrapper">
- <img className="CFTR-gif" src="https://static.igem.wiki/teams/5247/fanzor/cftr-wt.gif"></img>
- </div>
- <figcaption> <b>Figure x.</b></figcaption>
- </figure>
- </div>
-
+ <p>The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein is an ion channel that facilitates the movement of chloride ions across epithelial cell membranes [1][2]. This movement is essential for controlling the flow of water in tissues such as the lungs and intestines [3]. This increase in ion concentration in the extracellular space draws water out of the cells and into the surrounding mucus or fluid, ensuring it stays thin and mobile [4].</p>
+ <p>The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is a specialized protein that plays a crucial role in maintaining the balance of ions and water on the surface of certain cells, particularly in the lungs, pancreas, and other organs [2][5]. </p>
+ <H4 text="Structure of CFTR" id="structure-cftr" />
+ <p>CFTR is a large protein embedded in the cell membrane. It belongs to a family of proteins known as ABC transporters (ATP-Binding Cassette transporters), which typically move molecules across cell membranes [1]. CFTR, however, is unique because it functions as an ion channel rather than a transporter [3]. </p>
+ <p>The protein consists of several important regions: </p>
+ <ul>
+ <li><b>Two transmembrane domains (TMDs)</b>: These span the cell membrane and create the channel through which ions can flow [4].</li>
+ <li><b>Two nucleotide-binding domains (NBDs)</b>: Located on the cytoplasmic side of the membrane, these domains bind and hydrolyze ATP (adenosine triphosphate). ATP binding and hydrolysis regulate the opening and closing of the chloride channel [1] [5].</li>
+ <li><b>Regulatory (R) domain</b>: This domain is unique to CFTR and controls the activity of the protein. It requires phosphorylation by protein kinase A (PKA) to activate the ion channel [2][3].</li>
+ </ul>
+ <div className="row align-items-center">
+ <figure>
+ <div className="row">
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
+ </div>
+ <div className="col">
+ <img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
+ </div>
+ </div>
+ <figcaption><b>Figure 2: </b> 3D model of the CFTR protein in its wildtype form (left) and with the F580del mutation (right) </figcaption>
+ </figure>
+ <div className="col">
+ <div className="figure-wrapper">
+ <figure>
+ <div className="col gif-wrapper">
+ <img className="CFTR-gif" src="https://static.igem.wiki/teams/5247/fanzor/cftr-wt.gif"></img>
+ </div>
+ <figcaption> <b>Figure x.</b></figcaption>
+ </figure>
+ </div>
+ <H4 text="Function of CFTR" id="function-cftr" />
+ <p>CFTR functions primarily as a chloride ion channel. It is responsible for transporting chloride ions (Clâ») across epithelial cell membranes [1][4]. Here's how it works:</p>
+ <ol>
+ <li><b>Regulation by phosphorylation</b>: The R domain must first be phosphorylated by PKA to allow channel activation. This phosphorylation is often triggered by cyclic AMP (cAMP), a signaling molecule [5].</li>
+ <li><b>Opening the channel</b>: Once the R domain is phosphorylated, ATP binds to the NBDs, causing conformational changes that open the chloride channel [2].</li>
+ <li><b>Chloride transport</b>: With the channel open, chloride ions move from inside the cell to the outside. This movement of chloride helps draw water out of the cell, thinning mucus and maintaining proper hydration of the epithelial surfaces [3][4].</li>
+ <li><b>Closing the channel</b>: Hydrolysis of ATP causes the channel to close after a certain period, tightly regulating chloride transport [1].</li>
+ </ol>
+ <p>CFTR plays a critical role in maintaining the fluid balance on the surfaces of tissues such as the airways, digestive tract and sweat glands. By allowing chloride ions to flow out of the cells, CFTR ensures that water follows, preventing the accumulation of thick, sticky mucus [3].</p>
+ <H4 text="CFTR in Cystic Fibrosis" id="CFTR-in-cftr" />
+ <p>In the lungs, this water movement is crucial for maintaining a thin, slippery layer of mucus that can trap and remove particles like dust and bacteria. The mucus is then moved out of the lungs by the action of cilia, tiny hair-like structures on the surface of epithelial cells. When the CFTR protein is defective, as in cystic fibrosis, chloride cannot properly exit the cells. This disrupts the osmotic gradient, preventing water from entering the mucus. As a result, the mucus becomes thick and sticky, making it difficult to clear and creating an ideal environment for bacterial infections, which leads to chronic inflammation and lung damage over time.</p>
+ <p>In the intestines, CFTR regulates fluid secretion into the digestive tract, which is vital for the normal movement of digestive contents. Without proper CFTR function, water movement is reduced, leading to thickened digestive fluids, blockages, and impaired nutrient absorption. This contributes to malnutrition and other digestive complications in cystic fibrosis patients. </p>
+ <p>By correcting the genetic mutations that lead to CFTR malfunction, such as the ∆F508 mutation, we aim to restore the proper balance of chloride and water movement, which is crucial for preventing the buildup of mucus and ensuring normal function in both the respiratory and digestive systems.</p>
</div>
</div>
</Subesction>
<Subesction title="F508del" id="Cystic Fibrosis3">
- <p>A multitude of mutations in the CFTR gene, exceeding 1,000, are responsible for the development of cystic
- fibrosis. The most prevalent variant is F508del, observed in approximately 70% of affected individuals of
- Caucasian descent in Canada, Northern Europe, and the United States<SupScrollLink label="14"/>. It is estimated that around 90% of
- the European population and people of European heritage with cystic fibrosis carry at least one F508del
- variant <SupScrollLink label="15"/><sup>,</sup><SupScrollLink label="16"/>. Analyses have demonstrated that the F508del mutation originated in Western Europe at least
- 5,000 years ago <SupScrollLink label="15"/>. </p>
- <p>It is a deletion of the three nucleotides "CTT" at position 508, which removes the phenylalanine residue
- without causing a frameshift. This deletion leads to defects in the kinetic and thermodynamic folding
- of the NBD1 domain <SupScrollLink label="16"/>. However, this not only leads to misfolding of CFTR but also to defects in
- trafficking and premature degradation, resulting in reduced surface expression of CFTR <SupScrollLink label="17"/>. </p>
+ <p>More than 1,000 mutations in the CFTR gene are responsible for the development of cystic fibrosis. The most common variant is the F508del mutation, found in approximately 70% of affected individuals of Caucasian descent in Canada, Northern Europe, and the United States [18]. It is estimated that around 90% of people with cystic fibrosis in Europe and those of European heritage carry at least one F508del allele [19, 20]. Research suggests that this mutation originated in Western Europe at least 5,000 years ago [19].</p>
+ <p>The F508del mutation involves the deletion of three nucleotides, "CTT," at position 508, which removes a phenylalanine residue without causing a frameshift. This deletion impairs the kinetic and thermodynamic folding of the NBD1 domain [21]. As a result, the CFTR protein not only misfolds but also experiences defects in trafficking and premature degradation, leading to a reduction in its surface expression [22]. This specific mutation is particularly severe because it affects both the production and function of CFTR, resulting in a more aggressive disease course. Consequently, patients with the F508del mutation may respond better to CFTR modulators, which target these specific defects in protein folding and function.</p>
+ <Collapsible id="statistical-distribution-collapsible" title="Statistical distribution of F508del mutations">
+ <p>In 2023, a comprehensive analysis was conducted to assess the distribution of mutations in the CFTR gene associated with cystic fibrosis (CF) worldwide. Data was sourced from two reputable databases: the <a href="https://cftr.iurc.montp.inserm.fr/cgi-bin/variant_list.cgi" title="CFTR-database-1" >CFTR Mutation Database</a> and the <a href="https://cftr2.org/mutations_history" title="CFTR-database-2" >CFTR2 Database</a>. </p>
+ <p>The results indicate the following distribution of CFTR mutation types and their frequencies in percent: </p>
+ <div className="row align-items-center">
+ <div className="col" >
+ <ul>
+ <li><b>Insertions (ins)</b>: 0.00088%</li>
+ <li><b>Deletions (del)</b>: 72.64%</li>
+ <li><b>Substitutions (subs)</b>: 23.84%</li>
+ <li><b>Insertions/Deletions (indel)</b>: 0,00485%</li>
+ <li><b>Other mutations</b>: 0,00370%</li>
+ </ul>
+ </div>
+ <div className="col" >
+ <PieChart /> {/* Render the PieChart component */}
+ </div>
+ </div>
+ </Collapsible>
+ <p>Overall, the statistical distribution of CFTR mutations reveals significant variations in mutation types and their frequencies worldwide, with deletions (72.64%) being the most common mutation type. This underscores the need for continued research and monitoring of these genetic variations to improve patient care and treatment strategies. CF not only affects the directly affected organs, but also many other areas of the body that are indirectly affected by the extent of the disease, e.g. through the condition of diseased organs. </p>
<div className="row">
<div className="col">
- <img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"/>
- </div>
- <div className="col-4">
- <QuizQuestion name="schreibweise" front="What do the codes F508del and F508del stand for?" back="they..."/>
+ <PieChart/>
</div>
+ <div className="col"></div>
</div>
-
-
</Subesction>
<Subesction title="Symptoms" id="Cystic Fibrosis4">
<p>Since the CFTR gene is expressed in nearly all tissues of the human body, cystic fibrosis affects as a metabolic disease a wide range of vital organs.</p>
@@ -134,50 +157,79 @@ export function Description() {
</Collapsible>
</Subesction>
<Subesction title="Diagnosis" id="Cystic Fibrosis5">
- <p>About the ways one can be diagnosed </p> <LoremMedium/>
- <div className="row align-items-center">
- <div className="col" >
- <img src="https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg"/>
- </div>
- <div className="col" >
- How newbornscreening affected the numbers.
- <LoremMedium/>
- </div>
- </div>
+ <p>With Cystic Fibrosis being a hereditary disease, the diagnostic methods have evolved significantly [1][2]. Early diagnosis is crucial, as it allows for timely interventions that can improve the quality of life and longevity for CF patients [1][3]. With advancements in screening and diagnostic tools, many individuals are diagnosed shortly after birth, enabling early management of the disease [3][4].</p>
+ <p>Cystic fibrosis can be diagnosed through a variety of methods, often starting in infancy or even before birth [2][5]. The most common diagnostic test is the newborn screening, which involves a blood test that checks for elevated levels of a protein called immunoreactive trypsinogen (IRT) [4][6]. Elevated IRT levels can indicate potential CF, prompting further testing [6]. </p>
+ <Collapsible id="newborn-screening-collapsible" title="Newborn screening">
+ <p>Newborn screening for Cystic Fibrosis (CF) has been a major advancement in early detection and management, leading to significantly improved patient outcomes. This practice, which started in the late 1970s, became more widespread in the 1980s. The screening typically involves a blood test within the first few days of life, measuring immunoreactive trypsinogen (IRT), a marker that is elevated in newborns with CF. Elevated IRT levels prompt further genetic testing to identify CFTR mutations [25]. If mutations are found, a sweat chloride test is often conducted to confirm the diagnosis. </p>
+ <p>Many countries, including the United States, Canada, the United Kingdom, Australia, and several European nations, have implemented newborn screening programs for CF. However, a survey of CF screening in Europe revealed that the implementation of such programs varies widely, with some countries adopting more comprehensive protocols than others [26]. Early diagnosis through screening offers significant benefits, such as improved growth, better lung function, and overall enhanced health outcomes [27]. The discovery of the CFTR gene has further refined diagnostic techniques and underscored the crucial role of newborn screening in the early detection and management of CF. </p>
+ <p>Technological advancements and improved medical procedures have greatly transformed the diagnosis of cystic fibrosis. While newborn screening has revolutionized early detection and treatment, traditional methods such as the sweat test and symptom observation continue to play a vital role, particularly in regions where screening programs are not yet widely available.</p>
+ </Collapsible>
+ <p>Another widely used method is the sweat test, which measures the concentration of chloride in a person's sweat [2][7]. CF patients typically have higher-than-normal chloride levels due to defective CFTR protein function [2][7]. While the sweat test is non-invasive and reliable for indicating CF, it is limited in scope [7]. For definitive diagnosis and to guide specific treatments, a genetic analysis is usually required to identify the exact CFTR mutation, such as the F508del mutation [5][8].</p>
+ <Collapsible id="sweat-test-collapsible" title="Sweat test">
+ <p>Traditionally, Cystic Fibrosis (CF) has been diagnosed using the sweat test, which measures chloride levels in sweat. A chloride level below 40 mmol/L (millimoles of chloride per litre of sweat) is considered normal and unlikely to indicate CF. Levels between 40 and 60 mmol/L require further investigation, while levels above 60 mmol/L strongly suggest the presence of CF [28].</p>
+ <p>This quick and painless test has been the gold standard for CF diagnosis for many years. Despite its accuracy, the sweat test requires specialized lab personnel and can be difficult to perform on newborns. While diagnosing CF based on symptoms can be useful, it is not always reliable, particularly in mild or atypical cases.</p>
+ </Collapsible>
</Subesction>
<Subesction title="Treatment" id="Cystic Fibrosis6">
- <p>Cystic fibrosis therapy means inevitably a complex and customized treatment plan for each patient. It consists of a range of components. These include medication such as CFTR modulators and antibiotics as well as inhalation therapy and mucolytics, physiotherapy, nutritional therapy and sports therapy. It is therefore essential that CF patients receive treatment at a specialist centre <SupScrollLink label="1"/>.</p>
+ <p>Current Cystic fibrosis treatments focus on managing symptoms, slowing disease progression, and improving quality of life [1][2]. Since there is still no cure for CF, treatment is typically lifelong and involves multiple approaches, including medications, physical therapy, and dietary adjustments [3][4]. </p>
+ <p>The primary goal of CF treatment is to clear the thick mucus from the lungs to prevent infections and improve breathing [2][5]. Airway clearance techniques, such as chest physiotherapy, are often used alongside inhaled medications, like bronchodilators and mucolytics, to thin the mucus and open the airways [5][6]. Antibiotics are frequently prescribed to treat or prevent lung infections caused by trapped bacteria in the airways [2][4].</p>
+ <p>One of the most significant advances in CF treatment has been the development of CFTR modulators, which target the underlying protein dysfunction caused by mutations in the CFTR gene [7][8]. These drugs, such as ivacaftor, lumacaftor, and elexacaftor, work by improving the function of the defective CFTR protein, particularly in patients with specific mutations like F508del [7][8]. While CFTR modulators can dramatically improve lung function and overall health in many patients, they are not effective for all CFTR mutations and often are very expensive [7][8].</p>
+ <p>Digestive enzyme supplements are essential for CF patients who suffer from pancreatic insufficiency, helping them to absorb nutrients from food [3][9]. Additionally, high-calorie diets and vitamins are recommended to support growth and maintain body weight [3][9].</p>
+ <p>Although current treatments can significantly improve quality of life and life expectancy, managing CF remains a daily challenge for patients [1][2]. Continued research into gene therapy and other innovative treatments offers hope for more permanent solutions in the future [10].</p>
<Collapsible id="drugs-collapsible" title="Different types of drugs" >
<TabButtonRow data={medibuttonrowdata} opentype="symptabs" closing=""/>
<ButtonRowTabs data={medibuttonrowdata} cla="symptabs"/>
</Collapsible>
- <H2 text="CF treatment with gene therapy"></H2>
+ <H4 text="CF treatment with gene therapy"></H4>
<p>While mentioned medications have improved the quality of life for numerous CF patients, they only manage symptoms rather than cure the disease. Moreover, most of them are expensive and not world-wide accessible. Our research is focused on the development of a gene therapy that targets the underlying cause of CF by correcting the defective CFTR gene. <PreCyse/> aims to halt disease progression and reduce the treatment burden for patients.</p>
- <img src="https://static.igem.wiki/teams/5247/charts-maps/cfper10-000.png"/>
</Subesction>
</Section>
<Section title="Approach" id="Approach">
<Subesction title="Mechanism" id="Approach1">
- <p>To correct the mutation, we are utilizing Prime Editing technologies. Prime Editing is a genome editing technique that allows precise DNA modifications without causing double-strand breaks<SupScrollLink label="2"/>. Structurally, the Prime Editing complex consists of a Cas9 endonuclease fused to a reverse transcriptase (RT) and guided by a pegRNA, which directs the complex to the target site in the genome. </p>
+ <p>The development of an improved Prime Editing complex holds great promise for advancing gene editing technologies. Our enhanced system, Prime Guide, addresses key limitations of conventional Prime Editing by focusing on four main areas: editing efficiency, precision, size, and safety. Prime Guide has been designed to target the ΔF508 mutation in cystic fibrosis with high accuracy, while minimizing off-target effects. By optimizing the pegRNA, reverse transcriptase, and nickase components, we aim to deliver precise and efficient genetic modifications.</p>
+ <p>As shown in the image, we developed two potential configurations for Prime Guide, each using a different nickase: one based on the Fanzor (nSpuFz1) nickase and the other on a CasX (nPlmCasX) nickase. Both configurations are designed to improve the precision and stability of the Prime Editing system. The pegRNA scaffold, reverse transcriptase (PE6c), and primer binding site (PBS/RTT) work together in both systems to introduce precise edits, with the La(1-194) enhancing stability and function.</p>
<InfoBox title="Prime Editing" id="prime-editing">
<details>
<summary>Prime editing is a new method of gene editing based on an RNA-Protein complex. It was developed by a group of researchers revolving around Professor David Liu from Harvard University in 2019. <SupScrollLink label="9"/></summary>
- <p>Details</p>
- <LoremMedium/>
+ <p></p>
+ <p><b>How does Prime Editing work?</b></p>
+ <p>Prime Editing builds on the well-known CRISPR technology, offering a more precise and controlled approach to DNA modification. Traditional CRISPR-Cas9 methods typically involve creating double-strand breaks in DNA, which can be repaired by the cell in ways that might introduce unintended mutations. Prime Editing, by contrast, circumvents this issue by using a more refined method that avoids double strand breaks altogether [31].</p>
+ <p>At the heart of Prime Editing is a fusion protein, which combines two key components: a modified Cas9 enzyme, known as a "nickase," and a reverse transcriptase enzyme. The nickase is responsible for making a single strand cut in the DNA, unlike the traditional Cas9, which cuts both strands. This single strand cut minimizes the risk of unintended mutations or large-scale DNA damage. The reverse transcriptase attached to the nickase then modifies the DNA at the targeted site by incorporating new genetic information.</p>
+ <p>To guide this process, Prime Editing uses a specialized RNA molecule known as prime editing guide RNA (pegRNA). This pegRNA serves two functions: it directs the Cas9 nickase to the specific location on the genome, and it carries a template for the desired DNA modification. Now, let’s go through the process in more detail, referencing the image above.</p>
+ <ol>
+ <li><b>DNA Nicking</b>: In the first step (top left in the image), the Cas9 nickase, guided by the pegRNA, binds to the target genomic DNA and creates a single-strand break, or "nick," at the precise location. This is a key difference from standard CRISPR, where both DNA strands are cut, increasing the risk of unwanted mutations.</li>
+ <li><b>Primer Binding and Reverse Transcription</b>: Once the DNA is nicked, the primer binding site (PBS) on the pegRNA hybridizes with the exposed single-stranded DNA, as shown in the middle of the image. This alignment allows the reverse transcriptase (RT) enzyme, also fused to the nickase, to begin copying the edit into the target DNA. The reverse transcriptase uses the template encoded within the pegRNA to create a complementary DNA sequence (depicted as the new sequence in the image), ensuring the corrected genetic sequence is accurately inserted into the genome.</li>
+ <li><b>Flap Formation and Equilibration</b>: The process continues as the reverse transcriptase copies the new genetic sequence into the DNA strand, creating what is called a "3' flap" (as shown in the bottom part of the image). This flap contains the newly edited sequence. At this point, there is an equilibration between the new flap (which encodes the intended edit) and the unedited 5' flap, which still contains the original, unmodified DNA sequence. The cell's natural mechanisms typically degrade the unedited 5' flap, favoring the integration of the 3' flap encoding the edit.</li>
+ <li><b>Flap Resolution and Final Editing</b>: In some cases, an additional nick (seen in the PE3/PE5 systems in the image) is introduced in the non-target DNA strand to promote repair and favor the incorporation of the edit. This step increases the efficiency of Prime Editing by ensuring that the newly edited strand is preferentially used during the cell's DNA repair process. The mismatch repair (MMR) system of the cell also plays a role in determining whether the edit is retained or reverted to the original sequence. For systems like PE4 and PE5, inhibition of the mismatch repair system (e.g., by MLH1dn) further promotes the integration of the desired edit.</li>
+ <li><b>Final outcome</b>: Once the unedited flap is degraded and the new sequence is integrated, the cell completes the repair, and the edit becomes permanently incorporated into the DNA. As shown in the diagram, the result is a successful genetic modification, where the new, corrected sequence replaces the original faulty sequence.</li>
+ </ol>
+ <figure>
+ <img className="img" src="https://static.igem.wiki/teams/5247/project-description/illustration-primeediting.webp"/>
+ <figcaption>
+ <b>Figure 4: </b>
+ Illustration of the Prime Editing process and its possible outcomes
+ </figcaption>
+ </figure>
+ <p>Overall, there are many different Prime Editing systems with a variety of components and complexity, starting from PE2 up to PE7. Possible edits could integrate substitutions, inserts and deletions in the range of one base up to hundreds of nucleotides, with gradually decreasing editing efficiency. Therefore Prime Editing technology allows targeted modifications of specific genes. </p>
</details>
</InfoBox>
+ <p>However, the Prime Editing complex is relatively large, posing challenges for therapeutic delivery<SupScrollLink label="3"/>. Additionally, Prime Editing has been shown to be relatively inefficient in terms of gene editing rates, which could limit its therapeutic utility<SupScrollLink label="4"/>. Our project aims to enhance the Prime Editing approach by miniaturizing its components and enhancing its efficiency, as well as precision. </p>
<div className="row">
<div className="col">
- <p>However, the Prime Editing complex is relatively large, posing challenges for therapeutic delivery<SupScrollLink label="3"/>. Additionally, Prime Editing has been shown to be relatively inefficient in terms of gene editing rates, which could limit its therapeutic utility<SupScrollLink label="4"/>. Our project aims to enhance the Prime Editing approach by miniaturizing its components. Fanzor, a recently discovered eukaryotic endonuclease, performs functions similar to Cas9, a crucial part of the Prime Editing complex, but is significantly smaller. We aim to substitute Cas9 with Fanzor. </p>
- <p>Additionally, we plan to replace the reverse transcriptase in the Prime Editing complex with a smaller RT variant. Furthermore, MCP proteins will be added to the Prime Editing complex to increase its stability<SupScrollLink label="5"/>. </p>
+ <img className="img" src="https://static.igem.wiki/teams/5247/project-description/primeguide.png"/>
</div>
<div className="img-right img-half col"><PrimeEditingComplex/></div>
</div>
-
- <Collapsible id="fanzorcas-collapsible" title="Cas vs. Fanzor"> child </Collapsible>
- <p>The pegRNA is optimized via an extension by a stem loop, which stabilizes the RNA by protecting it from RNases and serves as a binding site for the MCP, which also supports the secondary RNA structure.
- This represents a major biosafety feature in that the complex is switched off after successful DNA editing and the subsequent increased influx of chloride ions into the cell. The pegRNA is combined with an optimized sgRNA resulting in higher on-target effect. Overall, its optimization leads to a longer shelf life and an increase in the biosafety of the complex. </p>
-
+ <p>To develop our innovative Prime Editing system, Prime Guide, we worked closely with several leading experts in the field. Among them were Mattijs Bulcaen, Makato Saito, Dr. Hammer, Jan-Phillipp Gerhard and Prof. Kristian Müller, whose insights helped guide our decisions. Prime Guide is a highly specialized Prime Editing complex, designed to target the F508del mutation in cystic fibrosis with precision and efficiency. </p>
+ <p>Our Prime Guide system consists of carefully selected components, each optimized for its role. For the nickase, we chose between SpuFz1 and CasX nickases due to their smaller size and structural advantages, which suggest increased stability for the pegRNA within the Prime Editing complex. Smaller nickases also provide benefits in terms of overall efficiency and ease of delivery, aligning with the compact design we aimed for.</p>
+ <Collapsible id="fanzorcas-collapsible" title="Cas vs. Fanzor">
+ <p>Fanzor is a recently discovered eukaryotic endonuclease, initially identified in fungi. Compared to the Cas9 endonuclease, Fanzor is significantly smaller and also RNA-guided. SpuFz1 and GtFz1 are specific variants of Fanzor that share similar properties and are also found in eukaryotic organisms.</p>
+ <p>CasX is a relatively new RNA-guided endonuclease, discovered in bacteria. It is smaller than the commonly used Cas9, making it an attractive option for applications where a more compact protein is beneficial. Like Cas9, CasX is capable of precise DNA targeting but offers advantages due to its reduced size and simpler structure.</p>
+ </Collapsible>
+ <p>In terms of the pegRNA, we opted for a pegRNA, including a 16-base primer binding site (PBS) and a 30-base reverse transcription template (RTT), with no silent edits and a structural motif, the tevopreQ1. After extensive screening using a reporter system, this pegRNA demonstrated the highest performance, leading us to select it as the best candidate for further development. While other pegRNAs also showed promise, pegRNA5 was ultimately chosen for its superior results in our testing.</p>
+ <p>For the reverse transcriptase, we selected the P6C variant, which has shown to provide the best editing efficiency and a more compact structure compared to alternatives. Its advanced development stage and ability to offer high precision and editing performance made it the ideal choice for Prime Guide. </p>
+ <p>Together, these components form a highly optimized Prime Editing system that balances size, stability, and efficiency. Our aim with Prime Guide is to create a robust and precise solution for correcting the F508del mutation in cystic fibrosis, building on the guidance from our expert collaborators and extensive testing of each individual component.</p>
+
</Subesction>
<Subesction title="Delivery" id="Approach2">
<div className='row align-items-center'>
@@ -238,10 +290,18 @@ export function Description() {
<p>To evaluate the <strong>delivery efficiency</strong>, we transfected HEK293 and CFBE41o- cells using fluorescent cargo and quantified the results through FACS analysis. We also ensured that AirBuddy meets the necessary standards for safety and efficacy since we conducted extensive <a onClick={() => goToPageAndScroll ('In-Depth Characterization of LNPsH', '/materials-methods')}> characterization of the LNPs </a>using techniques such as Zeta potential analysis, Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Cryogenic Electron Microscopy (cryo-EM). These methods confirmed the uniformity, stability, and optimal size distribution of the nanoparticles. Furthermore, <strong>cytotoxicity assessments</strong> including MTT and proliferation assays demonstrated that our LNPs are biocompatible and do not impede cell growth or function by the incorporation of <a onClick={() => goToPagesAndOpenTab('it4', '/engineering')}>PEG</a> and other ambivalent components. These findings reinforce AirBuddy's potential as a safe and effective tool for pulmonary delivery, with broad implications for gene therapies targeting lung diseases.</p>
</Subesction>
</Section>
+ <Section title="Our Achievement" id="Our Achievement">
+ <p>Lorem Ipsum hier kommt noch ein banger text hin </p>
+ </Section>
<Section title="Our Vision" id="Our Vision">
- <p>We are envisioning a potential integration into a broader therapeutic framework involving customized gene editing tools for various genetic disorders, that present similar difficulties to the F508del mutation, as well as other genetic diseases of different causes. This could include collaborations with pharmaceutical companies to develop new treatment modalities for genetic diseases beyond cystic fibrosis, utilizing advanced delivery systems and personalized medicine approaches. </p>
- <H2 text="Editing Statistics"/>
- <PieChart /> {/* Render the PieChart component */}
+ <p>At <b>PreCyse</b>, we envision a future where gene therapy for cystic fibrosis (CF) is as simple and user-friendly as using an inhaler. Our goal is to develop a fully integrated Prime Editing system, <b>PrimeGuide</b>, delivered via a cutting-edge lipid nanoparticle (LNP) platform, <b>AirBuddy</b>. The therapy would allow patients to inhale the therapeutic complex, targeting the underlying genetic mutation that causes CF—specifically, the F508del mutation in the CFTR gene. </p>
+ <p>The core of our vision is to create a highly efficient and safe Prime Editing complex, referred to as Prime Guide, that is delivered directly into lung epithelial cells. This complex will be packaged as mRNA into LNPs, with an optimal ratio of the Prime Editing components and its guide RNA (pegRNA). Once inside the cell, the mRNA will be translated, forming the active Prime Editing complex, which then translocates into the nucleus using nuclear localization sequences. There, the complex will precisely edit the genome to correct the F508del mutation. </p>
+ <p>To ensure safety, we are working on developing a robust mechanism that regulates the Prime Editing complex at the mRNA level. One concept we are exploring is using the XBP1 intron, which responds to cellular stress signals. Additionally, in the future, we aim to develop more mutation-specific control mechanisms, such as RNA riboswitches that activate the editing complex only in the presence of the target mutation, offering an even greater level of precision and safety. </p>
+ <p>The long-term vision for PreCyse is to provide a gene therapy that can be administered through inhalation, much like an asthma spray. The patient would simply inhale the LNPs, which then deliver the therapeutic mRNA to the lungs. This approach offers a user-friendly and minimally invasive treatment that could suppress the symptoms of CF for several months. By correcting the mutation in the top layers of lung epithelial cells, where mucus buildup is most problematic, we could offer relief from symptoms over an extended period. However, since these epithelial cells naturally regenerate over time, the therapy would need to be reapplied periodically, likely every few months, balancing long-lasting effects with the need for occasional re-administration. </p>
+ <p>Ultimately, our vision is to create a therapeutic approach that not only offers a cure that is safe and efficient but also maximizes convenience for the patient. With an easy-to-use inhaler, patients could administer their treatment with minimal disruption to their daily lives, inhaling the gene therapy in just a few breaths, leaving the rest of the process to the science we've built into PreCyse. By reducing the frequency of administration and simplifying the delivery method, we aim to make gene therapy for cystic fibrosis both accessible and practical for patients around the world. </p>
+ <div className="casettecontainer" >
+ <img className="img" src="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-del-mech.webp"/>
+ </div>
</Section>
<Section title="References" id="References">
<ol>
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 2d69497cf3e62a1fa1eaad5e996ceb2582508460..4002aa8998bbc58f2be311f4c617ab0eca289b0c 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -2,17 +2,20 @@
import { ButtonOneEngineering } from "../components/Buttons";
import { LoremShort } from "../components/Loremipsum";
import { openElement } from "../utils/openElement";
-import { H3, H4, H5 } from "../components/Headings";
+import { H2, H3, H4, H5 } from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
import { Collapsible } from "../components/Collapsible";
import { useNavigation } from "../utils";
import { TabScrollLink } from "../components/Link";
import { InfoBox } from "../components/Boxes";
import { DownloadLink } from "../components/Buttons";
-import EngPEsystems from "../sources/eng-pe-sources";
-import EngPoCsources from "../sources/eng-poc-sources";
import { Section } from "../components/sections";
-
+import EngTrfsources from "../sources/eng-trf-sources";
+import EngRepsources from "../sources/eng-reporter-sources";
+import EngPEsystems from "../sources/eng-pe-sources";
+import EngPegsources from "../sources/eng-peg-sources";
+import EngNicksources from "../sources/eng-nickases-sources";
+import EngDelsources from "../sources/eng-delivery-sources";
export function Engineering() {
@@ -28,7 +31,7 @@ export function Engineering() {
<div id="tab-our-cycle" className="enginneeringtab" style={{display: "block"}}>
<section > <br id="obenengineering"/>
<div className="eng-box box" >
- <H3 text="Our cycle" id="ourcycle"></H3>
+ <H4 text="Our cycle" id="our-cycle-header"></H4>
<p>Hallo Prime Editing diesdas</p>
</div>
@@ -38,22 +41,182 @@ export function Engineering() {
</div>
<div className="col button-left">
- <div className="right"><ButtonOneEngineering label="Next" open="proof-of-concept" scrollToId="Proof of Concept"/></div>
+ <div className="right"><ButtonOneEngineering label="Next" open="reporter" scrollToId="reporter-header"/></div>
</div>
</div>
</section>
</div>
- <div id="tab-proof-of-concept" className="enginneeringtab" style={{display: "none"}}>
+
+ <div className="enginneeringtab" id="tab-reporter" style={{display: "none"}}>
+ <section id="reporter sec" >
+ <div className="eng-box box" >
+ <H2 id="reporter-header" text="Prime Editing Reporter"></H2>
+ <p>Prime editing is a is a very precise and safe method. However, depending on the genomic locus targeted, the editing efficiency can be very low. The cystic fibrosis causing CFTR F508del mutation is, as Mattijs Bulcaen[link] stated in our interview, one of, if not the most obvious application of prime editing, considering the large amount of people affected. The lack of publications addressing CFTR target implied, that the mutation might be particularly hard to edit. At low editing efficiency, successful edits are hard, if not impossible to distinguish from the background noise using conventional methods like sanger sequencing or qPCR. As a basis to effectively test our approach and screen for working pegRNAs, we needed a highly sensitive method of detection with as little noise as possible to optimize our prime editing approach for genomic CFTR targeting.</p>
+ </div>
+ <div className="box" >
+ <p id="rep1">
+ <H3 text="A Fluorescence Reporter" id="rep1head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+ We reasoned that the easiest way of detecting DNA changes in a cell would be fluorescence. Our initial idea was to create pegRNAs targeting the coding sequence of a fluorescent protein, that would introduce a mutation resulting in a different emission, giving easily detectable feedback of correct editing. The original Aequorea victoria GFP protein differs from avGFP(Y66W), emitting light in a wavelength of around 509 nm (cyan), and avGFP(Y66H), emitting light in a wavelength of around 448 nm (blue) by only one amino acid substitution each.<TabScrollLink tab="tab-reporter" num="1" scrollId="desc-1"/> Prime editing could therefore be visualized by facilitating these substitutions with a prime editor.
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+ To this end, the wild-type and edited versions of the avGFP were put in contrast and we started searching for potential pegRNAs for editing one into the other.
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+ When trying to find protospacers for Cas9 and other possible nickases[link], we noticed, that the locus of the mutations is too far away from any SpuFz1 TAM sequences. Additionally, the applicability of insights gained through pegRNA optimization in this locus to CFTR editing would also be very limited due to the vast differences in the sequence of protospacer[link] and surrounding genomic region. Additionally, we learned from our interview with Mattijs Bulcaen[link] that the type of edit (insertion, substitution or deletion) significantly impacts editing efficiency. A mutation changing GFP to BFP would have to be a substitution instead of the three-nucleotide insertion needed to correct CFTR F508del.
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+ From our observations we learned that a reporter system is only of use, if it can really mimic the genomic target of choice. The adjustments to be made to create a pegRNA targeting the genomic target from a pegRNA targeting the reporter should be as minor as possible. This includes a similar spacer and a similar edit to be made.
+ </p>
+ </p>
+ </div>
+ <div className="box" >
+ <p id="rep2">
+ <H3 text="Proof of Concept for PEAR" id="rep2head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+ After extensive research we came across the prime editor activity reporter (PEAR) created by Simon et al. (2022)<TabScrollLink tab="tab-reporter" num="2" scrollId="desc-2"/>, which is the template our modified reporter plasmid is based on. The PEAR plasmid contains an eGFP coding sequence with an intron derived from the mouse Vim gene. If the intron is removed during RNA splicing, the two exons form a continuous open reading frame. By mutating the 5’ splicing signal, a target is created which, upon correct editing, leads to a gain-of-function. The resulting fluorescence can be imaged using confocal microscopy or quantified by means of fluorescence activated cell sorting (FACS). Notably, the area downstream of the 5’ splice signal is intronic, and thus can be edited without any impact on the coding sequence. Additionally, Simon et al. showed, that “efficiency of prime editing to modify PEAR plasmids is governed by the same factors as prime editing in genomic contextâ€. We reasoned that this system might be flexible, and sensitive enough to build our optimizations strategies upon.
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+ Since none of us had any experience in prime editing before our project, we wanted to test whether we can facilitate prime editing in the first place. To do this and also assess the functionality of the PEAR system, we set up a proof of concept using the PEAR 2in1 system. This plasmid includes not only the eGFP with and intron and disrupted 5’ splice site, but also a pegRNA expression cassette. The pegRNA is designed in a way that, in combination with a prime editing protein complex, corrects the disrupted splicing signal.
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+ In the experiment, we transfected HEK293 cells (as recommended by Mattijs Bulcaen[link]) with the pCMV-PE2 prime editor[link PE systems] plasmid and the pDAS12489_PEAR-GFP_2in1_2.0 mentioned above. Our first proof of concept succeeded as we could see fluorescent cells 72 h after transfection. In contrast, negative controls with only one of the plasmids transfected did not show any fluorescence. However, the transfection efficiency in our initial test runs was quite low, as indicated by a technical positive control.
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+ This proved, that not only we were able to use prime editing in our model, but also that the PEAR reporter system can report successful prime editing. Though this was a very promising start, further steps had to be taken to enable context specific testing of prime editing. Firstly, the transfection efficiency had to be improved (see Transfection Optimization[link]). Secondly, the reporter had to be modified in a way that resembles the genomic CFTR target.
+ </p>
+ </p>
+ </div>
+ <div className="box" >
+ <p id="rep3">
+ <H3 text="Contextualization of PEAR" id="rep3head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+ The original PEAR plasmid pDAS12124_PEAR-GFP-preedited that we bought from AddGene represents, as the name suggests, how the reporter should look like after successful editing and can thus be used as a positive control and for normalization. To alter the PEAR plasmid so that it mimics the mutated genomic CFTR target, we first analyzed the region surrounding CFTR F508del mutation. As the mutation is a three base pair deletion, we introduced the very same at the 5’ splicing signal. For this modification to reliably disrupt intron splicing and thus eGFP expression, we effectively removed the GT bases of the intronic 5’ splice donor site as well as the preceding, exonic G base of the 5’ flanking sequence. Secondly, we replaced the intronic region downstream of the four base pair 3’ flanking region with the respective sequence from the CFTR locus. This 27 bp substitute included a PAM sequence, an entire spacer as well as four additional base pairs in between present in the original gene sequence. Lastly, we introduced silent mutations upstream of the 5’ flanking sequence that lowered the GC content. This was to mimic the AT-rich region preceding the F508del mutation in the CFTR gene. This reveals one of the necessary shortcomings of this reporter: Edits upstream of the 5’ donor site are heavily restricted by the eGFP coding sequence.
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+ We constructed the reporter system by first analyzing the original plasmid to identify appropriate restriction sites. We then digested the plasmid backbone and cloned in a gene synthesis fragment ordered at IDT containing the edits via Gibson Assembly cloning. The correct cloning was validated first by colony PCR and then by sequencing the regions of the plasmid containing the cloning sites and our modifications.
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+ We evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE2 prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter (see pegRNA engineering cycle[link]) into HEK293 cells. After 72 h we saw a significant number of cells showing fluorescence.
+ </p>
+ <p>
+ Additionally, for positive controls we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid, which could be used to determine the transfection efficiency as well as normalize the editing efficiency. As negative controls, our modified plasmid, pCMV-PE2 and the pegRNA plasmid were transfected. The positive controls showed fluorescence, while the negative control did not.
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+ Our results demonstrate three things: Firstly, the original pDAS12124_PEAR-GFP-preedited plasmid leads to undisrupted expression of eGFP in the transfected cells. Secondly, the modifications that we made to create our own, context specific PEAR plasmid prevented proper expression of eGFP in transfected, unedited cells as planned and notably with no apparent noise. The last and most important insight gained was, that editing of the reporter plasmid using respective pegRNAs successfully restores eGFP expression, proving that our reporter works as intended.
+ </p>
+ <p>
+ <b>This achievement formed a convenient basis for the following optimization of prime editing in the CFTR F508del locus for us as well as other research groups.</b>
+ </p>
+ </p>
+ </div>
+ <div className="box" >
+ <p id="rep4">
+ <H3 text="Application in epithelial Cells" id="rep4head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+ Although we could show that our PEAR reporter plasmid works in a HEK cell model, according to Prof. Dr. Zoya Ignatova[link] insights gained here might still not entirely transfer to cells actively expressing CFTR. As recommended, we applied our reporter to a system closer to a therapeutic target CFBE41o-[link]. The cells are derived from bronchial epithelial cells of a cystic fibrosis patient and are homozygous for CFTR F508del.
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+ For experimenting in CFBE41o- cells, the same reporter construct was used as for the HEK293 test. However, we used a different prime editor (pCMV-PE6c, see prime editing systems engineering cycle[link]), and only pegRNAs were used, that proved the most efficient in preceding experiments (see pegRNA engineering cycle[link]).
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+ Similar to the previous cycle, we evaluated the functionality of our reporter system by co-transfecting our reporter construct with a pCMV-PE6c prime editor plasmid as well as a plasmid expressing pegRNA that targeted our reporter this time into CFBE41o- cells. After 72 h we saw a significant number of cells showing fluorescence.
+ </p>
+ <p>
+ Like with the experiments in HEK cells, we transfected a technical control plasmid as well the unmodified pDAS12124_PEAR-GFP-preedited plasmid as positive controls and our modified plasmid, pCMV-PE6c and the pegRNA plasmid individually as negative controls. Again, the positive controls showed solid fluorescence, while the negative control did not.
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+ This experiment confirms that our reporter can not only be used in cell lines distantly related to patient cells of interest, in our case HEK203 cells, but also works in cells actively expressing CFTR and carrying the mutation. The reporter still showed no noise.
+ </p>
+ </p>
+ </div>
+ <div className="box" >
+ <p id="rep5">
+ <H3 text="Application in Primary Cells" id="rep5head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+ The model closest to application in actual patient cells are human derived primary cells. For our last test of our modified PEAR reporter, we thus chose to use human nasal epithelial cells[link] derived from members of our team.
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+ For testing our reporter in the human nasal epithelial cells, the same constructs have been used as in the previous iteration with CFBE41o- cells.
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+ The experimental setup for this experiment was a scaled down version of the previous cycle with the only altered variable being the cells transfected. In this case, we did not observe any fluorescence, neither in the tested cells, nor the technical or pDAS12124_PEAR-GFP-preedited positive controls.
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+ In this last experiment, the negative technical positive control implies a failed transfection of the cells. Thus, this attempt did not allow to draw any conclusion regarding the function of our reporter in primary cells. The experiment is to be repeated in the future.
+ </p>
+ </p>
+ </div>
+ <div className="box" >
+ <p id="rep6">
+ <H3 text="Outlook" id="rep6head"/>
+ <p>
+ Our CFTR contextualized PEAR reporter proved to consistently allow detection of prime editing without notable noise, laying the foundation for optimization of existing and testing of new prime editing systems. Although very versatile in the context of targeting CFTR F508del with the spacer of our choice[link], a wider applicability to other genomic targets and other possible prime editor variants working differently than Cas9-based systems would be favorable. In the original PEAR plasmid however, modification of variable region is quite impractical. Also, as a part the eGFP is RCF[1000] but not RCF[10] BioBrick standard conform and hardly compatible with other parts like our PreCyse cassette[link].
+ </p>
+ <H4 text="Design" id="design-head"/>
+ <p>
+ This is why, as an outlook and contribution for future iGEM teams, we created a more modular and compatible part. For this we made use of the experience gained when cloning pegRNAs. An oligonucleotide-based golden gate cloning site in the region of interest surrounding the 5’ splice donor site allows for cheap and convenient modification of the sequence. The area between the TypeIIS restriction sites is designed as a dropout cassette coding for a fluorescence marker expressed in E. coli, that enables rapid screening for transformants containing correctly digested plasmid backbones.
+ </p>
+ {/* <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p> */}
+ </p>
+ </div>
+ <Section title="References" id="references">
+ <EngRepsources/>
+ </Section>
+ <br/>
+ <div className="row ">
+ <div className="col">
+ <div className="left"><ButtonOneEngineering label="Previous" open="our-cycle" scrollToId="our-cycle-header"/></div>
+ </div>
+ <div className="col button-left">
+ <div className="right"><ButtonOneEngineering label="Next" open="transfection" scrollToId="transfection-header"/></div>
+ </div>
+ </div>
+ </section>
+ </div>
+
+ <div id="tab-transfection" className="enginneeringtab" style={{display: "none"}}>
<section >
<div className="eng-box box" >
- <H3 id="Proof of Concept" text="Proof of Concept"></H3>
+ <H2 id="transfection-header" text="Optimization of Transfection"></H2>
<p>
- To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology<TabScrollLink tab="tab-proof-of-concept" num="1" scrollId="desc-1"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system<TabScrollLink tab="tab-proof-of-concept" num="2" scrollId="desc-2"/> and optimizing transfection protocols.
+ To test prime editors, a reliable model system is required. HEK293 cells are a human derived cell line and widely used in a variety of fields in biology<TabScrollLink tab="tab-transfection" num="1" scrollId="desc-1"/>. Apart from easy handling and comparatively easy transfection, they have, as we found out in our exchange with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}>Mattijs Bulcaen</a>, one advantage over other models: They are naturally impaired in DNA repair mechanisms and therefore easier to edit. To properly compare editing efficiencies, a high transfection efficiency is of utmost importance. This engineering cycle focuses on our work in simulating prime editing using the PEAR reporter system<TabScrollLink tab="tab-transfection" num="2" scrollId="desc-2"/> and optimizing transfection protocols.
</p>
</div>
<div className="box" >
- <p id="cyc1">
- <H3 text="Test of Lipofectamine 2000" id="cyc1head"/>
+ <p id="trf1">
+ <H3 text="Test of Lipofectamine 2000" id="trf1head"/>
<H4 text="Design" id="text"/>
<p>
Before testing any of our mechanistic approaches, we had to examine whether we can facilitate and detect prime editing in the first place. During our research we eventually stumbled upon the PEAR reporter system (see pegRNA engineering cycle[link]). The PEAR 2in1 plasmid reporter includes a GFP that is to be edited for sensitive prime editing detection, and a pegRNA expression cassette with a pegRNA targeting the plasmid itself. Having found a system capable of detecting even small-scale prime editing, the next step was to find transfection conditions that would work. In the literature, Lipofectamine is described as a common transfection agent.
@@ -63,7 +226,7 @@ export function Engineering() {
</p>
<H4 text="Build" id="text"/>
<p>
- Anzalone et al. 2019<TabScrollLink tab="tab-proof-of-concept" num="3" scrollId="desc-3"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
+ Anzalone et al. 2019<TabScrollLink tab="tab-transfection" num="3" scrollId="desc-3"/> describe a transfection of prime-editing complexes with Lipofectamine 2000.
</p>
<H4 text="Test" id="text"/>
<p>
@@ -77,8 +240,8 @@ export function Engineering() {
</div>
<div className="box" >
- <p id="cyc2">
- <H3 text="Initial Test with Lipofectamine 3000" id="cyc2head"/>
+ <p id="trf2">
+ <H3 text="Initial Test with Lipofectamine 3000" id="trf2head"/>
<H4 text="Design" id="text"/>
<p>
In light of the aforementioned findings, the decision was taken to test Lipofectamine 3000, given its reputation for greater efficiency. A new test design was devised, utilizing Lipofectamine 3000 with an equivalent quantity of DNA and modified transfection conditions.
@@ -101,8 +264,8 @@ export function Engineering() {
</p>
</div>
<div className="box" >
- <p id="cyc3">
- <H3 text="Optimization of DNA and Lipofectamine Volumes" id="cyc3head"/>
+ <p id="trf3">
+ <H3 text="Optimization of DNA and Lipofectamine Volumes" id="trf3head"/>
<H4 text="Design" id="text"/>
<p>
In order to optimize the transfection process, a new optimization test was designed, which incorporated a variable design with regard to the quantity of Lipofectamine 3000 and DNA.
@@ -125,8 +288,8 @@ export function Engineering() {
</p>
</div>
<div className="box" >
- <p id="cyc4">
- <H3 text="Validation of optimized Protocol" id="cyc4head"/>
+ <p id="trf4">
+ <H3 text="Validation of optimized Protocol" id="trf4head"/>
<H4 text="Design" id="text"/>
<p>
The results obtained were used to develop an optimized protocol that takes into account both the concentration of Lipofectamine and the amount of DNA.
@@ -146,15 +309,15 @@ export function Engineering() {
</p>
</div>
<Section title="References" id="references">
- <EngPoCsources/>
+ <EngTrfsources/>
</Section>
<br/>
<div className="row ">
<div className="col">
- <div className="left"><ButtonOneEngineering label="Previous" open="our-cycle" scrollToId="ourcycle"/></div>
+ <div className="left"><ButtonOneEngineering label="Previous" open="reporter" scrollToId="reporter-header"/></div>
</div>
<div className="col button-left">
- <div className="right"><ButtonOneEngineering label="Next" open="pe-systems" scrollToId="PE Systems"/></div>
+ <div className="right"><ButtonOneEngineering label="Next" open="pe-systems" scrollToId="pe-systems-header"/></div>
</div>
</div>
</section>
@@ -163,12 +326,12 @@ export function Engineering() {
<div id="tab-pe-systems" className="enginneeringtab" style={{display: "none"}}>
<section id="PE Systems sec" >
<div className="eng-box box" >
- <H3 id="PE Systems" text="Prime Editing Systems"></H3>
+ <H2 id="pe-systems-header" text="Prime Editing Systems"></H2>
<p>Different versions of the original prime editing system have been developed since its initial introduction. Deciding on what system to use for the application in therapeutic human gene editing, especially concerning the correction of F508del, was the goal of this engineering cycle.</p>
<p>
Since we aim to develop a therapy delivered to the human body, we wanted to obtain high editing efficiency while risking as little off-targets as possible and also reducing the size for improved packability.
</p>
- <InfoBox title="Prime Editing" id="pe-systeme">
+ <InfoBox title="Existing Prime Editing Systems" id="current-pe-systems">
<details>
<summary>
@@ -236,7 +399,6 @@ export function Engineering() {
</details>
</InfoBox>
</div>
-
<div className="box" >
<p id="pe1">
<H3 text="PE2 and PECO-Mini" id="pe1head"/>
@@ -250,7 +412,7 @@ export function Engineering() {
</p>
<H4 text="Test" id="text"/>
<p>
- To compare the prime editing performances of M-MLV RT (PE2) and PE<sup>CO</sup>-Mini RT, both were tested using a 2in1 prime editing reporter plasmid system<TabScrollLink tab="tab-pe-systems" num="12" scrollId="desc-12"/> (see <a onClick={() => goToPageWithTabAndScroll({scrollToId: 'Proof of Concept', path: '/engineering', tabId: 'tab-proof-of-concept' })}>Proof of Concept</a>) in HEK293 cells. Contrary to the findings of Gao et al., here the PE<sup>CO</sup>-Mini prime editor performed a lot worse than the PE2 prime editor.
+ To compare the prime editing performances of M-MLV RT (PE2) and PE<sup>CO</sup>-Mini RT, both were tested using a 2in1 prime editing reporter plasmid system<TabScrollLink tab="tab-pe-systems" num="12" scrollId="desc-12"/> (see <a onClick={() => goToPageWithTabAndScroll({scrollToId: 'Proof of Concept', path: '/engineering', tabId: 'tab-transfection' })}>Proof of Concept</a>) in HEK293 cells. Contrary to the findings of Gao et al., here the PE<sup>CO</sup>-Mini prime editor performed a lot worse than the PE2 prime editor.
</p>
<H4 text="Learn" id="text"/>
<p>
@@ -329,100 +491,153 @@ export function Engineering() {
<br/>
<div className="row ">
<div className="col">
- <div className="left"><ButtonOneEngineering label="Previous" open="proof-of-concept" scrollToId="Proof of Concept"/></div>
+ <div className="left"><ButtonOneEngineering label="Previous" open="transfection" scrollToId="transfection-header"/></div>
</div>
<div className="col button-left">
- <div className="right"><ButtonOneEngineering label="Next" open="nikase" scrollToId="Nikase"/></div>
+ <div className="right"><ButtonOneEngineering label="Next" open="pegrna" scrollToId="pegrna-header"/></div>
</div>
</div>
</section>
</div>
+
<div className="enginneeringtab" id="tab-pegrna" style={{display: "none"}}>
<section id="pegRNA sec" >
<div className="eng-box box" >
- <H3 id="pegRNA" text="pegRNA"></H3>
+ <H2 id="pegrna-header" text="pegRNA"></H2>
<p><LoremShort></LoremShort></p>
</div>
<div className="box" >
<p id="peg1">
<H3 text="peg1" id="peg1head"/>
- <LoremShort></LoremShort>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
</div>
<div className="box" >
<p id="peg2">
<H3 text="peg2" id="peg2head"/>
- <LoremShort></LoremShort>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
</div>
<div className="box" >
<p id="peg3">
<H3 text="peg3" id="peg3head"/>
- <LoremShort></LoremShort>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
</div>
<div className="box" >
<p id="peg4">
<H3 text="peg4" id="peg4head"/>
- <LoremShort></LoremShort>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
</div>
<div className="box" >
<p id="peg5">
<H3 text="peg5" id="peg5head"/>
- <LoremShort></LoremShort>
- </p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
- </div>
- <div className="box" >
- <p id="peg6">
- <H3 text="peg6" id="peg6head"/>
- <LoremShort></LoremShort>
- </p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
- </div>
- <div className="box" >
- <p id="peg7">
- <H3 text="peg7" id="peg7head"/>
- <LoremShort></LoremShort>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
- <p><LoremShort></LoremShort></p>
- <p><LoremShort></LoremShort></p>
</div>
+ <Section title="References" id="references">
+ <EngPegsources/>
+ </Section>
<br/>
<div className="row ">
<div className="col">
- <div className="left"><ButtonOneEngineering label="Previous" open="nikase" scrollToId="Nikase"/></div>
+ <div className="left"><ButtonOneEngineering label="Previous" open="pe-systems" scrollToId="pe-systems-header"/></div>
</div>
<div className="col button-left">
- <div className="right"><ButtonOneEngineering label="Next" open="delivery" scrollToId="Delivery"/></div>
+ <div className="right"><ButtonOneEngineering label="Next" open="nickase" scrollToId="nickase-header"/></div>
</div>
</div>
</section>
</div>
- <div className="enginneeringtab" id="tab-nikase" style={{display: "none"}}>
- <section id="Nikase sec" >
+ <div className="enginneeringtab" id="tab-nickase" style={{display: "none"}}>
+ <section id="Nickase sec" >
<div className="eng-box box" >
- <H3 id="Nikase" text="Nikase"></H3>
+ <H2 id="nickase-header" text="Alternative Nickases"></H2>
<p><LoremShort></LoremShort></p>
<img src="https://static.igem.wiki/teams/5247/fanzor/movie4-ezgif-com-video-to-gif-converter.gif"></img>
<img src="https://static.igem.wiki/teams/5247/fanzor/movie5-ezgif-com-video-to-gif-converter-2.gif"></img>
</div>
<div className="box" >
- <p id="nik1">
- <H3 text="SpuFz1 Zink Finger Mutation " id="nik1head"/>
+ <p id="nic1">
+ <H3 text="SpuFz1 Zink Finger Mutation " id="nic1head"/>
<H4 text="Design" id="text"/>
- <p>In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins, with SpuFz1 being a standout candidate due to its smaller size compared to Cas9. According to the paper "Fanzor is a Eukaryotic Programmable RNA-Guided Endonuclease"<TabScrollLink tab="tab-nikase" num="1" scrollId="desc-1"/>, SpuFz1 operates with two key domains: the RuvC domain, which cuts the non-target DNA strand, and the TNB domain (also called NUC domain), which helps facilitating the cleavage by pushing the target strand into the RuvC domain. We hypothesized that a zinc finger, which is part of the TNB domain and crucial for DNA coordination, could be a target for mutation to disrupt this process. Thus, our initial design strategy involved replacing the cysteine residues coordinating the zinc ion in the zinc finger with alanine to potentially impair its DNA-binding ability.</p>
+ <p>In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins, with SpuFz1 being a standout candidate due to its smaller size compared to Cas9. According to the paper "Fanzor is a Eukaryotic Programmable RNA-Guided Endonuclease"<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>, SpuFz1 operates with two key domains: the RuvC domain, which cuts the non-target DNA strand, and the TNB domain (also called NUC domain), which helps facilitating the cleavage by pushing the target strand into the RuvC domain. We hypothesized that a zinc finger, which is part of the TNB domain and crucial for DNA coordination, could be a target for mutation to disrupt this process. Thus, our initial design strategy involved replacing the cysteine residues coordinating the zinc ion in the zinc finger with alanine to potentially impair its DNA-binding ability.</p>
<H4 text="Build" id="text"/>
<p>We used the protein visualization software ChimeraX to explore the SpuFz1 protein structure and identify the specific cysteine residues involved in zinc ion coordination. Based on this visualization, we designed mutant sequences by substituting these cysteines with alanine.</p>
<H4 text="Test" id="text"/>
@@ -433,112 +648,129 @@ export function Engineering() {
</p>
</div>
<div className="box" >
- <p id="nik2">
- <H3 text="Fusion Protein from GtFz1 & SpuFz1" id="nik2head"/>
- <H4 text="Design" id="text"/>
- <p>In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in cystic fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature. To address this, we devised a strategy to combine the favorable TAM-binding region of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. The idea was to create a new endonuclease with the optimal TAM sequence for our application and a robust DNA cutting ability.</p>
- <p>Given that we were swapping entire domains rather than just single amino acids, we realized that the fusion protein might not retain the ideal TAM-binding efficiency or cutting efficiency of the original proteins. Our strategy was to create a fusion protein that could bind to the TAM site and perform DNA cutting to a certain extent, albeit weakly. We planned to use directed evolution techniques, such as Phage Assisted Continuous Evolution (PACE), to enhance these functionalities over time. This approach relies on having a starting point with some degree of the desired activity, which can then be incrementally improved through evolution.</p>
- <H4 text="Build" id="text"/>
- <p></p>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
- </p>
- </div>
- <div className="box" >
- <p id="nik3">
- <H3 text="nik3" id="nik3head"/>
- <H4 text="Design" id="text"/>
- <p></p>
- <H4 text="Build" id="text"/>
- <p></p>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
+ <p id="nic2">
+ <H3 text="Fusion Protein from GtFz1 & SpuFz1" id="nic2head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
</div>
<div className="box" >
- <p id="nik4">
- <H3 text="nik4" id="nik4head"/>
- <H4 text="Design" id="text"/>
- <p></p>
- <H4 text="Build" id="text"/>
- <p></p>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
+ <p id="nic3">
+ <H3 text="nic3" id="nic3head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
</div>
<div className="box" >
- <p id="nik5">
- <H3 text="nik5" id="nik5head"/>
- <H4 text="Design" id="text"/>
- <p></p>
- <H4 text="Build" id="text"/>
- <p></p>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
+ <p id="nic4">
+ <H3 text="nic4" id="nic4head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
</div>
<div className="box" >
- <p id="nik6">
- <H3 text="nik6" id="nik6head"/>
- <H4 text="Design" id="text"/>
- <p></p>
- <H4 text="Build" id="text"/>
- <p></p>
- <H4 text="Test" id="text"/>
- <p></p>
- <H4 text="Learn" id="text"/>
- <p></p>
+ <p id="nic5">
+ <H3 text="nic5" id="nic5head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
</div>
<div className="box" >
- <p id="nik6">
- <H3 text="References" id="nikref"/>
- <ol>
- {/*<!-- Citation num 1--> */}
- <li typeof="schema:ScolarlyArticle" role="doc-biblioentry" property="schema:citation" id="desc-1">
- <span property="schema:author" typeof="schema:Person">
- <span property="schema:Name"> Saito, M.</span>
- <span property="schema:Name"> Xu, P.</span>
- <span property="schema:Name"> Faure, G.</span>
- <span property="schema:Name"> Maguire, S.</span>
- <span property="schema:Name"> Kannan, S.</span>
- <span property="schema:Name"> Altae-Tran, H.</span>
- <span property="schema:Name"> Vo, S.</span>
- <span property="schema:Name"> et al.</span>
- </span>
- <span property="schema:name"> Fanzor is a eukaryotic programmable RNA-guided endonuclease</span>.
- <i property="schema:publisher" typeof="schema:Organization"> Nature</i>
- <b property="issueNumber" typeof="PublicationIssue"> 620</b>
- , <span property="schema:pageBegin"> 660</span>-<span property="schema:pageEnd">668</span>
- (<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2023">2023</time>).
- <a className="doi" href="https://doi.org/10.1038/s41586-023-06356-2"> doi: 10.1038/s41586-023-06356-2</a>
- </li>
- </ol>
+ <p id="nic6">
+ <H3 text="nic6" id="nic6head"/>
+ <H4 text="Design" id="design-head"/>
+ <p>
+
+ </p>
+ <H4 text="Build" id="build-head"/>
+ <p>
+
+ </p>
+ <H4 text="Test" id="test-head"/>
+ <p>
+
+ </p>
+ <H4 text="Learn" id="learn-head"/>
+ <p>
+
+ </p>
</p>
</div>
+ <Section title="References" id="references">
+ <EngNicksources/>
+ </Section>
<br/>
<div className="row ">
<div className="col">
- <div className="left"><ButtonOneEngineering label="Previous" open="pe-systems" scrollToId="PE Systems"/></div>
+ <div className="left"><ButtonOneEngineering label="Previous" open="pegrna" scrollToId="pegrna-header"/></div>
</div>
<div className="col button-left">
- <div className="right"><ButtonOneEngineering label="Next" open="pegrna" scrollToId="pegRNA"/></div>
+ <div className="right"><ButtonOneEngineering label="Next" open="delivery" scrollToId="delivery-header"/></div>
</div>
</div>
</section>
</div>
+
<div className="enginneeringtab" id="tab-delivery" style={{display: "none"}}>
<section id="Delivery sec" >
<div className="eng-box box" >
- <H3 id="Delivery" text="Delivery"></H3>
+ <H2 id="delivery-header" text="Delivery"></H2>
<p>The design path of our lipid nanoparticle (LNP) for mRNA delivery underwent multiple cycles of research and discussion, marked by important decision points and learnings along the way. By ongoing further improvement, we designed our lungs-specific LNP called AirBuddy with improved stability aspects, becoming more precise in the delivery of our therapeutic cargo LNP by LNP.</p>
<img src="https://static.igem.wiki/teams/5247/delivery/airbuddy.webp" style={{maxHeight: "80pt"}}/>
</div>
@@ -643,13 +875,15 @@ export function Engineering() {
Ultimately, through continuous cycles of experimentation, feedback, and optimization, a LNP formulation called AirBuddy was designed using SORT LNPs and a spray-drying process, achieving lung specificity and improved safety. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells [9] and ionocytes [11] as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision. In addition, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the mRNA with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization.
</p>
</div>
+ <Section title="References" id="references">
+ <EngDelsources/>
+ </Section>
<br/>
<div className="row">
<div className="col">
- <div className="left"><ButtonOneEngineering label="Previous" open="pegrna" scrollToId="pegRNA"/></div>
+ <div className="left"><ButtonOneEngineering label="Previous" open="nickase" scrollToId="nickase-header"/></div>
</div>
<div className="col button-left">
- <div className="right"><ButtonOneEngineering label="Next" open="references" scrollToId="References"/></div>
</div>
</div>
</section>
diff --git a/src/contents/methods.tsx b/src/contents/methods.tsx
index 8b12d84b5ba15cfb90d6f7fab5e9f6a2a874f194..7b63f4ad2581d9fa66c43539b1a834e73d94a325 100644
--- a/src/contents/methods.tsx
+++ b/src/contents/methods.tsx
@@ -1,6 +1,6 @@
import { Section, Subesction } from "../components/sections";
import { useTabNavigation } from "../utils/TabNavigation";
-import {H5} from "../components/Headings";
+import {H4} from "../components/Headings";
import MethodSources from "../sources/methods-sources";
import { useNavigation } from "../utils";
@@ -10,8 +10,9 @@ export function Methods() {
useTabNavigation();
return (
<>
+
<Section title="Introduction" id="Introduction">
- ...
+ <p>This section highlights the key materials and methods pivotal to advancing our project with the primary goal to develop an efficient prime editing technology to correct the F508del mutation in the CFTR gene by the delivery to lung epithelial cells using optimized lipid nanoparticles (LNPs) via pulmonary administration. We utilized patch clamp electrophysiology to precisely measure ion channel activity, providing crucial insights into cellular function and the impact of genetic modifications on CFTR performance. Additionally, our cell culture models of lung epithelial cells allowed us to test both the delivery and efficacy of our gene-editing system under conditions that closely mimic the <i>in vivo</i> environment. To ensure that our LNPs were both effective and safe, we performed extensive LNP cytotoxicity and characterization experiments, evaluating their biocompatibility, stability, and efficiency in delivering the editing technology. Each of these methodologies was carefully selected to optimize the delivery process and maximize the therapeutic potential of our approach.</p>
</Section>
<Section title="Patch Clamp" id="Patch Clamp">
<Subesction title="Patch Clamp: A Key Tool in Electrophysiology" id="Patch Clamp1">
@@ -32,23 +33,23 @@ export function Methods() {
</figure>
</Subesction>
<Subesction title="Application in CFTR gene prime editing validation" id="Patch Clamp3">
- <p>In our ongoing research project focusing on the treatment of cystic fibrosis (CF), our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
+ <p>In our ongoing research project focusing on the treatment of cystic fibrosis, our patch clamp measurements, performed in collaboration with Dr. Oliver Dräger from the Cellular Neurophysiology working group at Bielefeld University, serve as a powerful validation tool for the assessment of the functional correction of the CFTR gene, particularly the common F508del mutation, via prime editing. The patch clamp technique can be employed in this context to measure the resulting chloride ion channel activity which is altered by the mutation [4]. Whole-Cell recordings were performed to assess whether the corrected CFTR channels function similarly to those in healthy cells. If the chloride ion currents in the edited cells approach levels of healthy cells, this would strongly suggest successful gene editing and validate the functionality of our therapeutic approach.</p>
</Subesction>
</Section>
<Section title="Cell Culture" id="Cell Culture">
<Subesction title="HEK293 and HEK293T cell lines" id="Cell Culture1">
- <p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication.[2] Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[4], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction.1,3 </p>
+ <p>For testing our prime editing approach, we needed an easy-to-handle cell line with a measurable high expression of CFTR and the CFTR F508del mutation. When talking to Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven, he recommended to use HEK293T cell lines overexpressing CFTR they had used. HEK293 cells are a very common immortalized human cell line derived from the kidneys of a female embryo. They are particularly suited to research due to their convenient handling and transfection properties. Basic HEK293 cells were provided to us by the Cellular and Molecular Biotechnology working group at Bielefeld University led by Prof. Dr. Kristian Müller, who is also one of the Principal Investigators of our team. HEK293T cells express an additional tsA1609 allele of the SV40 large T-antigen, allowing for replication of vectors containing the SV40 origin of replication[5]. Besides the native CFTR gene, which is not expressed in HEK cells, the HEK293T cell lines used in Leuven carry another copy of the gene embedded in an expression cassette. The cassette includes a CMV promoter, which is a standard promoter used for gene overexpression in human cells derived from the human Cytomegalovirus[6], as well as a puromycin resistance co-expressed with the CFTR allowing for continuous selection of CFTR expressing cells. The whole construct was stably inserted into the genome using lentiviral transduction[7][8]. </p>
<figure>
<img src="https://static.igem.wiki/teams/5247/photos/for-wiki-texts/meth-used-cells/mikroskopie-hek293t.png" alt="Phase contrast image of HEK293T at 20x magnification"/>
<figcaption> <b>Figure 3.</b>Phase contrast image of HEK293T at 20x magnification</figcaption>
</figure>
</Subesction>
<Subesction title="CFBE41o- cell line" id="Cell Culture2">
- <p>The CFBE41o- cell line, derived from bronchial epithelial cells of a one-year-old cystic fibrosis patient, serves as a vital model for studying cystic fibrosis. These cells closely mimic the physiological environment of the airway epithelium, allowing for more accurate studies on how CFTR mutations affect cell function and response to treatments. They were immortalized through calcium-phosphate-mediated transfection using a replication-defective pSVori plasmid that carries the simian virus 40 large T-antigen (SV40-LT). The plasmid's defective origin of replication prevents viral propagation, thus preserving essential physiological characteristics of the cells while enabling them to develop differentiated morphologies. CFBE41o- cells are homozygous for the F508del-CFTR mutation [1]. We are happy we got this cell line with permission from <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Prof. Dr. Ignatova</a>, who is leader of a working group at the Institute for Biochemistry and Molecular Biology of Hamburg University and an iGEM supporter since a long time [6]. </p>
+ <p>The CFBE41o- cell line, derived from bronchial epithelial cells of a one-year-old cystic fibrosis patient, serves as a vital model for studying cystic fibrosis. These cells closely mimic the physiological environment of the airway epithelium, allowing for more accurate studies on how CFTR mutations affect cell function and response to treatments. They were immortalized through calcium-phosphate-mediated transfection using a replication-defective pSVori plasmid that carries the simian virus 40 large T-antigen (SV40-LT). The plasmid's defective origin of replication prevents viral propagation, thus preserving essential physiological characteristics of the cells while enabling them to develop differentiated morphologies. CFBE41o- cells are homozygous for the F508del CFTR mutation [9]. We are happy we got this cell line with permission from <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Prof. Dr. Ignatova</a>, who is leader of a working group at the Institute for Biochemistry and Molecular Biology of Hamburg University and an iGEM supporter since a long time [10]. </p>
</Subesction>
<Subesction title="Human nasal epithelial cells (hNECs)" id="Cell Culture3">
- <p>Human nasal epithelial cells were obtained by nasal brushing, a minimally invasive method. These cells function/act as primary cultures. Cultivated in air-liquid interface (ALI) cultures and apical-out airway organoids (AOAO), they serve as a suitable model to visualise the functional epithelium of the airways in a differentiated form. The in vivo aspects of an airway disease, such as CF, can be modelled using donors with those airway diseases (5) This model is therefore particularly suitable for testing our prime editing complex. </p>
+ <p>Human nasal epithelial cells were obtained by nasal brushing, a minimally invasive method. These cells function/act as primary cultures. Cultivated in air-liquid interface (ALI) cultures and apical-out airway organoids (AOAO), they serve as a suitable model to visualise the functional epithelium of the airways in a differentiated form. The <i>in vivo</i> aspects of an airway disease, such as CF, can be modelled using donors with those airway diseases [11]. This model is therefore particularly suitable for testing our prime editing complex. </p>
<div className="row">
<div className="col">
<figure>
@@ -69,31 +70,30 @@ export function Methods() {
</Section>
<Section title="LNPs" id="LNPs">
<Subesction title="Cytotoxicity Tests" id="Cytotoxicity Tests">
- <H5 text="Assessing the Safety of Our LNPs "></H5>
+ <H4 text="Assessing the Safety of Our LNPs "></H4>
<p>Ensuring the safety and thorough characterization of our lipid nanoparticles (LNPs) was a central part of our project, as these particles are intended for use in biological systems. We implemented a comprehensive range of assays and techniques to assess their biosafety and physical properties, ensuring their suitability for applications such as drug delivery and gene therapy. Below is an overview of the key steps we took in our assessment.</p>
- <H5 text="MTT Assay"></H5>
+ <H4 text="MTT Assay"></H4>
<p>To evaluate the cytotoxicity of our LNPs, we conducted an MTT assay, which measures the metabolic activity of cells. This assay is based on the ability of living cells to reduce MTT, a yellow tetrazolium salt, into purple formazan crystals through NAD(P)H-dependent enzymes. Cells were treated with various concentrations of LNPs, and after dissolving the formazan crystals with DMSO, we measured absorbance. Higher absorbance values indicate greater cell viability. Our results showed no significant reduction in cell viability across all LNP concentrations, demonstrating that the LNPs did not induce cytotoxic effects. This finding is crucial for ensuring that the LNPs are safe for biological use, supporting their potential in clinical applications such as drug delivery and gene therapy. Overall, the MTT assay provided strong evidence of the biocompatibility of our LNPs. </p>
- <H5 text="Proliferation Assay to Monitor Long-Term Safety"></H5>
+ <H4 text="Proliferation Assay to Monitor Long-Term Safety"></H4>
<p>In addition to assessing immediate cytotoxicity, we also evaluated the long-term safety of the LNPs by conducting a proliferation assay. This assay tracked cell division and growth over time to determine whether the LNPs impacted cellular function. Our results showed that LNP-treated cells had similar growth rates to untreated controls, indicating that the LNPs do not interfere with normal cell processes. This further confirms their biocompatibility and suitability for use in biological systems.</p>
</Subesction>
<Subesction title="Transfection Efficiency" id="Transfection Efficiency">
- <H5 text="Transfection with LNPs"></H5>
<p></p>
- <H5 text="Fluorescence-Activated Cell Sorting (FACS)"></H5>
+ <H4 text="Fluorescence-Activated Cell Sorting (FACS)"></H4>
<p>To assess the transfection efficiency of our LNPs, we used fluorescence-activated cell sorting (FACS). This method involved tagging the LNPs with fluorescent markers and measuring their ability to deliver genetic material into target cells. FACS provided quantitative insights into how effectively the LNPs transfected cells, helping us optimize their design for gene therapy applications. </p>
</Subesction>
<Subesction title="In-Depth Characterization of LNPs" id="In-Depth Characterization of LNPs">
- <H5 text="Dynamic Light Scattering (DLS) and Zeta Potential"></H5>
+ <H4 text="Dynamic Light Scattering (DLS) and Zeta Potential"></H4>
<p>We used dynamic light scattering (DLS) to measure the size distribution and polydispersity index (PDI) of our LNPs. This technique allowed us to confirm that the LNPs had a consistent size distribution with minimal aggregation, which is essential for their stability. Additionally, we measured the zeta potential of the LNPs to assess their surface charge. A high zeta potential confirmed that the LNPs were stable in suspension, which is critical for their effectiveness in biological environments. </p>
- <H5 text="Scanning Electron Microscopy (SEM) and Cryo-TEM for Structural Analysis"></H5>
- <p>To further characterize the morphology and surface structure of the LNPs, we employed scanning electron microscopy (SEM). SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs. Additionally, cryo-transmission electron microscopy (cryo-TEM) allowed us to investigate the internal structure of the LNPs, revealing the presence of lipid layers and encapsulated materials, which are crucial for understanding their function in drug delivery. </p>
- <H5 text="DNase Assay for Stability of Encapsulated Material "></H5>
- <p>Finally, we conducted a DNase assay to evaluate whether the LNPs could protect encapsulated nucleic acids, such as mRNA, from enzymatic degradation. This assay demonstrated that the LNPs successfully shielded the genetic material, ensuring its stability until it reaches target cells. </p>
+ <H4 text="SEM and Cryo-EM for Structural Analysis"></H4>
+ <p>To further characterize the morphology and surface structure of the LNPs, we employed scanning electron microscopy (SEM). SEM provided high-resolution images that confirmed the spherical shape and uniformity of the LNPs. Additionally, cryo-electron microscopy (cryo-EM) allowed us to investigate the internal structure of the LNPs, revealing the presence of lipid layers and encapsulated materials, which are crucial for understanding their function in drug delivery. </p>
+ {/* <H4 text="DNase Assay for Stability of Encapsulated Material "></H4>
+ <p>Finally, we conducted a DNase assay to evaluate whether the LNPs could protect encapsulated nucleic acids, such as mRNA, from enzymatic degradation. This assay demonstrated that the LNPs successfully shielded the genetic material, ensuring its stability until it reaches target cells. </p> */}
</Subesction>
<Subesction title="Conclusion" id="Conclusion">
- <H5 text="Importance of Safety in LNP Development"></H5>
+ <H4 text="Importance of Safety in LNP Development"></H4>
<p>Testing the safety of our LNPs was a critical step in their development. LNPs are increasingly being used in cutting-edge therapies, such as mRNA vaccines and targeted drug delivery systems. For these technologies to be viable, the nanoparticles must not harm the cells they are intended to interact with. The MTT and proliferation assays provided robust data, confirming the biocompatibility of our LNPs and reinforcing their potential for safe use in further research and clinical applications. </p>
</Subesction>
diff --git a/src/contents/notebook.tsx b/src/contents/notebook.tsx
index 4bed7559ce3a21e21a1bd176ed8fc0605f324721..0dba96b2f622ada1b06255394def1273aba878ac 100644
--- a/src/contents/notebook.tsx
+++ b/src/contents/notebook.tsx
@@ -1,5 +1,5 @@
-import { InfoBox } from "../components/Boxes";
+
import { DownloadImageButton } from "../components/Buttons";
import H2 from "../components/Headings";
import { useTabNavigation } from "../utils/TabNavigation";
@@ -16,7 +16,7 @@ export function Notebook() {
<p></p>
<div className='row'>
<div className="col">
- <DownloadImageButton url="" fileName="">
+ <DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/laboratory-notebook-1-proof-of-concept-for-pe.pdf" fileName="laboratory-notebook-1-proof-of-concept-for-pe.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-1-proof-of-concept-pe.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
@@ -29,7 +29,7 @@ export function Notebook() {
</div>
<div className='row'>
<div className="col">
- <DownloadImageButton url="" fileName="">
+ <DownloadImageButton url="https://static.igem.wiki/teams/5247/pdfs/lab-book-3-primary-cultures.pdf" fileName="lab-book-3-primary-cultures.pdf">
<img src="https://static.igem.wiki/teams/5247/lab-journals/titelseite-lab-book-3-primary-cell-culture.webp" style={{height: "75%", width: "auto"}}/>
</DownloadImageButton>
</div>
diff --git a/src/contents/safety.tsx b/src/contents/safety.tsx
index 550c0cc348c68f2c0eef5d6ad4a8ba5c40b20589..3de4b307a944c723bbf3a17063886f2241c26cdc 100644
--- a/src/contents/safety.tsx
+++ b/src/contents/safety.tsx
@@ -133,13 +133,13 @@ export const Safety: React.FC = () =>{
Our Cloning-laboratory is divided into different work areas to ensure that the experiments run smoothly and efficiently. These include the gel station, the PCR station, the transformation section and the measurement area. Each area is specially equipped for the respective method, and the corresponding experiments were carried out exclusively in the designated stations. In this way, we ensure that our work is carried out under optimal conditions and with the greatest possible precision.
<div className="figure-wrapper">
<figure>
- <img src="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2041.jpeg" style={{height: "10%"}}/>
+ <img src="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2041.jpeg" style={{height: "10%", width:"auto"}}/>
<figcaption> <b>Figure 1</b> Photo-gallery of laboratory. A: Key lock. B: Key-locked door. C: Alarm plan. D: Emergeny button for electriotion stop. E: Emergency telephone. F: First aid kit, cardiac defibrillaton and emergency exit and fire alarm plan. G: Wash bin with emergency eye wash. H: Emergency shower. I: Lockable cabinets for chemical storage. </figcaption>
</figure>
</div>
<div className="figure-wrapper">
<figure>
- <img src="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2037.jpeg" style={{height: "10%"}}/>
+ <img src="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2037.jpeg" style={{height: "10%", width: "auto"}}/>
<figcaption> <b>Figure 2</b> Photo-gallery of S1 laboratory. A: Autoclave. B: Refrigerator with chemicals. C: Weighing room with chemical storage. D: Clean bench work space with vortex, pipettes, heat block and bench top centrifuge. E: pH electrode in fume hood. F: Ice machine. G: Fire distinguisher and S1 waste. H: Fume hood with liquid waste.</figcaption>
</figure>
</div>
@@ -150,7 +150,7 @@ export const Safety: React.FC = () =>{
</p>
<div className="figure-wrapper">
<figure>
- <img src="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2040.jpeg" style={{height: "10%"}}/>
+ <img src="https://static.igem.wiki/teams/5247/photos/biosafety/kollage/new/img-2040.jpeg" style={{height: "10%", width: "auto"}}/>
<figcaption> <b>Figure 3</b> Photo-gallery of laboratory and chemical storage. A: Safety cabinets. B: Incubator. C: Safety cabinet.</figcaption>
</figure>
</div>
@@ -179,7 +179,7 @@ export const Safety: React.FC = () =>{
</p>
<div className="figure-wrapper">
<figure>
- <img src="https://static.igem.wiki/teams/5247/photos/biosafety/wiki/bild.png" style={{height: "10%"}}/>
+ <img src="https://static.igem.wiki/teams/5247/photos/biosafety/wiki/bild.png" style={{height: "10%", width:"auto"}}/>
<figcaption> <b>Figure 5</b>Illustration of the introduction of silent mutations leading to the PAM disrupt. </figcaption>
</figure>
</div>
@@ -189,7 +189,7 @@ export const Safety: React.FC = () =>{
</p>
<div className="figure-wrapper">
<figure>
- <img src="https://static.igem.wiki/teams/5247/photos/biosafety/wiki/bild-2.png" style={{height: "10%"}}/>
+ <img src="https://static.igem.wiki/teams/5247/photos/biosafety/wiki/bild-2.png" style={{height: "10%", width: "auto"}}/>
<figcaption> <b>Figure 6</b>Illustration of the mechanism of action of the riboswitch. </figcaption>
</figure>
</div>
diff --git a/src/data/hptimelinedata.tsx b/src/data/hptimelinedata.tsx
index 14c092dd9fca72a16eaeadc2c320a03cebfbbf48..4421417071ff311310f20e4f4e6f8c07225107cf 100644
--- a/src/data/hptimelinedata.tsx
+++ b/src/data/hptimelinedata.tsx
@@ -3,6 +3,7 @@ import { TabScrollLink } from "../components/Link";
import { ScrollLinkWithChild } from "../components/ScrollLink";
import JoshuaInterviewSources from "../sources/joshua-inv-sources";
import MattijsInterviewSources from "../sources/mattij-inv-sources";
+import LiuInterviewSources from "../sources/liu-inv-sources";
import RnhaleSources from "../sources/rnhale-sources";
import WischmeyerSources from "../sources/wimscheyer-sources";
import { useNavigation } from "../utils";
@@ -27,13 +28,13 @@ export interface TimelineDatenpunkt {
job?: string;
cardtext: string;
language?: Language;
- quote: string;
+ quote?: string;
quoteVorname?: string; /* Wenn die quote nicht von der Person ist über die der Text ist */
quoteNachname?: string;
- aimofcontact: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
- insights: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
+ aimofcontact?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
+ insights?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
clarification?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
- implementation: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
+ implementation?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
pictureurl_interview?: string; /* Picture that goes into the paragraph "Insights" */
pictureurl_aim?: string; /* Picture that goes into the paragraph "Aim of contact" */
pictureurl_implementation?: string; /* Picture that goes into the paragraph "Implementation" */
@@ -46,7 +47,7 @@ export interface TimelineDatenpunkt {
months: string,
}
-type StakeholderTag = 'Industry' | 'Academia' | 'Patient' | 'Medical Professional' | 'Milestone' | 'Other';
+type StakeholderTag = 'Industry' | 'Academia' | 'Patient' | 'Medical Professional' | 'Milestone' | 'Education'| 'Outreach'| 'Other';
type TypeTag = 'meta' | 'kaya' ;
type Language = 'en' | 'de';
@@ -55,13 +56,13 @@ type Language = 'en' | 'de';
const pics: { [key: string]: string } = {
placeholder: "https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg",
max: "https://static.igem.wiki/teams/5247/photos/hp/hp-max-portrait.jpg",
- kristian: "https://static.igem.wiki/teams/5247/photos/hp/kristian.jpeg",
+ kristian: "https://static.igem.wiki/teams/5247/photos/hp/kristian-interview.webp",
olariu: "https://static.igem.wiki/teams/5247/photos/hp/olariu-cristian.jpg",
westhoff: "https://static.igem.wiki/teams/5247/photos/hp/hp-katrin-portrait.jpg",
mattijs: "https://static.igem.wiki/teams/5247/photos/hp/mattijs.jpg",
julia: "https://static.igem.wiki/teams/5247/photos/hp/julia.jpg",
kolonko: "https://static.igem.wiki/teams/5247/photos/hp/kolonko-neu.jpg",
- svenja: "https://static.igem.wiki/teams/5247/placeholders/placehilderperson.jpeg",
+ svenja:"https://static.igem.wiki/teams/5247/photos/hp/svenja-vinke.webp",
berens: "https://static.igem.wiki/teams/5247/photos/hp/berens.jpg",
draeger: "https://static.igem.wiki/teams/5247/photos/hp/oliver-draeger-patch-clamp.jpeg",
winkeljann: "https://static.igem.wiki/teams/5247/photos/hp/rnhale-winkeljann.jpg",
@@ -80,7 +81,22 @@ const pics: { [key: string]: string } = {
kaihammer:"https://static.igem.wiki/teams/5247/photos/hp/interwiev-kai.jpeg",
nilshefe:"https://static.igem.wiki/teams/5247/photos/hp/nilshefe-hp.webp",
gxpexpert:"https://static.igem.wiki/teams/5247/photos/hp/gxpexpert.webp",
- gxpcourse:"https://static.igem.wiki/teams/5247/photos/hp/kayagxpexpert.webp"
+ gxpcourse:"https://static.igem.wiki/teams/5247/photos/hp/kayagxpexpert.webp",
+ linköping:"https://static.igem.wiki/teams/5247/photos/hp/liu2024-rund.webp",
+ biobank:"https://static.igem.wiki/teams/5247/photos/hp/biobank.webp",
+ bethel: "https://static.igem.wiki/teams/5247/photos/hp/logo-evangelisches-klinikum-bethel.webp",
+ saito:"https://static.igem.wiki/teams/5247/photos/hp/hp-makoto-saito.jpg",
+ physik:" https://static.igem.wiki/teams/5247/delivery/hp-uni-logo.webp",
+ hannovermesse: "https://static.igem.wiki/teams/5247/photos/hp/hannover-messe-svg.webp",
+ frankfurtmesse: "https://static.igem.wiki/teams/5247/photos/hp/achema.webp",
+ david:"https://static.igem.wiki/teams/5247/photos/hp/liu-talk.webp",
+ teuto: "https://static.igem.wiki/teams/5247/photos/edcation-and-outreach/teutoruft-experminet.jpeg",
+ mint: "https://static.igem.wiki/teams/5247/photos/hp/mintsommerlogo.png",
+ schueler: "https://static.igem.wiki/teams/5247/photos/edcation-and-outreach/schielerakademie-lisa-gruppe.jpg",
+ mukomove:"https://static.igem.wiki/teams/5247/photos/for-wiki-texts/po-mukomove/wir-plakat-mukomove.jpeg",
+ hakan:"https://static.igem.wiki/teams/5247/photos/hp/hakan.webp",
+ stemcell: "https://static.igem.wiki/teams/5247/photos/hp/stemcellquadrat.webp",
+
};
/* {
@@ -108,7 +124,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
vorname: "Building the team",
nachnname: "",
pictureurl: pics['placeholder'],
- tag: "Other",
+ tag: "Milestone",
heading: "Development of a multidisciplinary team structure",
interviewtabid: "recruiting",
cardtext: "",
@@ -124,7 +140,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
vorname: "Pitching ideas",
nachnname: "",
pictureurl: pics['placeholder'],
- tag: "Other",
+ tag: "Milestone",
heading: "Getting Acquainted with Cystic Fibrosis",
interviewtabid: "firstpresi",
cardtext: "",
@@ -134,13 +150,13 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
implementation: "",
type: "meta",
summary: "",
- months: ""
+ months: "March"
},
{
vorname: "Ideation",
nachnname: "",
pictureurl: pics['placeholder'],
- tag: "Other",
+ tag: "Milestone",
heading: "Brainstorming and selection of ideas and concepts",
interviewtabid: "ideas",
cardtext: "",
@@ -150,32 +166,13 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
implementation: "",
type: "meta",
summary: "",
- months: ""
- },
- {
- title: "Prof. Dr.",
- vorname: "Kristian",
- nachnname: "Müller",
- job: "Research Group Cellular and Molecular Biotechnology",
- affiliation: "Technical Faculty of Bielefeld University",
- pictureurl: pics['kristian'],
- tag: "Academia",
- heading: "Discussion about the delivery method- AVV vs. LNPs",
- interviewtabid: "kristian",
- cardtext: "",
- language: "de",
- quote: "X",
- aimofcontact: "X",
- insights: "X",
- implementation: "X",
- summary: "",
- months: ""
+ months: "March"
},
{
vorname: "Max",
nachnname: "Beckmann",
- job: "Patient",
- affiliation: "and Student of Bielefeld University",
+ job: "Patient and Student",
+ affiliation: "Bielefeld University",
pictureurl: pics['max'],
tag: "Patient",
heading: "Gathering valuable insights from the patient’s perspective",
@@ -243,8 +240,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="A therapy for which organ would benefit most people that have worse health than you do?" a="Probably the lung. The pancreas is important too, but stomach problems are usually less pressing than difficulty in breathing." />
<QaBox q="You mentioned that doing sport is difficult with CF, why?" a="Hygiene. In the lockers and the showers but also with the equipment." />
<QaBox q="Do you feel restricted in your free time activities?" a="No, I always had good alternatives. For example, going swimming at an open-air swimming pool instead of a lake. " />
- <QaBox q="Would you have more freedom when you are better protected from Pseudomonas spcc. and other potential infections? " a="text" />
- <QaBox q="text" a="Definitely. That is a big increase in the quality of life and that is a win. It also changes the picture people have of the illness. Of course being protected by prevention is good already but effective therapies for infections increase the sense of freedom even more. " />
+ <QaBox q="Would you have more freedom when you are better protected from Pseudomonas spcc. and other potential infections? "a="Definitely. That is a big increase in the quality of life and that is a win. It also changes the picture people have of the illness. Of course being protected by prevention is good already but effective therapies for infections increase the sense of freedom even more. " />
<QaBox q="You said you are afraid every time you must go for a swab, why is that? " a="I am afraid of getting an infection. That still could be a death sentence. " />
<QaBox q="Are rooms with air conditioning a problem due to the possible germs in the air conditioners? " a="No, there is usually enough movement. But humidifiers are bad because of the pond water. " />
<QaBox q="You mentioned going to the hairdresser is problematic. Could you elaborate? " a="There are many possible sources of ponding water and with that, infections. That and the hygiene aspect in general. I am visited by my hairdresser, and he only uses a specific spray bottle to wet my hair that I keep and dry thoroughly between uses." />
@@ -256,6 +252,58 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="Are most of the other patients you know in good health like you?" a="No. Another boy my age got a fungal infection and does not have long time left to live. " />
</>,
summary: "We reached out to Max, a friend of a teammate, to gain insights into the needs and experiences of cystic fibrosis (CF) patients. Our discussions revealed the challenges faced by CF patients, even those in relatively good health, and emphasized the ongoing need for new treatment options. Max's candid sharing of his experiences highlighted the limitations of current modulators, the importance of lung function, and the impact of treatments on quality of life. This meeting transformed our project perspective, urging us to prioritize safety and real-world benefits in our design. Ultimately, Max's influence led us to focus on lung-targeted gene therapy instead of a diagnostic approach, reinforcing our commitment to Integrated Human Practices.",
+ months: "April",
+ /*interview:<iframe title="Bielefeld-CeBiTec: Interview with Max Beckmann (2024) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/16501867-a687-4205-949a-51ead876e109" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>,*/
+ },
+ {
+ title: "Prof. Dr.",
+ vorname: "Kristian",
+ nachnname: "Müller",
+ job: "Research Group Cellular and Molecular Biotechnology",
+ affiliation: "Technical Faculty of Bielefeld University",
+ pictureurl: pics['kristian'],
+ tag: "Academia",
+ heading: "Discussion about the delivery method- AVV vs. LNPs",
+ interviewtabid: "kristian",
+ cardtext: "",
+ language: "de",
+ quote: "X",
+ aimofcontact: "X",
+ insights: "X",
+ implementation: "X",
+ summary: "",
+ months: "April",
+ /*interview:<iframe title="Bielefeld-CeBiTec: Interview Müller AAV vs LNP (2024) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/0613b6b8-7755-4373-9d86-9910fe30781f" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>,*/
+ },
+ {
+ vorname: "Visiting ",
+ nachnname: "the Hannover Messe",
+ pictureurl: pics['hannovermesse'],
+ tag: "Industry",
+ heading: "Two teammembers visited the fair to talk with potential stakeholder and sponsors",
+ interviewtabid: "hannovermesse",
+ cardtext: "",
+ quote: "x",
+ aimofcontact: "",
+ insights: "",
+ implementation: "",
+ summary: "",
+ months: "April"
+ },
+ {
+ vorname: "Looking for expertise",
+ nachnname: "",
+ pictureurl: pics['placeholder'],
+ tag: "Milestone",
+ heading: "Identifying key experts in cystic fibrosis and prime editing",
+ interviewtabid: "experts",
+ cardtext: "",
+ quote: "",
+ aimofcontact: "",
+ insights: "",
+ implementation: "",
+ type: "meta",
+ summary: "",
months: "April"
},
{
@@ -290,135 +338,6 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
summary: "The interview provided key insights into targeting ionocytes for CFTR-mRNA delivery, overcoming mucus barriers, and testing delivery effectiveness using Ussing chambers, organoids, and patch clamping, while also suggesting chitosan-based nanoparticles as a safer alternative to PEG-lipid systems.",
months: "may"
},
- {
- vorname: "Exploring new ideas",
- nachnname: "",
- pictureurl: pics['placeholder'],
- tag: "Other",
- affiliation: "",
- heading: "Further brainstorming on approaches",
- interviewtabid: "brainstorming",
- cardtext: "",
- quote: "",
- aimofcontact: "",
-
- insights: "",
- implementation: "",
- type: "meta",
- summary: "",
- months: ""
- },
- {
- title: "Dr.",
- vorname: "Michaela",
- nachnname: "Bienert",
- job: " Scientific Sales Representative for Cell Culture Products",
- affiliation: "Stemcell",
- pictureurl: pics['placeholder'],
- tag: "Industry",
- heading: "Determining the optimal cell media for experimentation",
- interviewtabid: "michaela",
- cardtext: "",
- language: "de",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
- },
- {
- vorname: "Looking for expertise",
- nachnname: "",
- pictureurl: pics['placeholder'],
- tag: "Other",
- heading: "Identifying key experts in cystic fibrosis and prime editing",
- interviewtabid: "experts",
- cardtext: "",
- quote: "",
- aimofcontact: "",
-
- insights: "",
- implementation: "",
- type: "meta",
- summary: "",
- months: ""
- },
- {
- vorname: "Documenting progress",
- nachnname: "",
- pictureurl: pics['placeholder'],
- tag: "Other",
- heading: "Tracking progress in expert search and idea development",
- interviewtabid: "progress",
- cardtext: "",
- quote: "",
- aimofcontact: "",
-
- insights: "",
- implementation: "",
- type: "meta",
- summary: "",
- months: ""
- },
- {
- vorname: "Jan-Phillipp",
- nachnname: "Gerhards",
- job: "Student and",
- affiliation: "Intern at Harvard/ Boston Childrens Hospital",
- pictureurl: "https://static.igem.wiki/teams/5247/photos/hp/hp-jpgerhards-potrats.webp",
- tag: "Academia",
- heading: "Discussion on optimizing our pegRNA Design to improve precision in prime editing",
- interviewtabid: "JPpegRNA",
- cardtext: "",
- language: "de",
- quoteNachname: "Lenger, Teammember",
- quoteVorname: "Malte",
- quote: "The interview proved invaluable in gaining an initial understanding of the principles of pegRNA design and optimisation, particularly in the context of silent edits.",
- aimofcontact: "The aim of the contact was to engage in a discussion about prime editing and pegRNAs, as the Jan-Phillip Gerhards had used these technologies in his internship. We sought to exchange ideas, gather insights, and explore potential improvements or strategies for our project, leveraging his experience with prime editing tools. His practical knowledge in this field was very valuable for refining our approach and ensuring we were aligned with the latest advancements and methodologies in prime editing. ",
- insights: "During our discussion we gained valuable insights that had a significant impact on our project. One of the most important findings was the effectiveness of silent edits, which will enable us to make our PrimeGuide safer. Silent edits changes the sequence of bases in the DNA in such a way that the resulting protein remains unchanged, because the genetic code is redundant. This means that different codons can code for the same amino acid. By making silent edits in addition to correcting the CFTR gene, we can prevent the pegRNA from rebinding. We have also learned that the length of the primer binding site (PBS) plays a crucial role in determining optimal results and that it is recommended to keep the PBS temperature close to 37°C. Specifically, PBS lengths of 17nt (38.3°C) and 16nt (36.4°C) were found to be ideal options. For our planned set of 12 samples, it was recommended to use three different PBS lengths (differing by +/- 1nt from that close to 37°C) in combination with four RTTs to achieve the best result. Another important finding was the use of non-annotated regions with overhangs for cloning, which could give better results in our experiments. However, we also encountered concerns that circRNA, a covalently closed circular RNA molecule, might be sterically hindered by Cas9, which we need to investigate further. When discussing cloning overhangs, we learned that a base-pair length close to 60°C is optimal. However, the use of a 15nt PBS was not recommended as it has a lower temperature range which could affect performance. Although we still need to confirm the oligonucleotide delivery time, these findings will help us to refine our cloning strategy, optimise PBS selection and improve our overall approach to primer editing, especially in terms of the pegRNA design.",
- implementation: "We incorporated the lessons learned from our discussions on prime editing and silent editing directly into our project by refining our approach to gene editing. Based on feedback about the optimal length of primer binding sequences (PBS) and RTTs, we adjusted the design of our pegRNAs to ensure greater precision and efficiency in our experiments. In particular, we learned that using PBS lengths close to 37°C melting temperatures (e.g. 16-17 nucleotides) increased stability, which led us to fine-tune these sequences for improved editing results. The concept of silent editing became an integral part of our safety strategy, allowing us to make changes to the DNA more precise. We also revised our cloning strategies by considering the appropriate overhang length, targeting a base pair length near the melting temperature of 60°C to improve cloning efficiency. We also reassessed the practicality of ordering shorter PBS sequences, concluding that lengths shorter than 15 nt were less advantageous due to reduced efficiency. By integrating these findings, we optimised our experimental workflow and made informed decisions about the tools and methods for our prime editing experiments. ",
- summary: "We engaged in a valuable discussion with Jan-Phillip Gerhards regarding prime editing and pegRNAs, leveraging his internship experience with these technologies. Key insights included the effectiveness of silent edits, which can enhance the safety of our PrimeGuide by modifying DNA sequences without altering the resultant protein, thereby preventing pegRNA rebinding. We also learned the importance of optimizing the primer binding site (PBS) length to achieve ideal temperatures close to 37°C, recommending lengths of 16-17 nucleotides. Additionally, we discovered the potential benefits of using non-annotated regions with overhangs for cloning, while also recognizing concerns about circRNA steric hindrance by Cas9. These insights directly informed our project, allowing us to refine our pegRNA design and cloning strategies, ultimately enhancing the precision and efficiency of our gene editing approach.",
- months: "May"
- },
- {
- vorname: "Katrin",
- nachnname: "Westhoff",
- job: "Physiotherapist",
- affiliation: "Independent",
- pictureurl: pics['westhoff'],
- tag: "Medical Professional",
- heading: "Interview with a specialized physiotherapist regarding breathing therapy for cystic fibrosis patients",
- interviewtabid: "westhoffinv",
- cardtext: "",
- language: "de",
- quote: "The more we know, the more opportunities we have.",
- aimofcontact: "The objective of the contact was to gain in-depth insights into the treatment and care of children with cystic fibrosis. The therapist's expertise was intended to help develop a better understanding of the challenges and necessary measures in the treatment of this chronic disease. In addition, the aim was to ascertain how the therapy is implemented in everyday life and which specific approaches and methods are particularly effective.",
- insights: "The interview yielded valuable insights into the regular implementation of the therapy, the use of aids and the adaptation of exercises to the individual needs of the patients. It was notable that the therapy has improved considerably thanks to better medication and adapted exercises, with a concomitant increase in life expectancy for children affected by cystic fibrosis. Of particular interest was the emphasis on the importance of sport and exercise, which should not only be therapeutically effective, but also increase quality of life. ",
- implementation: "The following statement by Katrin Westhoff had a particularly profound impact on our project: 'The more we know, the more opportunities we have.' We learned from the interview that the current medication is already helping many patients to a huge extent, but that there is still a significant opportunity for improvement. After all, successful gene therapy would markedly enhance the quality of life for those affected. The findings of this project will be disseminated to the relevant researchers in order to facilitate the rapid improvement of the quality of life of all cystic fibrosis patients, regardless of their mutation. ",
- pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/katrin-westhoff-zoom.webp",
- summary: "The objective of our discussion with a therapist was to gain a comprehensive understanding of the treatment and care of children with cystic fibrosis. The interview provided invaluable insights into the therapy's implementation, highlighting the significant advancements in medication and tailored exercises that have led to improved patient outcomes and increased life expectancy. A key takeaway was the emphasis on the role of sports and exercise, not just for therapeutic efficacy but also for enhancing overall quality of life. It underscored the potential for further enhancements in care through successful gene therapy, motivating us to share our findings with relevant researchers to help elevate the quality of life for all cystic fibrosis patients, regardless of their genetic mutations.",
- months: "May",
- interview:<>
- <QaBox q="From what age do the patients come to you? How long do they stay? How many patients do you treat?" a="The patients come to us at a very early age. A definite diagnosis is made after 6 weeks at the latest. Once diagnosed, the whole family is genetically tested, and children are sent for physiotherapy, often starting in the hospital. Currently, we have 8 children with cystic fibrosis in our practice, which is relatively small compared to other diseases. We have slightly more CF patients because we specialize in it."/>
- <QaBox q="What kind of exercises do you do?" a="We do a lot of breathing therapy and have attended special training courses for CF that introduced new techniques. The current gold standard is autogenous drainage according to Chevallier, which effectively removes mucus. We follow a general routine: 1. wet inhalation to bind mucus, 2. drainage to expel mucus, and 3. antibiotics to work optimally on clean lungs. We also use special belts for compressing 'magic points' to enhance lung ventilation."/>
- <QaBox q="When does drainage start?" a="We start drainage in newborns to prevent mucus from settling."/>
- <QaBox q="Are there special exercises that can also be done at home?" a="Yes, parents are instructed on exercises that can also be performed at home."/>
- <QaBox q="How often does the therapy take place?" a="Therapy usually occurs once a week or every two weeks. Thanks to improved medication, children are now better off. The therapy has evolved significantly, making it easier to cough up mucus and improving life expectancy. Exercise should be enjoyable and a part of daily life from the age of 8 or 9."/>
- <QaBox q="What would happen if no physiotherapy was performed?" a="It’s difficult to predict, but without therapy, children often become more mucousy, leading to worsened ventilation. Specific therapy is crucial, especially during infections."/>
- <QaBox q="How do you measure success (in terms of lung function test, exercise, etc.)?" a="Success is measured subjectively by listening to breathing and observing skin color. A well-ventilated lung shows a 'full barrel' appearance, while wheezing indicates poor ventilation. In clinics, lung function tests, CO2 measurements, and 'finger clip' tests are used, though results can be influenced by the child."/>
- <QaBox q="Do the exercises bring relief or are they preventative for further complaints?" a="The exercises serve both to relieve acute infections and to prevent further issues. Fewer lung infections reduce the likelihood of mucus adhesions."/>
- <QaBox q="Are there any tools to perform therapy?" a="Yes, devices like the 'flutter' or 'cornet' help with exhalation. They create vibrations that loosen mucus in the lungs and should be used by all children with lung diseases."/>
- <QaBox q="What complaints do patients bring with them?" a="Patients typically have lung problems, dry lung mucosa, and pancreatic issues leading to poor metabolism, requiring enzyme therapy before meals. Some children experience growth disorders and less commonly, excessive perspiration."/>
- <QaBox q="Are pancreatic complaints also treated by physiotherapists?" a="Pancreatic complaints are rarely treated with physiotherapy, except in cases of inflammation, where patients may be admitted to the hospital. Techniques like massage or kinesiology tape can help with constipation."/>
- <QaBox q="Are there any special hygiene guidelines for you when working with cystic fibrosis patients?" a="Hygiene is crucial when treating CF patients. We separate children with and without infections (e.g., Pseudomonas) and enforce strict disinfection protocols. Only children with similar infection statuses are treated on the same day."/>
- <QaBox q="Are the specific exercises customized? And if so, how do you know which therapy is the right one for which patient?" a="Exercises are tailored to each patient's situation, focusing on mucus removal and lung ventilation. Each therapist may have their own preferred exercises and techniques."/>
- <QaBox q="Do patients always go to the same physiotherapist?" a="Yes, if therapy is effective, patients tend to remain with the same physiotherapist."/>
- <QaBox q="How many physiotherapists offer muco-therapy?" a="The exact number is unknown, but several child therapists in the region provide cystic fibrosis therapy."/>
- <QaBox q="How are the relatives educated?" a="Education often begins in the maternity ward with a sweat test. In Gütersloh, all children are referred to Bethel for immediate CF care. Parents often experience trauma as children can be severely ill despite appearing healthy."/>
- <QaBox q="What are the limitations of individual medicine?" a="Drug effectiveness can vary, and some are only approved from a certain age. Improved medications can significantly enhance quality of life and life expectancy."/>
- </>
- },
{
title: "Dr.",
vorname: "Cristian-Gabriel",
@@ -479,70 +398,197 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
months: "may"
},
{
- vorname: "Mattijs",
- nachnname: "Bulcaen",
- job: "PhD Researcher at Laboratory for Molecular Virology & Gene Therapy",
- affiliation: "KU Leuven",
- pictureurl: pics['mattijs'],
- tag: "Academia",
- heading: "Discussion with a Prime Editing Expert on Similar Approaches for Different Mutations",
- interviewtabid: "mattijsinv",
+ vorname: "Exploring new ideas",
+ nachnname: "x",
+ pictureurl: pics['placeholder'],
+ tag: "Other",
+ affiliation: "",
+ heading: "Further brainstorming on approaches",
+ interviewtabid: "brainstorming",
cardtext: "",
- language: "en",
- quote: "[…] Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure.",
- aimofcontact: [<p>Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research <TabScrollLink tab="mattijsinv" scrollId="desc-1" num="1" />. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K).  </p>],
- insights: [<a>The interview with Mattijs was valuable for us in a lot of ways. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by <HPLinktoOtherHPTab tab="liu" text="David Liu" />, the principal
- investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a
- lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. </a>],
- implementation: "The inputs given by Mattijs directly impacted our design choices for multiple parts of the project. For the pegRNA design, we decided to use the same 3’ motif as Mattijs had used and also, like he suggested, checked our spacer candidates for predicted cleavage efficiency. Also we used HEK cells for screening our pegRNAs. We looked further into PE systems that influence cellular mismatch repair (such as PE4) and tried to include these into our design. ",
- interview: <>
- <QaBox q="We have prepared some questions for you. The first question is: You mentioned that it was quite challenging to target the F508 delta mutation. Could you provide more detailed reasons for why this is the case or explain why this mutation is particularly difficult to target compared to others?"
- a="Yes, that's the million-dollar question. First of all, let me clarify: our group has never directly worked on that mutation because we immediately focused on the drug-refractory mutations, such as nonsense mutations where the protein is not formed, indel mutations, or severe missense mutations that do not respond to modulator therapies. Of course, we know several groups in the field who either work on gene editing or focus on cystic fibrosis (CF). We've heard from some of them who attempted to target the F508 delta mutation. For example, some collaborators really tried to design different guides but were unable to find anything above the detection limit.
- F508del is probably one of the most logical mutations to try to correct, not just for CF but for the entire gene-editing field. If you look at the frequencies of mutations that cause genetic diseases, the F508 delta mutation is by far the most common deletion mutation causing a severe disease. This is because CF, along with sickle cell disease, is one of the most common deadly inherited diseases, and it's overrepresented within CF. So, it makes sense that they would have been trying to target it from the beginning.
- Interestingly, if you read the Prime Editing paper by Anzalone, F508 delta is mentioned in the introduction in connection with cystic fibrosis. So, it's somewhat surprising that after all this time—it's been almost five years now—they haven't published or released anything on F508 delta.
- However, last weekend, there was an online seminar where David Liu gave a talk, and he showed some unpublished data indicating that they managed to achieve quite good Prime Editing efficiency on F508 delta. It's worth noting that David Liu rarely presents unpublished data unless the publication is either accepted or very close to acceptance. So, we all kind of expect that the paper will be published soon, perhaps within the next week or at least within a month. From what I saw, it appears they leveraged many of the approaches available today to enhance Prime Editing.
- Now, regarding your question about why this mutation is so difficult to target with Prime Editing, I can't provide an exact answer. However, I can list some potential difficulties associated with the mutation, and it’s likely that F508 delta is challenging for several of these reasons. For instance, it could be related to the genomic region itself. Writing insertions can be more difficult; the easiest edits are single-point mutations, followed by deletions, and the most challenging are insertions. This difficulty arises because it involves writing a third strand and then relying on DNA damage repair mechanisms to fix it.
- It could also be that the region around the F508 delta mutation is challenging due to flap equilibration or that it attracts pathways such as mismatch repair that negatively impact Prime Editing. Additionally, the chromatin organization around that region could play a role. Over the past year, we’ve gathered clues that chromatin organization significantly affects Prime Editing capability, while this is much less of an issue for Cas9 and base editors.
- Studying this is not straightforward; you would need to conduct experiments like ATAC-seq to determine the chromatin organization around the mutation and how it might interfere. I also noticed on a slide that dsgRNAs were mentioned, though David Liu didn't discuss them in his talk. After looking them up online, I found that this technique, published a few years ago by other researchers, is specifically designed to open up chromatin. It seems they use different guides, without the three-prime extension, to open up the chromatin, which could be one way to overcome the limitations in Prime Editing efficiency.
- There could be other factors as well, and it’s often difficult to predict what will work and what won't. We have prediction tools for Prime Editing guides that work to some extent, but they are not as effective as the prediction tools available for regular CRISPR guide RNAs. This suggests that the Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure. I hope this answers your question somewhat. " />
- <QaBox q="That has already been very helpful, thank you for that. We'll consider this and might look into it a bit more.
- Perhaps we could quickly discuss which part of the prime editing complex you think plays the most significant role in making insertions much more challenging compared to deletions. Is it the reverse transcriptase or the RNA? "
- a="I don't think it's primarily the reverse transcriptase that's the issue. People have shown that longer insertions are definitely possible. I believe the challenge lies in the process when your cell has to repair the new DNA strand, which is generated and exists as a three-stranded intermediate. We don’t directly intervene in this process; it entirely depends on the cell and the DNA damage repair pathways active in those cells. Through expression of dominant negative DNA damage repair effectors, or by nicking the non-edited strand, the outcome can be steered to some extent.
- When you perform an insertion, the new strand must hybridize with the bottom strand, which remains intact. This creates a small loop that needs to be incorporated. At this point, the cell faces two options: it can either revert to the original state or incorporate the edit you’re trying to introduce. In certain circumstances, perhaps due to how the new DNA strand folds or the sequence context of the region of interest, the cell might heavily favor reverting to the original state, resulting in the absence of the intended edit.
- This process is extremely difficult to predict, but there are several indications pointing in this direction. For example, in the case of point mutations, it has been shown that it’s easier to convert a C to a G rather than the reverse, simply due to how these mismatches are recognized by the DNA damage repair mechanisms. This area is very complex, and I don’t think anyone fully understands it yet. It’s also difficult to study.
- I don't believe the rate of reverse transcription is the limiting factor here, although it could play a role for long or structured pegRNAs. You might have already come across this, but the PE6 generation of Prime Editors, which were released about half a year ago, involve engineered or evolved reverse transcriptases that are more processive and can more easily synthesize longer transcripts.
- Another factor that could play a role is the secondary structure of the guide RNA. Each prime editing guide RNA faces a common problem: it has a spacer that binds the bottom strand and a three-prime extension that binds the top strand. Since these two parts of the RNA bind complementary strands, they are also complementary to each other, meaning every prime editing guide has some tendency to bind itself. If the Gibbs free energy is too high, the guide RNA may fold in on itself, preventing it from binding to the prime editor, which then inhibits prime editing.
- Additionally, the three-prime extension itself can fold independently. I haven’t specifically examined this for the F508 delta guides, but it is something that can be predicted. There are tools available that can predict the secondary structure of an RNA sequence, and if there’s a significant hairpin structure, it might mean the three-prime extension remains closed, preventing the reverse transcriptase from using it as a template. The PE6 prime editors have been engineered to be more effective in such scenarios, being less affected by secondary structures and better able to read through them. " />
- <QaBox q="Yes, exactly, we noticed the same thing when predicting the secondary structure of our guide RNA. As you mentioned, the spacer and the binding site are complementary, so we end up with a really long complementary strand that binds to itself. We were also unsure whether it would open up or remain bound together."
- a="I think the Liu lab mentioned in the PE6 paper a threshold for the free energy of the guide RNA structure. They suggest that every guide will behave differently, but there’s often a more complex interaction at play than just a simple threshold. If the free energy is not too low, the guide RNA may still function efficiently and be incorporated into the prime editor, with everything remaining in equilibrium. However, if the free energy is too low, meaning high propensity for self-folding, it can cause problems.
- I also recently came across a paper from the group of Keith Joung, another prominent CRISPR scientist from the U.S., where they demonstrated that applying a heat shock to the guide RNA can help it refold. This is particularly relevant if you’re using RNP or mRNA with synthetic guide RNA. They linked this specifically to the self-binding capacity of the guide RNA, suggesting that heat shock can mitigate the issues caused by self-binding. " />
- <QaBox q="What would be the application? Would you administer the heat shock in vivo?"
- a="I believe they used it to engineer zebrafish embryos or something along those lines. It’s quite specific, of course. If you plan to deliver your guide RNA through a viral vector or similar method for human therapy, the application would differ significantly. You obviously can't administer a heat shock to humans, so it really depends on the context of your application." />
- <QaBox q="Okay, that's interesting. Given the time constraints, let's move on to the next question. Due to our limited resources, we are targeting a PE2 system, and we'd like to ask if you see any chances of success with this system. If so, how high do you think the chances of success are? We understand that the PE3 system, as shown in your paper, is much more advanced and performs significantly better. But given our situation, do you think our PE2 system could still be effective, or would you suggest that it only makes sense to use something like PE3?"
- a="PE2 can work, but it really depends on your application and the methods you have to assess the editing efficiency. If you can use NGS (Next-Generation Sequencing) for everything, you'll be able to detect edits even with PE2 systems. However, I would generally expect the efficiency to be low. Whenever possible, I would always recommend trying the PE3 system. Could you share what your specific application is, or is that confidential?" />
- <QaBox q="So our goal is to eventually use it in vivo, but for now, we're focusing on trying to correct the mutation first in regular cell cultures and then later in primary cells."
- a="Is your focus specifically on the F508 delta mutation? If so, we could potentially help you get you started, as we already have constructs and cells with that mutation. We would need to discuss the financial aspects, but we might be able to assist. However, are you fully committed to targeting F508, or are you also considering other diseases or mutations?" />
- <QaBox q="The timeframe of the project, combined with the fact that we’re all studying on the side, limits us to a certain scope. Since this is our first time tackling a project like this, it makes sense to stick to something more manageable. So, we're somewhat committed to focusing on F508 due to these constraints."
- a="That's understandable. It can be really tough to juggle a project like this along with exams and studies, especially if you're also involved in competitions. But it's definitely worth the effort, even if you don't achieve huge results right away. The experience and learning, as well as the connections you make, are incredibly valuable. I'm a big supporter of such projects. So, what resources do you currently have? Do you already have cells with the F508 delta mutation, or...? " />
- <QaBox q="We have one patient who is willing to provide us with cells, but we don't have them yet. "
- a="It sounds like you're aware of the challenges, and I don't want to discourage you, but just to be realistic, working with primary cells and getting everything ready could be tricky, especially considering the competition is in October. Experiments in human cells can take time, especially if you need to do multiple iterations or clone constructs—it could easily take a week or more per experiment.
- Regarding the cells we have, as mentioned in our paper, we screened all our guides on HEK cells with an integrated copy of the CFTR cDNA. HEK cells are easy to work with, but they don't naturally express CFTR, even though the gene is present in their genome. So, we introduced the mutation of interest into these cells, making it easier to screen.
- I'm not entirely sure if we can send over the cells due to ethical regulations, which can be complex and time-consuming to navigate. However, there's an alternative approach that might help you. Early on, we found that it's actually quite easy to screen guides using what we call a 'transient target'. In this method, you would transfect all your prime editing plasmids into HEK cells, along with a plasmid containing the CFTR cDNA with the mutation of interest. While this approach isn’t as physiological as editing the chromosome directly, our side-by-side comparisons showed almost equal efficiencies between transient and chromosomal targets. It's much easier and faster than working with patient-derived cells. I can definitely send you the plasmid, which would save you a lot of time and effort. This method is much simpler and could be a practical solution for your project. " />
- <QaBox q="Our initial plan is to work with a reporter plasmid that expresses eGFP, where we've removed a splice site, until we have patient cells or cell lines with CFTR mutations. This will allow us to screen easily without needing to sequence everything. Do you maybe have any suggestions or advice on this approach? "
- a="Is that the PEAR system? No, it’s a different one, but we also have a similar system. The advantage of this approach is that you can very easily see if it works, and it’s very sensitive—much easier than extracting and sequencing DNA. The downside, however, is that… actually, I’m not familiar with the 'flu PEAR system.'
- Actually, we use the exact same system in our lab. It’s very useful for optimizing delivery strategies because it’s easy to see results. The downside, of course, is that the guides you’re using for that system aren’t specific to the F508 delta mutation, right? So, these are scientific trade-offs. You could, for example, design a reporter that uses your F508 delta guide and also results in fluorescence, but you would need to design the reporter first. It’s challenging to prove that it works because you might not have a perfect guide for F508 delta.
- It really depends on what you want to achieve. If your goal is to first check if you can successfully perform prime editing, then using the reporter is definitely a good first step." />
- <QaBox q="We will edit the plasmid, specifically the vector, so that we have almost the same pegRNA. The only difference will be downstream, behind the edit."
- a="Is this approach based on a paper from the Netherlands, or is it something you came up with yourself? " />
- <QaBox q="Based on a paper. "
- a="Yeah, that sounds like a very good way to start. Do you already have the reporter plasmid ready? " />
- <QaBox q="Yeah, we bought the reporter, and now we’re making the necessary edits so we can use it. "
- a="Okay, so do you also already have guides targeting F508 right now? " />
- <QaBox q="We’ve designed some guides, but we haven’t tested them yet. That’s one of our next steps. So, at the moment, we’re just in the design phase, or we have already designed them, and..." a="Yeah, okay, cool. Good luck with that! And I suppose you’re starting off with HEK cells as well, right?"/>
- <QaBox q="We have HEK and HeLa cells, but we haven't decided yet which ones we'll use." a="I would start off in HEK cells because, by total accident or coincidence, they are much easier to achieve prime editing in. This is because the MLH1 gene, which negatively impacts prime editing outcomes, is naturally disabled in these cells—they don't produce the MLH1 protein. Of all cell lines available, HEK cells are the easiest to achieve editing with, so I would definitely recommend starting there. In terms of transfection, HEK cells are also very easily transfected. If I can offer another piece of advice, always include GFP controls—plasmids that simply express GFP without requiring editing—and use them to determine your transfection efficiency. It's crucial to have a very high transfection efficiency because you'll be working with a three-component system: your reporter, your prime editor, and your guides. All three plasmids need to be present in the same cell for the editing to occur, so you should aim for at least 70% transfection efficiency, preferably 80% or higher. I don't know what transfection method you're planning to use, but we've always used Lipofectamine 3000. It’s expensive, but it works very well. However, if you're looking for more cost-effective options, we recently discovered two other transfection reagents, Jet Optimus and Jet Prime, which are much cheaper and also work quite well. That said, I would advise against starting with any of the cheaper transfection reagents; you really need to aim for high transfection efficiency. Always make sure to measure and report transfection efficiency for every experiment because if it's low, the experiment might not yield useful results. If you have the funds or resources, I would also recommend designing P3 or even P3b guides, as they might offer better efficiency. When it comes to designing P3b guides, if you're primarily focused on P2 right now, there are some specific considerations to keep in mind. I'll provide you with a site that can help with this, and I'll give you the link in just a moment. So, it's very advisable to check the Doench score. Do you know what it is?"/>
- <QaBox q="No, not really." a="There are papers by John Doench, an American researcher, from quite a while ago that, in my opinion, are some of the best around. He developed a comprehensive scoring matrix specifically for regular Cas9 that can evaluate the quality of the spacer in your guide RNA. This is important because Cas9 tends to prefer certain sequences over others. For instance, a good spacer should have an appropriate GC content and should avoid hairpins that might cause it to fold in on itself, which would prevent it from functioning properly. You can use this matrix to give a score for the quality of a guide RNA. I’m going to pull up an example here. The site from Synthego, a commercial provider of CRISPR reagents, allows you to check the quality of your guide. When you validate it, the site gives a score based on various factors, including off-target effects, although that might not be your primary concern at the moment. If you hover over a specific area, it will show you the Doench Score, which is crucial. Ideally, you want a guide with a good Doench Score. A good score starts at around 0.4, indicated by a green check mark for good efficiency. If the score is very low, it means that the guide likely has low CRISPR-Cas9 activity and may not be very efficient. When designing prime editing guides, RNA, we always check the spacer for a good Doench Score. If we are designing nicking guides for a PE3 or PE3b strategy, we also ensure that they have a good score. This is one of the easiest tools to check for that. Whenever possible, try using PE3. In some cases, PE3 performs better than PE2, though not always. PE3b might not always work either, but for many mutations, we have seen significant increases in editing efficiency by including the PE3 guide."/>
- <QaBox q="Okay, yeah, that was quite clear from your results; the diagram illustrated that very well. Are there more off-target effects when using PE3 since you have to make another cut?" a="If you decide to use PE3, it's important to be aware that while it's not exactly an off-target issue, there is a risk of an undesired on-target outcome. The concern with regular PE3 is that both strands of DNA can be nicked simultaneously, which can lead to a staggered double-strand break. This can result in the formation of indels (insertions or deletions). In your case, this means that if the region around the F508 delta mutation is broken, the prime editor might not be able to repair it properly, leading to additional base pairs being removed or added, and thus, the sequence might be altered in an unintended way. The risk of on-target indels is definitely higher with PE3 compared to PE2. However, this risk is reduced when using PE3b, which employs sequential nicking. The PE3b nicking guides are designed to recognize the wild-type sequence, and they can only nick the opposite strand if the correction has already been made on the top strand. This sequential action helps to avoid the generation of indels. Introducing a second guide into the system also brings the possibility of off-target editing by that guide however, since only a Cas9 nickase is used, off-target indels should be limited."/>
- <QaBox q="Yes, okay, thank you. Do you have time left, or are we out of time?"a=" It's fine."/>
+ quote: "x",
+ quoteNachname:"x",
+ quoteVorname: "d",
+ aimofcontact: "d",
+ insights: "d",
+ implementation: "d",
+ type: "meta",
+ summary: "d",
+ months: "may"
+ },
+ {
+ vorname: "Katrin",
+ nachnname: "Westhoff",
+ job: "Physiotherapist",
+ affiliation: "Independent",
+ pictureurl: pics['westhoff'],
+ tag: "Medical Professional",
+ heading: "Interview with a specialized physiotherapist regarding breathing therapy for cystic fibrosis patients",
+ interviewtabid: "westhoffinv",
+ cardtext: "",
+ language: "de",
+ quote: "The more we know, the more opportunities we have.",
+ aimofcontact: "The objective of the contact was to gain in-depth insights into the treatment and care of children with cystic fibrosis. The therapist's expertise was intended to help develop a better understanding of the challenges and necessary measures in the treatment of this chronic disease. In addition, the aim was to ascertain how the therapy is implemented in everyday life and which specific approaches and methods are particularly effective.",
+ insights: "The interview yielded valuable insights into the regular implementation of the therapy, the use of aids and the adaptation of exercises to the individual needs of the patients. It was notable that the therapy has improved considerably thanks to better medication and adapted exercises, with a concomitant increase in life expectancy for children affected by cystic fibrosis. Of particular interest was the emphasis on the importance of sport and exercise, which should not only be therapeutically effective, but also increase quality of life. ",
+ implementation: "The following statement by Katrin Westhoff had a particularly profound impact on our project: 'The more we know, the more opportunities we have.' We learned from the interview that the current medication is already helping many patients to a huge extent, but that there is still a significant opportunity for improvement. After all, successful gene therapy would markedly enhance the quality of life for those affected. The findings of this project will be disseminated to the relevant researchers in order to facilitate the rapid improvement of the quality of life of all cystic fibrosis patients, regardless of their mutation. ",
+ pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/katrin-westhoff-zoom.webp",
+ summary: "The objective of our discussion with a therapist was to gain a comprehensive understanding of the treatment and care of children with cystic fibrosis. The interview provided invaluable insights into the therapy's implementation, highlighting the significant advancements in medication and tailored exercises that have led to improved patient outcomes and increased life expectancy. A key takeaway was the emphasis on the role of sports and exercise, not just for therapeutic efficacy but also for enhancing overall quality of life. It underscored the potential for further enhancements in care through successful gene therapy, motivating us to share our findings with relevant researchers to help elevate the quality of life for all cystic fibrosis patients, regardless of their genetic mutations.",
+ months: "May",
+ interview:<>
+ <QaBox q="From what age do the patients come to you? How long do they stay? How many patients do you treat?" a="The patients come to us at a very early age. A definite diagnosis is made after 6 weeks at the latest. Once diagnosed, the whole family is genetically tested, and children are sent for physiotherapy, often starting in the hospital. Currently, we have 8 children with cystic fibrosis in our practice, which is relatively small compared to other diseases. We have slightly more CF patients because we specialize in it."/>
+ <QaBox q="What kind of exercises do you do?" a="We do a lot of breathing therapy and have attended special training courses for CF that introduced new techniques. The current gold standard is autogenous drainage according to Chevallier, which effectively removes mucus. We follow a general routine: 1. wet inhalation to bind mucus, 2. drainage to expel mucus, and 3. antibiotics to work optimally on clean lungs. We also use special belts for compressing 'magic points' to enhance lung ventilation."/>
+ <QaBox q="When does drainage start?" a="We start drainage in newborns to prevent mucus from settling."/>
+ <QaBox q="Are there special exercises that can also be done at home?" a="Yes, parents are instructed on exercises that can also be performed at home."/>
+ <QaBox q="How often does the therapy take place?" a="Therapy usually occurs once a week or every two weeks. Thanks to improved medication, children are now better off. The therapy has evolved significantly, making it easier to cough up mucus and improving life expectancy. Exercise should be enjoyable and a part of daily life from the age of 8 or 9."/>
+ <QaBox q="What would happen if no physiotherapy was performed?" a="It’s difficult to predict, but without therapy, children often become more mucousy, leading to worsened ventilation. Specific therapy is crucial, especially during infections."/>
+ <QaBox q="How do you measure success (in terms of lung function test, exercise, etc.)?" a="Success is measured subjectively by listening to breathing and observing skin color. A well-ventilated lung shows a 'full barrel' appearance, while wheezing indicates poor ventilation. In clinics, lung function tests, CO2 measurements, and 'finger clip' tests are used, though results can be influenced by the child."/>
+ <QaBox q="Do the exercises bring relief or are they preventative for further complaints?" a="The exercises serve both to relieve acute infections and to prevent further issues. Fewer lung infections reduce the likelihood of mucus adhesions."/>
+ <QaBox q="Are there any tools to perform therapy?" a="Yes, devices like the 'flutter' or 'cornet' help with exhalation. They create vibrations that loosen mucus in the lungs and should be used by all children with lung diseases."/>
+ <QaBox q="What complaints do patients bring with them?" a="Patients typically have lung problems, dry lung mucosa, and pancreatic issues leading to poor metabolism, requiring enzyme therapy before meals. Some children experience growth disorders and less commonly, excessive perspiration."/>
+ <QaBox q="Are pancreatic complaints also treated by physiotherapists?" a="Pancreatic complaints are rarely treated with physiotherapy, except in cases of inflammation, where patients may be admitted to the hospital. Techniques like massage or kinesiology tape can help with constipation."/>
+ <QaBox q="Are there any special hygiene guidelines for you when working with cystic fibrosis patients?" a="Hygiene is crucial when treating CF patients. We separate children with and without infections (e.g., Pseudomonas) and enforce strict disinfection protocols. Only children with similar infection statuses are treated on the same day."/>
+ <QaBox q="Are the specific exercises customized? And if so, how do you know which therapy is the right one for which patient?" a="Exercises are tailored to each patient's situation, focusing on mucus removal and lung ventilation. Each therapist may have their own preferred exercises and techniques."/>
+ <QaBox q="Do patients always go to the same physiotherapist?" a="Yes, if therapy is effective, patients tend to remain with the same physiotherapist."/>
+ <QaBox q="How many physiotherapists offer muco-therapy?" a="The exact number is unknown, but several child therapists in the region provide cystic fibrosis therapy."/>
+ <QaBox q="How are the relatives educated?" a="Education often begins in the maternity ward with a sweat test. In Gütersloh, all children are referred to Bethel for immediate CF care. Parents often experience trauma as children can be severely ill despite appearing healthy."/>
+ <QaBox q="What are the limitations of individual medicine?" a="Drug effectiveness can vary, and some are only approved from a certain age. Improved medications can significantly enhance quality of life and life expectancy."/>
+ </>
+ },
+ {
+ title: "Dr.",
+ vorname: "Michaela",
+ nachnname: "Bienert",
+ job: " Scientific Sales Representative for Cell Culture Products",
+ affiliation: "Stemcell",
+ pictureurl: pics['stemcell'],
+ tag: "Industry",
+ heading: "Longstanding Support from Stemcell Expert in Cell Culture Media and Primary Cell Cultivation",
+ interviewtabid: "michaela",
+ cardtext: "",
+ language: "de",
+ quote: "x",
+ aimofcontact: "Our primary goal in reaching out to her was to gain insights into optimizing cell culture media, explore potential sponsorship opportunities for our project, and seek expert advice on handling primary cells in the lab.",
+ insights: "Throughout our multiple discussions, she offered detailed advice on selecting and optimizing cell culture media for our specific needs, while also connecting us with opportunities for sponsorship. Later in the project, she provided technical guidance on the cultivation and handling of primary cells, ensuring that we followed best practices for cell culture.",
+ implementation: "Based on her feedback, we refined our approach to cell culture media, enhancing our experimental workflows. Additionally, her advice was critical during the transition to primary cell culture, helping us secure the necessary resources and expertise for our experiments.",
+ summary: "We have had the privilege of collaborating with a dedicated expert from Stemcell Technologies, who has consistently supported iGEM Bielefeld. She provided valuable guidance on cell culture media, sponsorship opportunities, and later, practical advice for cultivating primary cells. Her contributions have been instrumental in advancing our project.",
+ months: "several times",
+ pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/daniela.webp",
+ },
+ {
+ vorname: "Documenting progress",
+ nachnname: "",
+ pictureurl: pics['placeholder'],
+ tag: "Other",
+ heading: "Tracking progress in expert search and idea development",
+ interviewtabid: "progress",
+ cardtext: "",
+ quote: "",
+ aimofcontact: "",
+ insights: "",
+ implementation: "",
+ type: "meta",
+ summary: "",
+ months: "may"
+ },
+ {
+ vorname: "MukoMove",
+ nachnname: "",
+ pictureurl: pics['mukomove'],
+ tag: "Outreach",
+ heading: "Moving together for health and hope, our participation in CF awarness month",
+ interviewtabid: "mukomovehp",
+ type: "meta",
+ cardtext: "",
+ quote: "x",
+ summary: "",
+ months: "May"
+ },
+ {
+ title: "M.Sc.",
+ vorname: "Jan-Phillipp",
+ nachnname: "Gerhards",
+ job: "Intern",
+ affiliation: " Harvard/ Boston Childrens Hospital",
+ pictureurl: "https://static.igem.wiki/teams/5247/photos/hp/hp-jpgerhards-potrats.webp",
+ tag: "Academia",
+ heading: "Discussion on optimizing our pegRNA Design to improve precision in prime editing",
+ interviewtabid: "JPpegRNA",
+ cardtext: "",
+ language: "de",
+ quoteNachname: "Lenger, Teammember",
+ quoteVorname: "Malte",
+ quote: "The interview proved invaluable in gaining an initial understanding of the principles of pegRNA design and optimisation, particularly in the context of silent edits.",
+ aimofcontact: "The aim of the contact was to engage in a discussion about prime editing and pegRNAs, as the Jan-Phillip Gerhards had used these technologies in his internship. We sought to exchange ideas, gather insights, and explore potential improvements or strategies for our project, leveraging his experience with prime editing tools. His practical knowledge in this field was very valuable for refining our approach and ensuring we were aligned with the latest advancements and methodologies in prime editing. ",
+ insights: "During our discussion we gained valuable insights that had a significant impact on our project. One of the most important findings was the effectiveness of silent edits, which will enable us to make our PrimeGuide safer. Silent edits changes the sequence of bases in the DNA in such a way that the resulting protein remains unchanged, because the genetic code is redundant. This means that different codons can code for the same amino acid. By making silent edits in addition to correcting the CFTR gene, we can prevent the pegRNA from rebinding. We have also learned that the length of the primer binding site (PBS) plays a crucial role in determining optimal results and that it is recommended to keep the PBS temperature close to 37°C. Specifically, PBS lengths of 17nt (38.3°C) and 16nt (36.4°C) were found to be ideal options. For our planned set of 12 samples, it was recommended to use three different PBS lengths (differing by +/- 1nt from that close to 37°C) in combination with four RTTs to achieve the best result. Another important finding was the use of non-annotated regions with overhangs for cloning, which could give better results in our experiments. However, we also encountered concerns that circRNA, a covalently closed circular RNA molecule, might be sterically hindered by Cas9, which we need to investigate further. When discussing cloning overhangs, we learned that a base-pair length close to 60°C is optimal. However, the use of a 15nt PBS was not recommended as it has a lower temperature range which could affect performance. Although we still need to confirm the oligonucleotide delivery time, these findings will help us to refine our cloning strategy, optimise PBS selection and improve our overall approach to primer editing, especially in terms of the pegRNA design.",
+ implementation: "We incorporated the lessons learned from our discussions on prime editing and silent editing directly into our project by refining our approach to gene editing. Based on feedback about the optimal length of primer binding sequences (PBS) and RTTs, we adjusted the design of our pegRNAs to ensure greater precision and efficiency in our experiments. In particular, we learned that using PBS lengths close to 37°C melting temperatures (e.g. 16-17 nucleotides) increased stability, which led us to fine-tune these sequences for improved editing results. The concept of silent editing became an integral part of our safety strategy, allowing us to make changes to the DNA more precise. We also revised our cloning strategies by considering the appropriate overhang length, targeting a base pair length near the melting temperature of 60°C to improve cloning efficiency. We also reassessed the practicality of ordering shorter PBS sequences, concluding that lengths shorter than 15 nt were less advantageous due to reduced efficiency. By integrating these findings, we optimised our experimental workflow and made informed decisions about the tools and methods for our prime editing experiments. ",
+ summary: "We engaged in a valuable discussion with Jan-Phillip Gerhards regarding prime editing and pegRNAs, leveraging his internship experience with these technologies. Key insights included the effectiveness of silent edits, which can enhance the safety of our PrimeGuide by modifying DNA sequences without altering the resultant protein, thereby preventing pegRNA rebinding. We also learned the importance of optimizing the primer binding site (PBS) length to achieve ideal temperatures close to 37°C, recommending lengths of 16-17 nucleotides. Additionally, we discovered the potential benefits of using non-annotated regions with overhangs for cloning, while also recognizing concerns about circRNA steric hindrance by Cas9. These insights directly informed our project, allowing us to refine our pegRNA design and cloning strategies, ultimately enhancing the precision and efficiency of our gene editing approach.",
+ months: "May"
+ },
+ {
+ vorname: "Mattijs",
+ nachnname: "Bulcaen",
+ job: "PhD Researcher at Laboratory for Molecular Virology & Gene Therapy",
+ affiliation: "KU Leuven",
+ pictureurl: pics['mattijs'],
+ tag: "Academia",
+ heading: "Discussion with a Prime Editing Expert on Similar Approaches for Different Mutations",
+ interviewtabid: "mattijsinv",
+ cardtext: "",
+ language: "en",
+ quote: "[…] Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure.",
+ aimofcontact: [<p>Shortly after we decided to use prime editing as the gene editing method for our cystic fibrosis therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research <TabScrollLink tab="mattijsinv" scrollId="desc-1" num="1" />. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K).  </p>],
+ insights: [<a>The interview with Mattijs was valuable for us in a lot of ways. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by <HPLinktoOtherHPTab tab="liu" text="David Liu" />, the principal
+ investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a
+ lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. </a>],
+ implementation: "The inputs given by Mattijs directly impacted our design choices for multiple parts of the project. For the pegRNA design, we decided to use the same 3’ motif as Mattijs had used and also, like he suggested, checked our spacer candidates for predicted cleavage efficiency. Also we used HEK cells for screening our pegRNAs. We looked further into PE systems that influence cellular mismatch repair (such as PE4) and tried to include these into our design. ",
+ interview: <>
+ <QaBox q="We have prepared some questions for you. The first question is: You mentioned that it was quite challenging to target the F508 delta mutation. Could you provide more detailed reasons for why this is the case or explain why this mutation is particularly difficult to target compared to others?"
+ a="Yes, that's the million-dollar question. First of all, let me clarify: our group has never directly worked on that mutation because we immediately focused on the drug-refractory mutations, such as nonsense mutations where the protein is not formed, indel mutations, or severe missense mutations that do not respond to modulator therapies. Of course, we know several groups in the field who either work on gene editing or focus on cystic fibrosis (CF). We've heard from some of them who attempted to target the F508 delta mutation. For example, some collaborators really tried to design different guides but were unable to find anything above the detection limit.
+ F508del is probably one of the most logical mutations to try to correct, not just for CF but for the entire gene-editing field. If you look at the frequencies of mutations that cause genetic diseases, the F508 delta mutation is by far the most common deletion mutation causing a severe disease. This is because CF, along with sickle cell disease, is one of the most common deadly inherited diseases, and it's overrepresented within CF. So, it makes sense that they would have been trying to target it from the beginning.
+ Interestingly, if you read the Prime Editing paper by Anzalone, F508 delta is mentioned in the introduction in connection with cystic fibrosis. So, it's somewhat surprising that after all this time—it's been almost five years now—they haven't published or released anything on F508 delta.
+ However, last weekend, there was an online seminar where David Liu gave a talk, and he showed some unpublished data indicating that they managed to achieve quite good Prime Editing efficiency on F508 delta. It's worth noting that David Liu rarely presents unpublished data unless the publication is either accepted or very close to acceptance. So, we all kind of expect that the paper will be published soon, perhaps within the next week or at least within a month. From what I saw, it appears they leveraged many of the approaches available today to enhance Prime Editing.
+ Now, regarding your question about why this mutation is so difficult to target with Prime Editing, I can't provide an exact answer. However, I can list some potential difficulties associated with the mutation, and it’s likely that F508 delta is challenging for several of these reasons. For instance, it could be related to the genomic region itself. Writing insertions can be more difficult; the easiest edits are single-point mutations, followed by deletions, and the most challenging are insertions. This difficulty arises because it involves writing a third strand and then relying on DNA damage repair mechanisms to fix it.
+ It could also be that the region around the F508 delta mutation is challenging due to flap equilibration or that it attracts pathways such as mismatch repair that negatively impact Prime Editing. Additionally, the chromatin organization around that region could play a role. Over the past year, we’ve gathered clues that chromatin organization significantly affects Prime Editing capability, while this is much less of an issue for Cas9 and base editors.
+ Studying this is not straightforward; you would need to conduct experiments like ATAC-seq to determine the chromatin organization around the mutation and how it might interfere. I also noticed on a slide that dsgRNAs were mentioned, though David Liu didn't discuss them in his talk. After looking them up online, I found that this technique, published a few years ago by other researchers, is specifically designed to open up chromatin. It seems they use different guides, without the three-prime extension, to open up the chromatin, which could be one way to overcome the limitations in Prime Editing efficiency.
+ There could be other factors as well, and it’s often difficult to predict what will work and what won't. We have prediction tools for Prime Editing guides that work to some extent, but they are not as effective as the prediction tools available for regular CRISPR guide RNAs. This suggests that the Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure. I hope this answers your question somewhat. " />
+ <QaBox q="That has already been very helpful, thank you for that. We'll consider this and might look into it a bit more.
+ Perhaps we could quickly discuss which part of the prime editing complex you think plays the most significant role in making insertions much more challenging compared to deletions. Is it the reverse transcriptase or the RNA? "
+ a="I don't think it's primarily the reverse transcriptase that's the issue. People have shown that longer insertions are definitely possible. I believe the challenge lies in the process when your cell has to repair the new DNA strand, which is generated and exists as a three-stranded intermediate. We don’t directly intervene in this process; it entirely depends on the cell and the DNA damage repair pathways active in those cells. Through expression of dominant negative DNA damage repair effectors, or by nicking the non-edited strand, the outcome can be steered to some extent.
+ When you perform an insertion, the new strand must hybridize with the bottom strand, which remains intact. This creates a small loop that needs to be incorporated. At this point, the cell faces two options: it can either revert to the original state or incorporate the edit you’re trying to introduce. In certain circumstances, perhaps due to how the new DNA strand folds or the sequence context of the region of interest, the cell might heavily favor reverting to the original state, resulting in the absence of the intended edit.
+ This process is extremely difficult to predict, but there are several indications pointing in this direction. For example, in the case of point mutations, it has been shown that it’s easier to convert a C to a G rather than the reverse, simply due to how these mismatches are recognized by the DNA damage repair mechanisms. This area is very complex, and I don’t think anyone fully understands it yet. It’s also difficult to study.
+ I don't believe the rate of reverse transcription is the limiting factor here, although it could play a role for long or structured pegRNAs. You might have already come across this, but the PE6 generation of Prime Editors, which were released about half a year ago, involve engineered or evolved reverse transcriptases that are more processive and can more easily synthesize longer transcripts.
+ Another factor that could play a role is the secondary structure of the guide RNA. Each prime editing guide RNA faces a common problem: it has a spacer that binds the bottom strand and a three-prime extension that binds the top strand. Since these two parts of the RNA bind complementary strands, they are also complementary to each other, meaning every prime editing guide has some tendency to bind itself. If the Gibbs free energy is too high, the guide RNA may fold in on itself, preventing it from binding to the prime editor, which then inhibits prime editing.
+ Additionally, the three-prime extension itself can fold independently. I haven’t specifically examined this for the F508 delta guides, but it is something that can be predicted. There are tools available that can predict the secondary structure of an RNA sequence, and if there’s a significant hairpin structure, it might mean the three-prime extension remains closed, preventing the reverse transcriptase from using it as a template. The PE6 prime editors have been engineered to be more effective in such scenarios, being less affected by secondary structures and better able to read through them. " />
+ <QaBox q="Yes, exactly, we noticed the same thing when predicting the secondary structure of our guide RNA. As you mentioned, the spacer and the binding site are complementary, so we end up with a really long complementary strand that binds to itself. We were also unsure whether it would open up or remain bound together."
+ a="I think the Liu lab mentioned in the PE6 paper a threshold for the free energy of the guide RNA structure. They suggest that every guide will behave differently, but there’s often a more complex interaction at play than just a simple threshold. If the free energy is not too low, the guide RNA may still function efficiently and be incorporated into the prime editor, with everything remaining in equilibrium. However, if the free energy is too low, meaning high propensity for self-folding, it can cause problems.
+ I also recently came across a paper from the group of Keith Joung, another prominent CRISPR scientist from the U.S., where they demonstrated that applying a heat shock to the guide RNA can help it refold. This is particularly relevant if you’re using RNP or mRNA with synthetic guide RNA. They linked this specifically to the self-binding capacity of the guide RNA, suggesting that heat shock can mitigate the issues caused by self-binding. " />
+ <QaBox q="What would be the application? Would you administer the heat shock in vivo?"
+ a="I believe they used it to engineer zebrafish embryos or something along those lines. It’s quite specific, of course. If you plan to deliver your guide RNA through a viral vector or similar method for human therapy, the application would differ significantly. You obviously can't administer a heat shock to humans, so it really depends on the context of your application." />
+ <QaBox q="Okay, that's interesting. Given the time constraints, let's move on to the next question. Due to our limited resources, we are targeting a PE2 system, and we'd like to ask if you see any chances of success with this system. If so, how high do you think the chances of success are? We understand that the PE3 system, as shown in your paper, is much more advanced and performs significantly better. But given our situation, do you think our PE2 system could still be effective, or would you suggest that it only makes sense to use something like PE3?"
+ a="PE2 can work, but it really depends on your application and the methods you have to assess the editing efficiency. If you can use NGS (Next-Generation Sequencing) for everything, you'll be able to detect edits even with PE2 systems. However, I would generally expect the efficiency to be low. Whenever possible, I would always recommend trying the PE3 system. Could you share what your specific application is, or is that confidential?" />
+ <QaBox q="So our goal is to eventually use it in vivo, but for now, we're focusing on trying to correct the mutation first in regular cell cultures and then later in primary cells."
+ a="Is your focus specifically on the F508 delta mutation? If so, we could potentially help you get you started, as we already have constructs and cells with that mutation. We would need to discuss the financial aspects, but we might be able to assist. However, are you fully committed to targeting F508, or are you also considering other diseases or mutations?" />
+ <QaBox q="The timeframe of the project, combined with the fact that we’re all studying on the side, limits us to a certain scope. Since this is our first time tackling a project like this, it makes sense to stick to something more manageable. So, we're somewhat committed to focusing on F508 due to these constraints."
+ a="That's understandable. It can be really tough to juggle a project like this along with exams and studies, especially if you're also involved in competitions. But it's definitely worth the effort, even if you don't achieve huge results right away. The experience and learning, as well as the connections you make, are incredibly valuable. I'm a big supporter of such projects. So, what resources do you currently have? Do you already have cells with the F508 delta mutation, or...? " />
+ <QaBox q="We have one patient who is willing to provide us with cells, but we don't have them yet. "
+ a="It sounds like you're aware of the challenges, and I don't want to discourage you, but just to be realistic, working with primary cells and getting everything ready could be tricky, especially considering the competition is in October. Experiments in human cells can take time, especially if you need to do multiple iterations or clone constructs—it could easily take a week or more per experiment.
+ Regarding the cells we have, as mentioned in our paper, we screened all our guides on HEK cells with an integrated copy of the CFTR cDNA. HEK cells are easy to work with, but they don't naturally express CFTR, even though the gene is present in their genome. So, we introduced the mutation of interest into these cells, making it easier to screen.
+ I'm not entirely sure if we can send over the cells due to ethical regulations, which can be complex and time-consuming to navigate. However, there's an alternative approach that might help you. Early on, we found that it's actually quite easy to screen guides using what we call a 'transient target'. In this method, you would transfect all your prime editing plasmids into HEK cells, along with a plasmid containing the CFTR cDNA with the mutation of interest. While this approach isn’t as physiological as editing the chromosome directly, our side-by-side comparisons showed almost equal efficiencies between transient and chromosomal targets. It's much easier and faster than working with patient-derived cells. I can definitely send you the plasmid, which would save you a lot of time and effort. This method is much simpler and could be a practical solution for your project. " />
+ <QaBox q="Our initial plan is to work with a reporter plasmid that expresses eGFP, where we've removed a splice site, until we have patient cells or cell lines with CFTR mutations. This will allow us to screen easily without needing to sequence everything. Do you maybe have any suggestions or advice on this approach? "
+ a="Is that the PEAR system? No, it’s a different one, but we also have a similar system. The advantage of this approach is that you can very easily see if it works, and it’s very sensitive—much easier than extracting and sequencing DNA. The downside, however, is that… actually, I’m not familiar with the 'flu PEAR system.'
+ Actually, we use the exact same system in our lab. It’s very useful for optimizing delivery strategies because it’s easy to see results. The downside, of course, is that the guides you’re using for that system aren’t specific to the F508 delta mutation, right? So, these are scientific trade-offs. You could, for example, design a reporter that uses your F508 delta guide and also results in fluorescence, but you would need to design the reporter first. It’s challenging to prove that it works because you might not have a perfect guide for F508 delta.
+ It really depends on what you want to achieve. If your goal is to first check if you can successfully perform prime editing, then using the reporter is definitely a good first step." />
+ <QaBox q="We will edit the plasmid, specifically the vector, so that we have almost the same pegRNA. The only difference will be downstream, behind the edit."
+ a="Is this approach based on a paper from the Netherlands, or is it something you came up with yourself? " />
+ <QaBox q="Based on a paper. "
+ a="Yeah, that sounds like a very good way to start. Do you already have the reporter plasmid ready? " />
+ <QaBox q="Yeah, we bought the reporter, and now we’re making the necessary edits so we can use it. "
+ a="Okay, so do you also already have guides targeting F508 right now? " />
+ <QaBox q="We’ve designed some guides, but we haven’t tested them yet. That’s one of our next steps. So, at the moment, we’re just in the design phase, or we have already designed them, and..." a="Yeah, okay, cool. Good luck with that! And I suppose you’re starting off with HEK cells as well, right?"/>
+ <QaBox q="We have HEK and HeLa cells, but we haven't decided yet which ones we'll use." a="I would start off in HEK cells because, by total accident or coincidence, they are much easier to achieve prime editing in. This is because the MLH1 gene, which negatively impacts prime editing outcomes, is naturally disabled in these cells—they don't produce the MLH1 protein. Of all cell lines available, HEK cells are the easiest to achieve editing with, so I would definitely recommend starting there. In terms of transfection, HEK cells are also very easily transfected. If I can offer another piece of advice, always include GFP controls—plasmids that simply express GFP without requiring editing—and use them to determine your transfection efficiency. It's crucial to have a very high transfection efficiency because you'll be working with a three-component system: your reporter, your prime editor, and your guides. All three plasmids need to be present in the same cell for the editing to occur, so you should aim for at least 70% transfection efficiency, preferably 80% or higher. I don't know what transfection method you're planning to use, but we've always used Lipofectamine 3000. It’s expensive, but it works very well. However, if you're looking for more cost-effective options, we recently discovered two other transfection reagents, Jet Optimus and Jet Prime, which are much cheaper and also work quite well. That said, I would advise against starting with any of the cheaper transfection reagents; you really need to aim for high transfection efficiency. Always make sure to measure and report transfection efficiency for every experiment because if it's low, the experiment might not yield useful results. If you have the funds or resources, I would also recommend designing P3 or even P3b guides, as they might offer better efficiency. When it comes to designing P3b guides, if you're primarily focused on P2 right now, there are some specific considerations to keep in mind. I'll provide you with a site that can help with this, and I'll give you the link in just a moment. So, it's very advisable to check the Doench score. Do you know what it is?"/>
+ <QaBox q="No, not really." a="There are papers by John Doench, an American researcher, from quite a while ago that, in my opinion, are some of the best around. He developed a comprehensive scoring matrix specifically for regular Cas9 that can evaluate the quality of the spacer in your guide RNA. This is important because Cas9 tends to prefer certain sequences over others. For instance, a good spacer should have an appropriate GC content and should avoid hairpins that might cause it to fold in on itself, which would prevent it from functioning properly. You can use this matrix to give a score for the quality of a guide RNA. I’m going to pull up an example here. The site from Synthego, a commercial provider of CRISPR reagents, allows you to check the quality of your guide. When you validate it, the site gives a score based on various factors, including off-target effects, although that might not be your primary concern at the moment. If you hover over a specific area, it will show you the Doench Score, which is crucial. Ideally, you want a guide with a good Doench Score. A good score starts at around 0.4, indicated by a green check mark for good efficiency. If the score is very low, it means that the guide likely has low CRISPR-Cas9 activity and may not be very efficient. When designing prime editing guides, RNA, we always check the spacer for a good Doench Score. If we are designing nicking guides for a PE3 or PE3b strategy, we also ensure that they have a good score. This is one of the easiest tools to check for that. Whenever possible, try using PE3. In some cases, PE3 performs better than PE2, though not always. PE3b might not always work either, but for many mutations, we have seen significant increases in editing efficiency by including the PE3 guide."/>
+ <QaBox q="Okay, yeah, that was quite clear from your results; the diagram illustrated that very well. Are there more off-target effects when using PE3 since you have to make another cut?" a="If you decide to use PE3, it's important to be aware that while it's not exactly an off-target issue, there is a risk of an undesired on-target outcome. The concern with regular PE3 is that both strands of DNA can be nicked simultaneously, which can lead to a staggered double-strand break. This can result in the formation of indels (insertions or deletions). In your case, this means that if the region around the F508 delta mutation is broken, the prime editor might not be able to repair it properly, leading to additional base pairs being removed or added, and thus, the sequence might be altered in an unintended way. The risk of on-target indels is definitely higher with PE3 compared to PE2. However, this risk is reduced when using PE3b, which employs sequential nicking. The PE3b nicking guides are designed to recognize the wild-type sequence, and they can only nick the opposite strand if the correction has already been made on the top strand. This sequential action helps to avoid the generation of indels. Introducing a second guide into the system also brings the possibility of off-target editing by that guide however, since only a Cas9 nickase is used, off-target indels should be limited."/>
+ <QaBox q="Yes, okay, thank you. Do you have time left, or are we out of time?"a=" It's fine."/>
<QaBox q="We have more or less one last question. If it’s not possible, that’s completely fine. We just wanted to ask if you could possibly forward the contact details for the Ussing chamber setup in Paris that you mentioned in your email. Would that be possible?" a="You can certainly try to contact them, but I actually know that there are quite good labs in Germany that work on similar things.
One major drawback for you might be the time it takes to differentiate cells. If you harvest stem cells or basal cells from patients, they will have the CFTR gene, but they don’t express it immediately. It takes about four weeks for them to differentiate and start producing the CFTR protein. Without this differentiation, you can't measure the currents, which could slow you down significantly. I'm not sure if you have that kind of time.
If I can give you one piece of advice: it’s less physiological, but it’s still an accepted assay—try it on organoids. We could actually perform both assays here. If you find guides that work really well, we could consider doing those tests here. Someone could come over, or we could do the experiments if they’re not too expensive and have a good chance of working. I think we wouldn’t mind adding the F508 delta mutation to our list of editable mutations.
@@ -563,10 +609,88 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
summary: "Our discussion with Mattijs Bulcaen from KU Leuven provided critical insights into the complexities of using prime editing for cystic fibrosis therapy. As we began designing our prime editor, Mattijs highlighted challenges specific to targeting the CFTR F508del deletion, including the influence of mismatch repair systems and chromatin organization on editing efficiency. He introduced us to advanced techniques, such as PE3b systems and dsgRNAs, and recommended using the 3’ stem loop motif from his research to enhance our pegRNA design. Additionally, he advised utilizing HEK cell lines for screening due to their ease of handling and reduced mismatch repair activity. These insights directly influenced our design choices and helped refine our approach to developing an effective prime editing strategy.",
months: "june"
},
+ {
+ vorname: "'Der Teuto ruft'",
+ nachnname: "",
+ pictureurl: pics['teuto'],
+ tag: "Education",
+ heading: "Educational city tour for young and old",
+ interviewtabid: "teuto",
+ type: "meta",
+ cardtext: "",
+ quote: "x",
+ summary: "",
+ months: "June"
+ },
+ {
+ vorname: "Julia",
+ nachnname: "",
+ job: "parent",
+ affiliation: "independent",
+ pictureurl: pics['julia'],
+ tag: "Patient",
+ heading: "Interview with a CF Parent about their experience and treatment needs",
+ interviewtabid: "julia",
+ cardtext: "",
+ language: "de",
+ quote: "At first, our world fell apart. I still remember the conversation with the doctor. ",
+ aimofcontact: [<p>We learned from our discussion with <HPLinktoOtherHPTab tab="maxfirst" text="Max" /> that cystic fibrosis (CF) has a profound impact on the whole family – not just the patient. In order to gain further insight into this subject, we sought to engage with the next of kin of CF patients.
+ We were able to make contact with Julia through the self-help group of <a href="https://www.muko.info/ " >Mukviszidose e.V. </a> of which Max is a member. She subsequently reached out to us following Max's request for potential candidates for an interview with a patient group.
+ She and her husband have a six-year-old daughter carrying the F508del mutation in the CFTR gene and a toddler without CF. </p>],
+ insights: [<p> The interview with Julia shifted our focus to a new group of stakeholders: The patient’s support systems. Most people do not get genetically tested before having children and due to that, many people could get in the position of having a loved one with CF.
+ We considered the societal impacts, such as the rising health care costs, which Nicole Friedlein emphasized during our interview. She explained how the long-term nature of treatment, frequent hospital visits, and the need for specialized medications place a significant
+ financial burden on both patients and the health care system. This insight shaped our understanding of the broader economic challenges faced by families and institutions involved in managing chronic illnesses. Meanwhile, Julia brought attention to the psychological impact,
+ stressing the emotional strain that accompanies not only the illness itself but also the financial pressures. She also showed us more perspectives on parenting of children with CF, than we heard before, and told us about the way from the first diagnosis to growing accustomed
+ to and living with a child with CF. Julia also confirmed that most children will have no issue using an inhalative therapy like we envision our gene therapy to be and shone light onto the comparatively very good situation for CF patients in Germany. </p>],
+ implementation: [<p> This interview helped us confirm the delivery method we planned to use as we were previously concerned how and if children would be able to use the inhalative therapy. Besides that, Julia gave us further insights into the emotional side of
+ dealing with CF and we were able to discuss the situation for patients in Germany in comparison to other countries better in later interviews <HPLinktoOtherHPTab tab="joshua" text="Joshua" />. </p>],
+ interview: <>
+ <QaBox q="Can you tell us a bit about your family? How old are your children and yourselves?" a="I’m 37, my husband is 44, and our daughter is six, turning seven soon. We also have a son who’s about a year and a half." />
+ <QaBox q="Does your son also have cystic fibrosis?" a="No, he doesn’t." />
+ <QaBox q="When was your daughter diagnosed with cystic fibrosis?" a="Right after birth. She was transferred to a bigger hospital due to an intestinal blockage and had surgery. After about two to three weeks in intensive care, the cystic fibrosis diagnosis came through newborn screening. At that time, the results took longer to process than they do now." />
+ <QaBox q="That intestinal issue can happen for many reasons, right?" a="Yes, it was all new to us. The beginning was difficult, but things have gotten better since then, and we’re very grateful." />
+ <QaBox q="How did you feel when you first heard the diagnosis?" a="It felt like our world was falling apart. I still remember the moment—it was like being in a movie. We were told in a separate room, and it felt overwhelming. One doctor even suggested we go home to think about it in peace, but all I could think about was returning to my child. It was a lot to take in, especially thinking about how we’d tell our family." />
+ <QaBox q="That sounds incredibly hard. How did you handle it as time passed?" a="It was tough, but we were fortunate to have a doctor who really understood what we were going through, as he had a disabled child himself. He never scared us unnecessarily and guided us step by step, which made a big difference. We know many families who live in constant fear, but since those first months, we’ve learned to manage the situation without being overwhelmed by fear." />
+ <QaBox q="Did any particular support help your family adjust to the diagnosis?" a="Yes, the rehab program we attended was a huge help. It was a family-oriented program, so my husband could be there too, which was important since I manage most things day-to-day. It really helped our daughter realize she’s not alone—she met other kids with similar conditions, which was a huge comfort." />
+ <QaBox q="How did you explain the illness to your daughter?" a="We try to give it as little attention as possible in daily life. She’s been inhaling medication since she was eight weeks old, and it’s just part of her routine now. Thankfully, she doesn’t fight it or question it much, and her school and kindergarten haven’t made a big deal of it either, which is what we wanted." />
+ <QaBox q="Does she ever ask about her illness compared to her younger brother, who doesn’t have cystic fibrosis?" a="She does sometimes ask why she’s sick and he’s not, but she’s not upset by it. We’ve made sure not to give her any special treatment because of her illness, which can be hard at times, but we want her to understand that her illness doesn’t define her." />
+ <QaBox q="That sounds like a good balance. What about medications—did she start on any special treatments?" a="Yes, she started on Orkambi at around three years old but had to stop briefly due to high liver values. Now she’s on Kaftrio, which she started shortly before her sixth birthday, and it’s been going well." />
+ <QaBox q="Did you face any issues with the health insurance for covering these medications?" a="Fortunately, no. We have statutory health insurance, and they’ve covered everything without any issues. We’ve heard it can be more complicated for those with private insurance." />
+ <QaBox q="Have you ever had difficulties with access to medication?" a="Yes, there have been times when we’ve had to wait a few days for certain medications, like Kreon or antibiotics, especially in the winter. But we always plan ahead and keep a buffer, so we’ve never been without what we need." />
+ <QaBox q="What would you say has been the most affected area for your daughter?" a="Her intestines are the most affected. Before she started Kaftrio, she had fatty stools and frequent bowel movements, even with the right Kreon dosage. Since starting Kaftrio, this has improved significantly." />
+ <QaBox q="What kind of support would you have liked to receive earlier?" a="We wish we had been given more information about available services early on. We found out about Mukoviszidose e.V. from another family, not from our doctor. It would have been helpful to know about these resources right from the start." />
+ <QaBox q="How about psychosocial support?" a="Initially, we didn’t have any psychological support—our doctor took care of everything. Now, where we live, there are more resources, and we think it’s a good thing. The rehab helped a lot in coming to terms with everything. We wish we had known about such services sooner." />
+ <QaBox q="Does your daughter do physiotherapy?" a="Yes, once a week for about an hour. She’s been going since she was discharged from the hospital, and she has a close bond with her physiotherapist. They’ve been working together since she was a baby, and she goes by herself now." />
+ <QaBox q="Are there any restrictions for her in terms of physical activities?" a="No, not really. She does dancing once a week, physiotherapy, and she’s even done a swimming course without any problems." />
+ <QaBox q="How do you handle communicating about her illness?" a="We try not to make a big deal of it. When I looked for information, I found what we needed. There’s nothing we’ve really felt was missing." />
+ </>,
+ summary: "Julia's insights shifted our focus to the support systems surrounding CF patients. She highlighted the societal implications of CF, including rising healthcare costs due to the long-term nature of treatment and the financial burdens faced by families. Additionally, Julia emphasized the emotional strain that accompanies the illness, alongside the complexities of parenting a child with CF. Importantly, she affirmed that most children adapt well to inhalative therapies, reinforcing our planned delivery method for gene therapy. This interview enriched our understanding of the challenges faced by families and enabled us to better compare the experiences of CF patients in Germany to those in other countries.",
+ months: "june"
+ },
+ {
+ title: "Prof. Dr.",
+ vorname: "David",
+ nachnname: "Liu",
+ job: " Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare",
+ affiliation: "vice chair of the faculty at the Broad Institute of MIT and Harvard",
+ pictureurl: pics['david'],
+ tag: "Academia",
+ language: "en",
+ heading: "Influence of research by David Liu on our design decisions ",
+ interviewtabid: "liu",
+ cardtext: "",
+ quote: "X",
+ aimofcontact: [<p>David Liu is the principal investigator responsible for the development of the prime editing systems and his laboratory is actively working on improving prime editors, also for application in CFTR mutation F508del. </p>],
+ insights: [<p>The talk and papers from David Liu not only introduced advances for prime editors, but also included valuable data for us to decide on which method might work best for our project. Because of this we decided to use an advanced system based on the PE6c and PE4 systems, both of which were published by the laboratory of David Liu. </p>],
+ implementation: "",
+ summary: "",
+ months: "June",
+ references: <LiuInterviewSources/>,
+ },
{
vorname: "Nicole",
nachnname: "Friedlein",
- job: "Research group on fundamental rights at",
+ job: "Research group on fundamental rights",
affiliation: "University Potsdam",
pictureurl: pics['nicole'],
tag: "Academia",
@@ -577,7 +701,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
quote: "Public health insurance operates under an economic efficiency principle, meaning the most cost-effective treatments are preferred. But if gene therapies become the only treatment option for certain conditions, they will likely have to be included in the coverage, which could be a challenge for the system.",
aimofcontact: "The main objective of the contact was to learn from the discussion on issues related to cystic fibrosis (CF), gene therapy, health insurance processes and regulatory pathways. In particular, we wanted to understand the real-world challenges and technical aspects of gene editing, especially prime editing, as well as the complexities of approval and reimbursement of gene therapies for CF patients.",
insights: "The regulatory approval process, particularly by the European Medicines Agency (EMA) for advanced medical devices, has highlighted the bureaucratic hurdles that gene therapies must overcome. We learned that such therapies for cystic fibrosis have to navigate complex European and German regulatory systems. The discussion on the AMNOG process was crucial. We learnt that the additional benefit of a therapy is assessed for reimbursement by the statutory health insurance funds. We implemented this insight in our project by considering the long-term regulatory and economic effects as important milestones for therapy development. We also gained insight into how public and private health insurers may differ in their reimbursement of such therapies. Public insurers have stricter guidelines, while private insurers can be more flexible, but both require strict justification, especially for rare diseases such as cystic fibrosis. Information on newborn screening and genetic counselling covered by public health insurance was crucial to understanding how preventive measures for CF are managed. This underlines the importance of early intervention and diagnosis in our project. Atypical forms of CF, where health insurance companies do not cover treatment due to non-standardised test results, were identified as a key problem. This helped us to recognise the need for more adaptable insurance policies and clearer pathways for the treatment of atypical cases in our project plans. The debate about whether healthcare systems can afford the high costs of gene therapies highlighted an important issue in the current medical landscape. We have incorporated this insight into our project by discussing possible cost-effective alternatives and the need for thorough cost-benefit analysis in the development of treatments.",
- implementation: [<p>After the interview, we further tailored our project to focus on a simple delivery method to reduce the therapeutic effort. To gain an overview of the regulatory requirements and to better deliver the project, one of our team members attended a GxP course[Link Gxp course] to ensure we met all the necessary standards. To deepen our knowledge of entrepreneurship, we conducted further interviews with start-ups and industrial companies [Link entrpeuneur], which gave us important insights into practical implementation. These steps ensure that our project is not only based on scientific research, but also takes into account the practical, regulatory and social aspects that are crucial to bringing new CF therapies to the market. We are currently developing strategies to successfully implement our ideas and the project in the future.</p>],
+ implementation: [<p>After the interview, we further tailored our project to focus on a simple delivery method to reduce the therapeutic effort. To gain an overview of the regulatory requirements and to better deliver the project, one of our team members attended a <HPLinktoOtherHPTab tab="gxpcourse" text="GxP course"/> to ensure we met all the necessary standards. To deepen our knowledge of entrepreneurship, we conducted further interviews with start-ups and industrial companies [Link entrpeuneur], which gave us important insights into practical implementation. These steps ensure that our project is not only based on scientific research, but also takes into account the practical, regulatory and social aspects that are crucial to bringing new CF therapies to the market. We are currently developing strategies to successfully implement our ideas and the project in the future.</p>],
interview: <>
<QaBox q="To start with this interview. Do you have any questions about this project?"
a="Are you writing a paper on this, or are you conducting actual laboratory research? Or is it primarily literature review? How does your work look?" />
@@ -621,6 +745,23 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
a="It’s mainly interdisciplinary. A lot of funding comes from industry, like BioNTech, or foundations like Mukoviszidose e.V., which funds research on cystic fibrosis. But in terms of practical research, it’s usually biologists or biotechnologists. Without industry support, research can struggle due to a lack of funding, so having backing is essential." />
</>,
summary: "Our discussion addressed the complexities of cystic fibrosis (CF) treatments, focusing on gene therapy and health insurance processes. We learned about the regulatory challenges gene therapies face, particularly regarding the European Medicines Agency (EMA) and the AMNOG process for reimbursement assessments. Public insurers impose stricter guidelines than private insurers, emphasizing the importance of early intervention in CF and the need for adaptable policies for atypical cases. We recognized the high costs associated with gene therapies and incorporated cost-benefit analysis into our project planning. Following the interview, we refined our approach to include straightforward delivery methods and attended a GxP course for regulatory compliance. Engaging with start-ups further informed our practical implementation strategies, ensuring our project aligns with both scientific and regulatory needs.",
+ months: "june",
+ pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/zoom-nicole.webp",
+ },
+ {
+ vorname: "Visiting Achema ",
+ nachnname: "in Frankfurt",
+ pictureurl: pics['frankfurtmesse'],
+ tag: "Industry",
+ heading: "Some of our team members attended the fair in Frankfurt to promote co-operation with sponsors and stakeholders",
+ interviewtabid: "frankfurtmesse",
+ cardtext: "",
+ quote: "x",
+ aimofcontact: "",
+ insights: "",
+ implementation: "",
+ type: "meta",
+ summary: "",
months: "june"
},
{
@@ -661,102 +802,35 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
months: "june"
},
{
- vorname: "Julia",
- nachnname: "XXX",
- job: "parent",
- affiliation: "independent",
- pictureurl: pics['julia'],
- tag: "Patient",
- heading: "Interview with a CF Parent About Their Experience and Treatment Needs",
- interviewtabid: "julia",
+ title: "Dr.",
+ vorname: "Marco",
+ nachnname: "Radukic",
+ job: "Postdoc at AG Cellular and Molecular Biotechnology",
+ affiliation: "University Bielefeld",
+ pictureurl: pics['marco'],
+ tag: "Academia",
+ heading: "Optimizing LNP Transfection: Insights into working with LNP Kits",
+ interviewtabid: "radukic",
+ language:"de",
cardtext: "",
- language: "de",
- quote: "At first, our world fell apart. I still remember the conversation with the doctor. ",
- aimofcontact: [<p>We learned from our discussion with <HPLinktoOtherHPTab tab="maxfirst" text="Max" /> that cystic fibrosis (CF) has a profound impact on the whole family – not just the patient. In order to gain further insight into this subject, we sought to engage with the next of kin of CF patients.
- We were able to make contact with Julia through the self-help group of <a href="https://www.muko.info/ " >Mukviszidose e.V. </a> of which Max is a member. She subsequently reached out to us following Max's request for potential candidates for an interview with a patient group.
- She and her husband have a six-year-old daughter carrying the F508del mutation in the CFTR gene and a toddler without CF. </p>],
- insights: [<p> The interview with Julia shifted our focus to a new group of stakeholders: The patient’s support systems. Most people do not get genetically tested before having children and due to that, many people could get in the position of having a loved one with CF.
- We considered the societal impacts, such as the rising health care costs, which Nicole Friedlein emphasized during our interview. She explained how the long-term nature of treatment, frequent hospital visits, and the need for specialized medications place a significant
- financial burden on both patients and the health care system. This insight shaped our understanding of the broader economic challenges faced by families and institutions involved in managing chronic illnesses. Meanwhile, Julia brought attention to the psychological impact,
- stressing the emotional strain that accompanies not only the illness itself but also the financial pressures. She also showed us more perspectives on parenting of children with CF, than we heard before, and told us about the way from the first diagnosis to growing accustomed
- to and living with a child with CF. Julia also confirmed that most children will have no issue using an inhalative therapy like we envision our gene therapy to be and shone light onto the comparatively very good situation for CF patients in Germany. </p>],
- implementation: [<p> This interview helped us confirm the delivery method we planned to use as we were previously concerned how and if children would be able to use the inhalative therapy. Besides that, Julia gave us further insights into the emotional side of
- dealing with CF and we were able to discuss the situation for patients in Germany in comparison to other countries better in later interviews <HPLinktoOtherHPTab tab="joshua" text="Joshua" />. </p>],
- interview: <>
- <QaBox q="Can you tell us a bit about your family? How old are your children and yourselves?" a="I’m 37, my husband is 44, and our daughter is six, turning seven soon. We also have a son who’s about a year and a half." />
- <QaBox q="Does your son also have cystic fibrosis?" a="No, he doesn’t." />
- <QaBox q="When was your daughter diagnosed with cystic fibrosis?" a="Right after birth. She was transferred to a bigger hospital due to an intestinal blockage and had surgery. After about two to three weeks in intensive care, the cystic fibrosis diagnosis came through newborn screening. At that time, the results took longer to process than they do now." />
- <QaBox q="That intestinal issue can happen for many reasons, right?" a="Yes, it was all new to us. The beginning was difficult, but things have gotten better since then, and we’re very grateful." />
- <QaBox q="How did you feel when you first heard the diagnosis?" a="It felt like our world was falling apart. I still remember the moment—it was like being in a movie. We were told in a separate room, and it felt overwhelming. One doctor even suggested we go home to think about it in peace, but all I could think about was returning to my child. It was a lot to take in, especially thinking about how we’d tell our family." />
- <QaBox q="That sounds incredibly hard. How did you handle it as time passed?" a="It was tough, but we were fortunate to have a doctor who really understood what we were going through, as he had a disabled child himself. He never scared us unnecessarily and guided us step by step, which made a big difference. We know many families who live in constant fear, but since those first months, we’ve learned to manage the situation without being overwhelmed by fear." />
- <QaBox q="Did any particular support help your family adjust to the diagnosis?" a="Yes, the rehab program we attended was a huge help. It was a family-oriented program, so my husband could be there too, which was important since I manage most things day-to-day. It really helped our daughter realize she’s not alone—she met other kids with similar conditions, which was a huge comfort." />
- <QaBox q="How did you explain the illness to your daughter?" a="We try to give it as little attention as possible in daily life. She’s been inhaling medication since she was eight weeks old, and it’s just part of her routine now. Thankfully, she doesn’t fight it or question it much, and her school and kindergarten haven’t made a big deal of it either, which is what we wanted." />
- <QaBox q="Does she ever ask about her illness compared to her younger brother, who doesn’t have cystic fibrosis?" a="She does sometimes ask why she’s sick and he’s not, but she’s not upset by it. We’ve made sure not to give her any special treatment because of her illness, which can be hard at times, but we want her to understand that her illness doesn’t define her." />
- <QaBox q="That sounds like a good balance. What about medications—did she start on any special treatments?" a="Yes, she started on Orkambi at around three years old but had to stop briefly due to high liver values. Now she’s on Kaftrio, which she started shortly before her sixth birthday, and it’s been going well." />
- <QaBox q="Did you face any issues with the health insurance for covering these medications?" a="Fortunately, no. We have statutory health insurance, and they’ve covered everything without any issues. We’ve heard it can be more complicated for those with private insurance." />
- <QaBox q="Have you ever had difficulties with access to medication?" a="Yes, there have been times when we’ve had to wait a few days for certain medications, like Kreon or antibiotics, especially in the winter. But we always plan ahead and keep a buffer, so we’ve never been without what we need." />
- <QaBox q="What would you say has been the most affected area for your daughter?" a="Her intestines are the most affected. Before she started Kaftrio, she had fatty stools and frequent bowel movements, even with the right Kreon dosage. Since starting Kaftrio, this has improved significantly." />
- <QaBox q="What kind of support would you have liked to receive earlier?" a="We wish we had been given more information about available services early on. We found out about Mukoviszidose e.V. from another family, not from our doctor. It would have been helpful to know about these resources right from the start." />
- <QaBox q="How about psychosocial support?" a="Initially, we didn’t have any psychological support—our doctor took care of everything. Now, where we live, there are more resources, and we think it’s a good thing. The rehab helped a lot in coming to terms with everything. We wish we had known about such services sooner." />
- <QaBox q="Does your daughter do physiotherapy?" a="Yes, once a week for about an hour. She’s been going since she was discharged from the hospital, and she has a close bond with her physiotherapist. They’ve been working together since she was a baby, and she goes by herself now." />
- <QaBox q="Are there any restrictions for her in terms of physical activities?" a="No, not really. She does dancing once a week, physiotherapy, and she’s even done a swimming course without any problems." />
- <QaBox q="How do you handle communicating about her illness?" a="We try not to make a big deal of it. When I looked for information, I found what we needed. There’s nothing we’ve really felt was missing." />
- </>,
- summary: "Julia's insights shifted our focus to the support systems surrounding CF patients. She highlighted the societal implications of CF, including rising healthcare costs due to the long-term nature of treatment and the financial burdens faced by families. Additionally, Julia emphasized the emotional strain that accompanies the illness, alongside the complexities of parenting a child with CF. Importantly, she affirmed that most children adapt well to inhalative therapies, reinforcing our planned delivery method for gene therapy. This interview enriched our understanding of the challenges faced by families and enabled us to better compare the experiences of CF patients in Germany to those in other countries.",
- months: "june"
- },
- {
- vorname: "Joshua",
- nachnname: "Bauder",
- job: "parent and activist",
- affiliation: "CF vests worldwide",
- pictureurl: pics['joshua'],
- tag: "Patient",
- heading: "Interview with a CF Parent and Global Advocate on Worldwide Support and Perspectives",
- interviewtabid: "joshua",
- cardtext: "",
- language: "en",
- quote: "We’ve had to sit by and watch people die, knowing that better treatment exists but is inaccessible. ",
- aimofcontact: [<p>We contacted the organization <a href="https://www.cfvww.org/">CF vests worldwide</a> with the aim to hear more diverse perspectives beyond Germany.
- After the founder Rod connected us with Joshua, Joshua was so kind to conduct an interview with us not only about the perspectives and
- stories he heard but also about his personal experiences with his daughter and living in a country where CF care is very hard to get. Joshua
- (from the USA) and his family live in Thailand where he and his wife run a children’s home. Their daughter is the only child with CF.</p>,
- <p>It is possible to learn more about Joshua and his family though the <a href="https://thebonnellfoundation.org/cf-vests-worldwide/">
- podcast of the Bonnel foundation</a>.</p>],
- insights: [<p> Joshua showed us just how dire the situation is for CF patients is in some regions. It was shocking to hear there is only one doctor
- knowledgeable about CF in Thailand and that many doctors dismiss the possibility of CF due to racial bias and misinformation. Additionally, we confirmed how much the accessibility
- of care depends on the healthcare system, as we already touched on during the interview with <HPLinktoOtherHPTab tab="nicole" text="Nicole Friedlein" />,. On the parenting level, Joshua brought in many perspectives contrary to what we previously heard. In the interview with <HPLinktoOtherHPTab tab="maxfirst" text="Max" />,, we learned he vehemently avoids ponding water while Joshua’s daughter is allowed to roam around with no such restrictions. Neither have chronic infections.</p>],
- implementation: [<p>The interview with Josh made us realize we too needed to look at the reason why we chose F508del. Did we, too, fall for bias?
- Despite a change of target not being feasible anymore, we looked into it and traced back our steps that led to our decision. We did not find as much
- information about other mutations when first researching cystic fibrosis, especially in the context of prime editing. Mattijs Bulceans's paper on
- targeting the mutations L227R and N1303K <TabScrollLink tab="joshua" scrollId="desc-1" num="1" /> was one of few papers. After explicitly searching for cystic fibrosis records for specific countries and
- regions, we uncovered a moderate number of papers examining CF in Asia and other regions we previously did not know much about. The very first article
- supported Joshua's hypotheses and painted a sad picture: Among other things, it describes the case of a four-month-old boy who was diagnosed with cystic
- fibrosis. Nothing unusual in itself, but the circumstances are depressing. Two of the three siblings born before him died within months of birth and had
- previously presented with symptoms of cystic fibrosis. He was the first to be diagnosed. A sweat test aimed at cystic fibrosis was not available at the
- hospital, so one was improvised. Later on, a genetic test revealed the presence of 508del. <TabScrollLink tab="joshua" scrollId="desc-2" num="2" /> We found ourselves and our lack of knowledge in good
- company as we found papers as new as from 2020 (14 years after the previously mentioned paper) containing statements such as “recent reports suggest
- that CF does occur in Asia†<TabScrollLink tab="joshua" scrollId="desc-3" num="3" />. Fortunately, there is a rising number of cystic fibrosis experts for Asia and other previously overlooked regions
- such as Africa. <TabScrollLink tab="joshua" scrollId="desc-4" num="4" /> We chose to not only look at the scientific data but also into anecdotal evidence. To find the latter, we searched official
- and private websites and chatrooms for information and experiences of patients. In the end, we found narratives from most ethnic backgrounds
- about being dismissed and often misdiagnosed. Of course, this is not an occurrence unique to cystic fibrosis. Our conclusion is that yes,
- we did fall for bias. But regardless of ethnicity, 508del occurs and is overall the most prevalent mutation as was confirmed in our interview
- with CF expert Sriram .... This experience was uncomfortable as we felt the pressure to be thorough and deliver a perfect project. What would
- have been more devastating than realizing we made a wrong choice at the very core? We made the conscious decision to invest our resources into
- figuring out if we indeed made a mistake and we want to encourage other teams to do the same. iGem stands for innovation – but also for growth.
- Especially in the context of Integrated Human Practices, it is important to examine both the positive and the negative to create a project with a
- future. </p>],
- pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/joshua-zoom.webp",
- references: <JoshuaInterviewSources />,
- summary: "Joshua, a CF parent living in Thailand, shared his experiences about the severe challenges of accessing CF care in regions like Southeast Asia. His story highlighted the racial bias and lack of medical knowledge about CF in these areas. This interview prompted the team to reflect on their focus on the F508del mutation, questioning if their research was biased towards more commonly studied mutations. After revisiting their research process, they found that the F508del mutation remains globally relevant, yet the experience reinforced the importance of addressing gaps in healthcare and research for underrepresented regions.",
- months: "July"
+ quote: "x",
+ aimofcontact: [<p>The primary objective was to tackle challenges in LNP transfection related to manufacturing and cell transfection methods. The focus was on improving LNP formulation and application protocols to enhance gene delivery effectiveness,
+ and on acquiring specialized expertise to optimize these processes. Dr. Radukic from Bielefeld University provided crucial insights for troubleshooting and protocol optimization to enhance LNP efficacy. </p>],
+ insights: [<p> Dr. Radukic told us that the efficiency of LNPs is significantly affected by lipid-to-nucleic acid ratios and that optimizing ratios like 22:1 versus 10:1 can improve transfection.
+ pH adjustments and buffer composition (e.g., modifying sodium acetate solutions) are also crucial for LNP performance. Proper storage at 4 °C, precise pipetting, and thorough mixing are essential to maintain LNP functionality.
+ In addition, he suggested quality control measures such as fluorescence testing, zeta potential, and light scattering analyses help ensure our LNP is stable. Additionally, spray drying was evaluated for potential use in long-term LNP stabilization. </p>],
+ implementation: [<p>Incorporating the insights Dr. Radukic and advice from recent consultations, we adjusted the lipid-to-nucleic acid ratio from 22:1 to 10:1 to enhance efficiency and modified the pH and concentration of sodium acetate solutions for better packaging.
+ Storage conditions were strictly managed at 4 °C, and pipetting/mixing techniques were refined to ensure quality consistency. Quality control was expanded to include fluorescence testing, zeta potential measurements, and light scattering, alongside cytotoxicity tests.
+ These improvements not only address transfection challenges but also strengthen our foundation for future LNP applications. </p>],
+ summary: "The aim of the contact was to address challenges in LNP transfection and improve formulation protocols for enhanced gene delivery. Dr. Radukic highlighted the importance of lipid-to-nucleic acid ratios, recommending adjustments like 10:1 for better transfection efficiency. He also emphasized optimizing pH and buffer composition, as well as strict storage and mixing practices. Additionally, quality control measures such as fluorescence testing and zeta potential analysis were suggested to ensure LNP stability. These insights were implemented into the project, improving transfection efficiency and paving the way for future LNP applications.",
+ months: "June"
},
{
title: "Prof. Dr.",
vorname: "Erhard",
nachnname: "Wischmeyer",
job: "Research Group Cellular Neurophysiology",
- affiliation: "Universität Bielefeld",
+ affiliation: "University Bielefeld",
pictureurl: pics['wischmeyer'],
tag: "Academia",
heading: "Discussion on Techniques for Measuring CFTR Channel Functionality",
@@ -800,8 +874,8 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
Oliver Dräger, we gained valuable insights and optimized our approach to effectively investigate and
measure the functionality of the CFTR ion channel, thereby determining the efficiency of our Prime
Editing strategy. </p></>],
- pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/for-wiki-texts/hp-patch-clamp/bild-patch-clamp-isi-oliver.webp",
- pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/for-wiki-texts/hp-patch-clamp/bild-interssierte-wissenschaftler-oho.webp",
+ pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/hp/bild-interssierte-wissenschaftler-oho.webp",
+ pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/hp/bild-patch-clamp-isi-oliver.webp",
references: <WischmeyerSources />,
interview: <>
<QaBox q="Can you educate us about your academic career?" a="I did my doctorate 30 years ago at Bielefeld University and then worked at the Max Planck Institute in Göttingen a lot with the patch-clamp technique. Today, I’m head of the working group Cellular Neurophysiology of the medicine faculty of Bielefeld University." />
@@ -814,88 +888,160 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="Who could help us with the patch-clamp measurements?" a="The patch-clamp devices are heavily utilized in our working group, so you probably cannot perform measurements on your own. However, postdocs could support you for some measurements. Dr. Oliver Dräger is available as a contact person of my working group." />
</>,
- summary: "n summary, through the interview with Prof. Dr. Wischmeyer and the collaboration with his employee Dr. Oliver Dräger, we gained valuable insights and optimized our approach to effectively investigate and measure the functionality of the CFTR ion channel, thereby determining the efficiency of our prime editing strategy.",
+ summary: "In summary, through the interview with Prof. Dr. Wischmeyer and the collaboration with his employee Dr. Oliver Dräger, we gained valuable insights and optimized our approach to effectively investigate and measure the functionality of the CFTR ion channel, thereby determining the efficiency of our prime editing strategy.",
months: "june"
},
+ {
+ vorname: "Joshua",
+ nachnname: "Bauder",
+ job: "parent and activist",
+ affiliation: "CF vests worldwide",
+ pictureurl: pics['joshua'],
+ tag: "Patient",
+ heading: "Interview with a CF Parent and Global Advocate on Worldwide Support and Perspectives",
+ interviewtabid: "joshua",
+ cardtext: "",
+ language: "en",
+ quote: "We’ve had to sit by and watch people die, knowing that better treatment exists but is inaccessible. ",
+ aimofcontact: [<p>We contacted the organization <a href="https://www.cfvww.org/">CF vests worldwide</a> with the aim to hear more diverse perspectives beyond Germany.
+ After the founder Rod connected us with Joshua, Joshua was so kind to conduct an interview with us not only about the perspectives and
+ stories he heard but also about his personal experiences with his daughter and living in a country where CF care is very hard to get. Joshua
+ (from the USA) and his family live in Thailand where he and his wife run a children’s home. Their daughter is the only child with CF.</p>,
+ <p>It is possible to learn more about Joshua and his family though the <a href="https://thebonnellfoundation.org/cf-vests-worldwide/">
+ podcast of the Bonnel foundation</a>.</p>],
+ insights: [<p> Joshua showed us just how dire the situation is for CF patients is in some regions. It was shocking to hear there is only one doctor
+ knowledgeable about CF in Thailand and that many doctors dismiss the possibility of CF due to racial bias and misinformation. Additionally, we confirmed how much the accessibility
+ of care depends on the healthcare system, as we already touched on during the interview with <HPLinktoOtherHPTab tab="nicole" text="Nicole Friedlein" />,. On the parenting level, Joshua brought in many perspectives contrary to what we previously heard. In the interview with <HPLinktoOtherHPTab tab="maxfirst" text="Max" />,, we learned he vehemently avoids ponding water while Joshua’s daughter is allowed to roam around with no such restrictions. Neither have chronic infections.</p>],
+ implementation: [<p>The interview with Josh made us realize we too needed to look at the reason why we chose F508del. Did we, too, fall for bias?
+ Despite a change of target not being feasible anymore, we looked into it and traced back our steps that led to our decision. We did not find as much
+ information about other mutations when first researching cystic fibrosis, especially in the context of prime editing. Mattijs Bulceans's paper on
+ targeting the mutations L227R and N1303K <TabScrollLink tab="joshua" scrollId="desc-1" num="1" /> was one of few papers. After explicitly searching for cystic fibrosis records for specific countries and
+ regions, we uncovered a moderate number of papers examining CF in Asia and other regions we previously did not know much about. The very first article
+ supported Joshua's hypotheses and painted a sad picture: Among other things, it describes the case of a four-month-old boy who was diagnosed with cystic
+ fibrosis. Nothing unusual in itself, but the circumstances are depressing. Two of the three siblings born before him died within months of birth and had
+ previously presented with symptoms of cystic fibrosis. He was the first to be diagnosed. A sweat test aimed at cystic fibrosis was not available at the
+ hospital, so one was improvised. Later on, a genetic test revealed the presence of 508del. <TabScrollLink tab="joshua" scrollId="desc-2" num="2" /> We found ourselves and our lack of knowledge in good
+ company as we found papers as new as from 2020 (14 years after the previously mentioned paper) containing statements such as “recent reports suggest
+ that CF does occur in Asia†<TabScrollLink tab="joshua" scrollId="desc-3" num="3" />. Fortunately, there is a rising number of cystic fibrosis experts for Asia and other previously overlooked regions
+ such as Africa. <TabScrollLink tab="joshua" scrollId="desc-4" num="4" /> We chose to not only look at the scientific data but also into anecdotal evidence. To find the latter, we searched official
+ and private websites and chatrooms for information and experiences of patients. In the end, we found narratives from most ethnic backgrounds
+ about being dismissed and often misdiagnosed. Of course, this is not an occurrence unique to cystic fibrosis. Our conclusion is that yes,
+ we did fall for bias. But regardless of ethnicity, 508del occurs and is overall the most prevalent mutation as was confirmed in our interview
+ with CF expert Sriram .... This experience was uncomfortable as we felt the pressure to be thorough and deliver a perfect project. What would
+ have been more devastating than realizing we made a wrong choice at the very core? We made the conscious decision to invest our resources into
+ figuring out if we indeed made a mistake and we want to encourage other teams to do the same. iGem stands for innovation – but also for growth.
+ Especially in the context of Integrated Human Practices, it is important to examine both the positive and the negative to create a project with a
+ future. </p>],
+ pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/joshua-zoom.webp",
+ references: <JoshuaInterviewSources />,
+ summary: "Joshua, a CF parent living in Thailand, shared his experiences about the severe challenges of accessing CF care in regions like Southeast Asia. His story highlighted the racial bias and lack of medical knowledge about CF in these areas. This interview prompted the team to reflect on their focus on the F508del mutation, questioning if their research was biased towards more commonly studied mutations. After revisiting their research process, they found that the F508del mutation remains globally relevant, yet the experience reinforced the importance of addressing gaps in healthcare and research for underrepresented regions.",
+ months: "July"
+ },
{
title: "Prof. Dr.",
vorname: "Stefan",
nachnname: "Hammer",
job: "Junior Professor of Organic Chemistry and Biocatalysis",
- affiliation: "Universität Bielefeld",
+ affiliation: "University Bielefeld",
pictureurl: pics['hammer'],
tag: "Academia",
- heading: "Safety Briefing and Laboratory Practices Advice",
+ heading: "Insights from the discussion on enzyme engineering",
interviewtabid: "hammer",
cardtext: "",
language: "de",
- quote: "",
- aimofcontact: "",
-
- insights: "",
- implementation: "",
- summary: "",
- months: ""
+ quote: "x",
+ aimofcontact: [<p>After we had developed a number of theroetic elaborations, feedback with corresponding expertise in the field of enzyme engineering was of exceptional importance to us. For this reason, we wanted to discuss our approaches with Prof. Dr. Hammer.</p>],
+ insights: [<p>In our discussion with Prof. Dr. Hammer discussion with Prof. Dr. Hammer about different theoretical approaches we had developed:</p>,
+ <ul>
+ <li>PAM engineering</li>
+ <li>Chimeric protein</li>
+ <li>Phage-Assisted Continuous Evolution (PACE)</li>
+ <li>Rational Design: Mutation of zinc-finger</li>
+ </ul>,
+ <p> We discussed whether a rational design, supported by structural simulations with AlphaFold 3, or the construction of a chimeric protein would be the superior approach. The possibility of using protein evolution via PACE or conventional methods was demonstrated, even if these approaches are very time-consuming. An important topic was the analysis of homologous mechanisms by sequence alignments to identify structural similarities in catalytic centers. In addition, we discussed the role of zinc finger domains and targeted mutations, such as substitution to alanine, in the function of optimization. It was also emphasized that mutations should be evaluated in enzymes with nickase activity, although the screening for this is very laborious. The reliability of AlphaFold predictions was evaluated positively despite the complexity of the enzymes. One promising approach could be the transfer of mutations to homologous proteins. </p>],
+ implementation: [<p>We learned from the discussion that we need to take a rational approach and consider the following conclusions:</p>,
+ <ul>
+ <li>A rational approach could be the transfer of mutations to homologous proteins</li>
+ <li>For rational design, structural simulation using AlphaFold 3 could be used to model and understand specific protein structures</li>
+ <li>Homologous mechanisms & sequence alignments</li>
+ <li>Analysis of structural similarities in catalytic centers by sequence comparisons</li>
+ <li>Using Ala or Gly for substitution</li>
+ <li>Development of a nickase assay</li>
+ </ul>,
+ <p>We developed our mutation candidates on this basis [link zum Cycle oder New Part ?]</p>
+ ],
+ summary: "In our discussion with Prof. Dr. Hammer, we explored several theoretical approaches in enzyme engineering, including PAM engineering, chimeric proteins, Phage-Assisted Continuous Evolution (PACE), and rational design involving zinc-finger mutations. We evaluated the potential of using structural simulations with AlphaFold 3 and the construction of chimeric proteins. The conversation highlighted the importance of analyzing homologous mechanisms through sequence alignments to identify structural similarities in catalytic centers. We also discussed targeted mutations, such as substitutions to alanine, and the development of a nickase assay. Overall, we concluded that transferring mutations to homologous proteins is a promising rational approach for optimizing enzyme function.",
+ months: "July"
},
{
- title: "Dr.",
- vorname: "Katharina",
- nachnname: "Kolonko",
- job: "Expert for nanocapsules",
- affiliation: "Biologist",
- pictureurl: pics['kolonko'],
- tag: "Academia",
- heading: "Optimizing our mRNA Delivery Systems",
- interviewtabid: "kolonkofirst",
+ vorname: "Steffen Bira and",
+ nachnname: "Serra Gürcan from Corden Pharma",
+ job: "Associate director",
+ affiliation: "Corden Pharma",
+ pictureurl: pics['corden'],
+ tag: "Industry",
+ heading: "Lipid Nanoparticles in Gene Therapy: perspectives from Corden Pharma ",
+ interviewtabid: "corden",
cardtext: "",
- language: "de",
- quote: "Wow, you’re already further along than I was! That’s a really good approach, especially since dry powder formulations can help with stability.",
- aimofcontact: [<p>Our goal in reaching out to Dr. Katharina Kolonko, who earned her PhD working on chitosan-based nanoparticles for delivering nucleic acids to human respiratory cells in the context of cystic fibrosis, was to seek her advice on the design, stability, and application of nanoparticles. We wanted to learn from her experience with chitosan-capsaicin nanoparticles, especially the challenges she encountered, and apply her insights to improve our own project. Specifically, we aimed to better understand nanoparticle stability, transfection methods, and how to effectively design our experiments.
- </p>],
- insights: [<p>Dr. Kolonko provided us with valuable insights into working with nanoparticles, particularly emphasizing the advantages of nanocapsules. She highlighted that nanocapsules are more stable than nano-complexes, which is crucial for experiments involving complex environments and high shear forces. Additionally, her use of capsaicin wasn’t aimed at improving transfection efficiency but was part of a broader strategy targeting specific channels. She also shared practical methods for measuring particle stability and cytotoxicity, giving us clear guidance on tools and techniques that we can apply to our project.
- Furthermore, Dr. Kolonko discussed the use of chitosan as a component in nanoparticle formulations. Chitosan, with its positive charge, can interact with mRNA, potentially enhancing the stability of the cargo. As an outlook, we plan to explore modifications using chitosan to improve the stability and performance of our mRNA delivery system. This approach may provide a more robust solution for optimizing nanoparticle formulations in future experiments. </p>],
- implementation:[<p>We directly applied Katharina’s insights to improve our nanoparticle design and testing methods. Her recommendations on using nano-capsules and OptiMEM as the transfection medium helped refine our experimental approach. She emphasized using a medium with fewer additives, like OptiMEM, and suggested removing antibiotics 24 hours before transfection to avoid interference, ensuring more controlled and effective conditions. We also explored new stability testing ideas, including nasal spray solutions and dry powder formulations.
- We incorporated her insights as follows: </p>,
+ language: "en",
+ quote: "The stability of LNPs depends on the specific lipid and RNA components used, but ensuring the overall stability of a new formulation requires rigorous empirical testing under various conditions.",
+ aimofcontact: [<p>The primary aim of the communication with Steffen Bira and Serra Gürcan from Corden Pharma was to explore the technical aspects and practical applications of Lipid Nanoparticles (LNPs) in advanced medical therapies, including gene therapy and inhalation treatments. The conversation focused on the possibility of using Corden Pharma’s LNP starter kits, understanding the factors affecting the stability of LNPs, and exploring options for incorporating antibodies into LNPs to target specific cells. </p>],
+ insights: [<p>The discussion with <a href="https://cordenpharma.com/">Corden Pharma</a>, led by Steffen Bira and Serra Gürcan, offered key insights into LNPs and their applications. While Corden Pharma hasn't extensively explored spray drying for LNPs, they recommended consulting specialists to evaluate its feasibility, especially concerning lipid stability during the process. Stability was highlighted as crucial for inhalation therapies, requiring thorough testing of entire LNP formulations, possibly aided by cryoprotectants and controlled temperatures.
+ Corden Pharma's LNP starter kits are based on well-researched lipid combinations designed for stability and encapsulation efficiency, making them suitable for multiple experiments. They suggested that modifying lipid components, such as incorporating cholesterol derivatives, could enhance cellular uptake and overall efficacy. Additionally, they confirmed the possibility of incorporating antibodies into LNPs and emphasized the importance of considering intellectual property when selecting lipids for commercial use. They also showed openness to collaboration,
+ including offering discounts in exchange for recognition in publications.
+ The interaction with Corden Pharma provided several key insights:</p>,
<ul>
- <li><strong>Nano-Capsules Focus:</strong> Based on Katharina’s advice, we prioritized nano-capsules for their enhanced stability over nano-complexes.</li>
- <li><strong>Chitosan for Stability:</strong> We're exploring chitosan to improve mRNA delivery system stability due to its positive charge, which binds mRNA to the nanoparticle surface.</li>
- <li><strong>OptiMEM for Transfection:</strong> OptiMEM is now our chosen transfection medium, with the suggestion to remove antibiotics 24 hours prior.</li>
- <li><strong>MTT Test for Cytotoxicity:</strong> We adopted the MTT test for cytotoxicity due to its simplicity and reliability.</li>
- <li><strong>Nasal Spray and Dry Powder Testing:</strong> We are considering testing nanoparticle stability using nasal spray solutions and exploring dry powder formulations.</li>
- <li><strong>Capsaicin Exclusion:</strong> As capsaicin did not significantly impact transfection efficiency in Katharina’s research, we decided not to include it in our project.</li>
+ <li><strong>Spray Drying Feasibility:</strong> Corden Pharma hasn’t explored spray drying extensively; consultation with specialists is recommended for assessing feasibility and lipid stability.</li>
+ <li><strong>LNP Stability:</strong> Stability of LNPs, particularly for inhalation therapies, needs empirical testing, considering shear forces and the potential use of cryoprotectants or temperature control.</li>
+ <li><strong>Lipid Selection in Kits:</strong> Starter kits use well-researched lipid combinations, tested for stability, encapsulation efficiency, and potency. They provide materials for multiple experimental batches.</li>
+ <li><strong>Lipid Modifications:</strong> Exploring alternative lipids (e.g., cholesterol derivatives) could enhance stability and cellular uptake, tailored to project needs.</li>
+ <li><strong>Antibody Incorporation:</strong> Antibodies can be incorporated into LNPs during preparation or afterward, depending on targeting requirements.</li>
+ <li><strong>Intellectual Property:</strong> IP considerations are crucial when selecting lipids for LNP formulations, as many lipids are patented.</li>
+ <li><strong>Collaboration Opportunities:</strong> Corden Pharma is open to offering discounts or forming partnerships, with recognition in publications or acknowledgments.</li>
</ul>
- ] ,
- interview:<>
- <QaBox q="How did you approach the design of Lipid Nanoparticles (LNPs)? What were the first steps you took at that time? Were you already familiar with LNPs, or was that a completely new experience for you?" a="I started working with nanoparticles during my bachelor’s thesis. I continued with nanoparticles into my master’s thesis, working on a project related to cystic fibrosis. Initially, I worked with nano-complexes, but later switched to nano-capsules due to their stability, especially in cell culture media." />
- <QaBox q="Since you’re focused on stability and applying high shear forces, could you explain why nano-capsules are more stable than complexes in this context?" a="Nano-capsules are generally more stable in cell culture media compared to nano-complexes, which often react with additives and proteins. However, I didn’t explore shear forces much further. My main goal was to stabilize the particles in cell culture media for testing on cells." />
- <QaBox q="What kind of cell culture medium did you use for these experiments?" a="For transfection, we used Optimem as the medium, after removing antibiotics from the culture medium 24 hours prior to transfection." />
- <QaBox q="How long did it take you to get to the point where you used nano-capsules?" a="I only started working with nano-capsules towards the end of my PhD. I spent much of my time with nano-complexes, but when I visited a lab in Leeds, I shifted to nano-capsules. This transition happened quite late, just months before I finished my thesis." />
- <QaBox q="You mentioned capsaicin in your recent paper. Does it significantly affect transfection efficiency, and is it worth including in our experiments?" a="No, capsaicin didn’t affect transfection efficiency in our experiments. It was included to inhibit the Ina-channel as part of a dual strategy targeting both CFTR and Ina-channels, but it might not be necessary for your project." />
- <QaBox q="Were there any critical components in the formulation of your nanoparticles that you couldn’t do without?" a="No, the main comparison was between nano-complexes and nano-capsules. Nano-complexes were inconsistent in size and stability, while nano-capsules were stable and smaller, which I believed would work better in later experiments." />
- <QaBox q="Is there a way to check if the mRNA sticks to the outside of the nanoparticle or ends up inside?" a="I believe the mRNA sticks to the outside. The process involved forming nano-capsules using lecithin and oil, and after the ethanol was evaporated, mRNA was added last. The mRNA likely adhered to the positively charged chitosan on the outside of the capsule." />
- <QaBox q="Do you remember the ratio of mRNA to nano-capsule?" a="I don’t remember the exact ratio offhand, but it’s documented in my dissertation. I optimized the amount of mRNA that needed to enter the cell for effective results, but didn’t do extensive testing with nano-capsules." />
- <QaBox q="Was determining the optimal amount of nanoparticles trial and error?" a="Yes, definitely. It involved a lot of optimization." />
- <QaBox q="You used the MTT test for cytotoxicity. Would you recommend it for us, or are there better alternatives?" a="Yes, the MTT test is simple and reliable. You just need to pipette accurately. We used it frequently, and it worked well." />
- <QaBox q="How did you assess the stability of the nano-capsules? Did you use microscopy or another method?" a="We used a device called a Zetasizer, which measures size, zeta potential, and polydispersity index (PDI). We used it to assess stability in cell culture medium over time, from half an hour to 24 hours." />
- <QaBox q="Do you have any advice for our project or anything we might have missed?" a="If you’re planning to use a diffuser for nasal administration, you might want to test the stability of the nanoparticles in a saline solution or standard nasal spray solution. It could be useful to see how they behave in such a medium. Otherwise, it seems like you’re well ahead of where I was!" />
- <QaBox q="Thank you so much for your time and insights!" a="You’re welcome! I’m glad I could help." />
+ ],
+ implementation: [<p>The insights from Corden Pharma had a major impact on our project, especially in selecting lipids critical for LNP stability and optimizing gene therapy applications. Initially, we used the Cayman kit, but it was suboptimal for delivering our Primeguide. After receiving feedback, we switched to Corden Pharma’s kit #2, which includes advanced lipid components like cationic lipids that improve cellular uptake and enhance LNP stability. This shift has significantly boosted the efficiency and robustness of our formulations.
+ Additionally, Corden Pharma's guidance on lipid modifications and antibody incorporation opened new possibilities for targeted therapies. These insights not only improved our technical approach but also paved the way for potential collaborations, offering cost benefits and increased scientific recognition. The feedback will continue to shape our testing process and improve therapeutic delivery. </p>],
+ interview: <>
+ <QaBox q="Is it possible to dry the LNPs designed by Corden Pharma, such as through spray drying?" a="It has not been confirmed whether LNPs have been successfully dried using spray drying. Further investigation or consultation with a specialized company would be required to determine feasibility."/>
+ <QaBox q="How is stability ensured in LNPs, particularly for use in inhalation therapy?" a="The stability largely depends on the specific lipid and RNA components used in the formulation. While the stability of individual lipids can be assessed, the overall stability of a new LNP formulation requires empirical testing under various conditions."/>
+ <QaBox q="How are lipid combinations selected for inclusion in the LNP starter kits, and what testing is conducted?" a="Lipid combinations in the LNP starter kits are selected based on known interactions, particularly in formulations containing RNA. Testing is conducted to assess physical-chemical properties, encapsulation efficiency, and overall potency. The kits are designed to provide sufficient material for multiple experimental batches."/>
+ <QaBox q="Is it advisable to modify the lipid components in an existing LNP formulation?" a="It is generally advisable to consider alternative lipid components, as different lipids may offer improved stability or efficacy. However, the specific needs of the project will dictate whether changes are necessary."/>
+ <QaBox q="Is it possible to incorporate antibodies into LNPs, and what is the recommended approach?" a="Yes. Incorporation of antibodies into LNPs is possible. This can be achieved either during the initial preparation phase or by incubating antibodies with LNPs after their formation, depending on whether surface or internal localization of antibodies is desired. Reference to specific studies may provide further guidance."/>
+ <QaBox q="What is Corden Pharma's position on projects involving gene therapy?" a="Corden Pharma operates as a service provider, focusing on the manufacturing of active pharmaceutical ingredients (APIs) and excipients rather than developing therapeutic products. Consideration should be given to the intellectual property status of the lipids used in LNP formulations, particularly for commercial applications."/>
+ <QaBox q="Is it possible to obtain a discount on LNP starter kits or establish a collaborative relationship with Corden Pharma?" a="We would need to discuss this internally but generally we would be open to potential collaborations that could involve recognition in publications or other forms of acknowledgment, pending approval from relevant management."/>
</>,
- summary: "We identified several crucial insights to guide our project development. Nano-capsules were found to be more stable than nano-complexes, making them our preferred formulation choice. We will utilize chitosan to enhance mRNA stability due to its positive charge, while capsaicin was deemed irrelevant for our purposes. For transfection, we will use OptiMEM as the medium, removing antibiotics 24 hours prior to the procedure. We will assess stability with a Zetasizer and evaluate cytotoxicity using the MTT test. Additionally, we are exploring nasal spray and dry powder formulations to improve nanoparticle delivery. These insights will significantly shape our approach to optimizing mRNA delivery systems.",
- months: ""
+ summary: "The primary goal of the communication with Steffen Bira and Serra Gürcan from Corden Pharma was to explore the use of Lipid Nanoparticles (LNPs) in gene therapy and inhalation treatments. Corden Pharma recommended consulting specialists for assessing spray drying feasibility, while highlighting the importance of testing LNP stability under various conditions. Their LNP starter kits are optimized for stability and encapsulation efficiency, with potential for lipid modifications to enhance cellular uptake. They also confirmed that antibodies can be incorporated into LNPs and emphasized considering intellectual property when selecting lipids. Based on Corden Pharma’s insights, we switched to their kit #2 for our project, hoping to improve the stability and efficiency of our LNP formulations. Their guidance also opened opportunities for targeted therapies and collaborations. This feedback will continue to enhance our testing and therapeutic approaches.",
+ months: "july",
+ pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/hp/hp-corden-zoom.png"
+ },
+ {
+ vorname: "'MINT Sommer'",
+ nachnname: "",
+ pictureurl: pics['mint'],
+ tag: "Education",
+ heading: "Innovative minds unite: iGEM's participation in 'MINT Sommer'",
+ interviewtabid: "mint",
+ type: "meta",
+ cardtext: "",
+ quote: "x",
+ summary: "",
+ months: "July"
},
{
+ title: "Dr.",
vorname: "Svenja",
nachnname: "Vinke",
job: "PostDoc",
- affiliation: "Harvard Medical School",
+ affiliation: "at Department of Genetics, Harvard Medical School, former iGEMer",
pictureurl: pics['svenja'],
tag: "Academia",
- heading: "",
+ heading: "Insights on the Feasibility of PACE for Prime Editing Optimization",
interviewtabid: "svenja",
cardtext: "",
language: "de",
- quote: "",
- aimofcontact: [<p>We contacted Svenja Vinke, a former iGEMer from Bielefeld, to get her insight and her opinion regarding the use of phage assisted continuous evolution
+ quote: "X",
+ aimofcontact: [<p>We contacted Svenja Vinke, a former iGEMer from Bielefeld, to get her insight and her opinion regarding the use of phage assisted continuous evoluWe contacted Svenja Vinke, a former iGEMer from Bielefeld, to get her insight and her opinion regarding the use of phage assisted continuous evolution
(PACE, see engineering cycle 1[Link]) for our prime editing optimizations. Svenja works at the iGEM Safety and Security Committee. Additionally, she was part of the Biosafety and Security Award Team of Bielefeld University in 2016.</p>],
insights: [<p>Svenja explained, that a PACE approach is not feasible to use for optimization of our nickase candidates because of several reasons:</p>,
<ul>
@@ -906,25 +1052,8 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
</ul>
],
implementation: [<p>On the basis of Svenja’s and other opinions on the topic, we decided not to try implementing a PACE system. </p>],
- summary: "",
- months: ""
- },
- {
- vorname: "Max",
- nachnname: "Beckmann",
- job: "Bielefeld University",
- pictureurl: pics['max'],
- tag: "Patient",
- heading: "Consultation on University Hygiene Risks and Improvement for Hygiene Concept",
- interviewtabid: "maxhygiene",
- cardtext: "",
- language: "de",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
+ summary: "We reached out to Svenja Vinke, a former iGEMer and Postdoc, to gain insights on using phage-assisted continuous evolution (PACE) for optimizing our prime editing. Svenja explained that a PACE approach is not feasible for our nickase candidates due to several reasons: it requires too much time, endonucleases are likely too large for optimization, unspecific cutting can kill bacterial cells, and prime editing is less effective in E. coli than in human cells. Based on Svenja's feedback and other expert opinions, we decided against implementing a PACE system for our project.",
+ months: "July"
},
{
title: "Dr.",
@@ -942,32 +1071,153 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
aimofcontact: "The aim of the interview was to get an answer to the question of whether we need an ethics vote for our project or not and to obtain guidelines for dealing with patient cells regarding ethical issues and data protection. ",
insights: "The discussion was very informative in terms of how we should approach this topic and focused primarily on the important factors that need to be considered when planning the handling of patient cells. These include which legal principles need to be observed, data protection, ethical considerations and, above all, detailed and specific information for the donor. It also made us look at the situation from many different angles and consider the risks of worst-case scenarios. Overall, this interview was very useful to us, and we were able to use the information we gained to develop a kind of guideline that allowed us to approach this sensitive topic, which was new to us, with a certain degree of confidence. ",
implementation: "Based on the knowledge we have gained, we have drawn up guidelines for our handling of the cells. We used this guide when handling the patient cells, to ensure they were handled in an ethically correct manner.",
- summary: "",
- months: ""
+ summary: "This interview focused on the ethical and legal considerations of handling patient cells, we sought to determine whether our project required an ethics vote and to gather guidelines on data protection and patient consent. The expert emphasized the importance of providing patients with a detailed consent letter and privacy policy, clearly explaining how their cells and data will be used, who will have access, and the time span involved. This conversation helped us understand key legal and ethical principles, especially regarding transparency with donors. We used these insights to develop guidelines for handling patient cells, ensuring we approached this sensitive process with confidence and ethical care.",
+ months: "July",
+ pictureurl_interview:"https://static.igem.wiki/teams/5247/integrated-human-practices/interview-berens.webp",
},
{
- vorname: "Collaboration",
- nachnname: "",
- job: "",
- affiliation: "",
- pictureurl: pics['placeholder'],
+ title: "M.Sc.",
+ vorname: "Mattijs",
+ nachnname: "Bulcaen",
+ job: "PhD Researcher at Laboratory for Molecular Virology & Gene Therapy",
+ affiliation: "KU Leuven",
+ pictureurl: pics['mattijs'],
tag: "Academia",
- heading: "LNP Handbook",
+ heading: "Visit Mattijs Bulcaen in Leuven and the Laboratory for Molecular Virology & Gene Therapy",
+ interviewtabid: "mattijsvisit",
+ cardtext: "",
+ language: "en",
+ quote: "x",
+ aimofcontact: [<p>After our first interview with Mattijs Bulcaen we stayed in contact via email and eventually visited him in Leuven at his laboratory. Here we wanted to gain further information about the CFTR F508del models and editing confirmation.</p>],
+ insights: [<p>We spoke about approaches for testing CFTR F508del correction in models and methods of confirmation. In this context we talked about HEK293T cell lines[Link] established in his laboratory that stably overexpress CFTR wild-type and F508del. We also discussed how to handle these cell lines. He explained, that the CFTR is fused with a 3HA tag, that in wild-type CFTR would be exposed to the extracellular space and therefore can be used for immunohistochemical staining of the protein, showing correct protein processing and channelling. Also, this allows for a western blot to be made using 3HA antibodies. Functional recovery of CFTR can also be visualized using halide sensitive eYFP or organoid assay, the ladder Mattijs had established an automated readout. Furthermore, we talked about how to handle Sanger sequencing data to analyse edits and discussed the possibility to avoid the weaknesses of Sanger sequencing by using Nanopore sequencing instead. We asked about the applicability of patch clamp analysis in the context of CFTR and Mattijs said that, to his knowledge, it has not been used to test for successful editing in CFTR.
+ Lastly Mattijs explained how he plans to deliver the prime editing complex to the patient, and we evaluated the advantages and disadvantages of delivery strategies, including our very own LNP approach.</p>],
+ implementation: [<p>When planning how to test and confirm editing by our own constructs, we were largely inspired by the information given to us by Mattijs. For example, we tested prime editing in the HEK293 cell lines we spoke about with Mattijs and used halide sensitive eYFP to check for CFTR function. Also, we tried differentiating wild-type and F508del cells using patch clamp. Unfortunately, a lot of the methods mentioned were not usable for us because of time constraints, but are still valuable for future projects and research built upon PreCyse. </p>],
+ summary: "We visited Mattijs Bulcaen in Leuven to enhance our understanding of CFTR F508del models and editing confirmation. During the visit, we examined HEK293T cell lines that stably overexpress wild-type CFTR and the F508del variant. Mattijs demonstrated how to use a 3HA tag for visualizing protein processing and discussed methods for assessing CFTR functional recovery. We also compared Sanger and Nanopore sequencing techniques and evaluated delivery strategies for our prime editing complex. The insights from this visit guided our project, leading us to test prime editing in HEK293T cells using halide-sensitive eYFP. While some methods were not feasible due to time constraints, they laid the groundwork for future research in the PreCyse project.",
+ months: "july"
+ },
+ {
+ vorname: "Collaborations",
+ nachnname: "iGEM Team Linköping ",
+ type: "meta",
+ pictureurl: pics['linköping'],
+ tag: "Milestone",
+ heading: "Cooperation to create a Lipid Delivery System Handbook",
interviewtabid: "handbook",
cardtext: "",
+ quoteVorname: "Kaya",
+ quoteNachname: "Lange",
+ quote: "We were genuinely excited when Linköping University approached us for collaboration. From the very beginning, their ideas resonated with us, and our shared enthusiasm laid a strong foundation for a productive partnership. We're happy to work together, also with the other teams, and explore new possibilities.",
+ aimofcontact: [<p>The initial contact for our collaboration came from the iGEM team 2024 of Linköping, Sweden, who approached us with a proposal to create a “Delivery-Based Handbookâ€[link Handbook]. Their goal was to reduce the steep learning curve associated with these technologies by sharing collective knowledge from multiple teams, including ours. We were excited to contribute and help future teams navigate these challenges more easily. The handbook would serve as a valuable tool. </p>],
+ insights: [<p>Throughout the collaboration, we gained significant insights, both scientific and collaborative. Initially, our meetings with the Linköping team and other participating teams - Patras, Radboud-University and TERMOSZ-Selye-HUN - were invaluable. These sessions allowed us to exchange ideas and learn how each team planned to use lipid-based delivery systems in their own projects. This mutual sharing of knowledge opened our eyes to new methodologies and potential applications of LNPs and liposomes. We also gained a deeper appreciation for the interdisciplinary nature of these systems. From the challenges of formulating stable particles to optimizing their efficiency in targeting cells, we realized the complexity of the field and how collaboration could help overcome many of these obstacles. By discussing our respective approaches, we were able to pool our expertise, which not only improved our understanding but also ensured that the handbook would be comprehensive and valuable for various iGEM teams, regardless of their specific project focus.
+ In summary: </p>,
+ <ul>
+ <li>Learned different approaches to using LNPs and liposomes in iGEM projects.</li>
+ <li>Discovered new methods for optimizing LNPs.</li>
+ <li>Recognized challenges in particle stability and targeted delivery.</li>
+ <li>Gained appreciation for the interdisciplinary complexity of these systems.</li>
+ <li>Focused on documenting work to benefit future iGEM teams.</li>
+ </ul>
+ ],
+ implementation: [<p>The collaboration expanded our understanding of what's possible, inspiring us to consider new ideas for how we might integrate advanced techniques into our nanoparticle systems in future projects. The collaborative process also encouraged us to document our work more thoroughly, ensuring that future iGEM teams could benefit from both our successes and the challenges we encountered along the way. Beyond the technical improvements, the experience taught us the value of teamwork across borders and disciplines. Each team brought a unique perspective, and by working together, we were able to develop a resource that was far greater than the sum of its parts</p>],
+ summary: "This collaboration with Linköping and the other iGEM teams was an incredibly enriching experience. Together, we developed a “Delivery-Based Handbookâ€[link Handbook] that will serve as a valuable resource for future teams working with LNPs and liposomes. The knowledge we gained not only enhanced our project but also strengthened our sense of community within iGEM. We are excited to present the handbook at the Grand Jamboree, where we will finally meet our collaborators in person and celebrate the culmination of our collective efforts. This partnership has shown us the immense power of collaboration, and we are proud to have been part of such a meaningful initiative.",
+ months: "several times",
+ pictureurl_aim: "https://static.igem.wiki/teams/5247/photos/hp/linkoping-handbook-lnp.webp",
+ pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/hp/linkoping-cooperation-photo.png",
+ },
+ {
+ title: "M.Sc.",
+ vorname: "Kai",
+ nachnname: "Schülke",
+ job: "PhD student Working group: Organic chemistry and biocatalysis  ",
+ affiliation: "University of Bielefeld",
+ pictureurl: pics['kaihammer'],
+ tag: "Academia",
+ heading: "First insights of Enzym Engineering",
+ interviewtabid: "hammerkai",
language: "de",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
+ cardtext: "",
+ quote: "x",
+ aimofcontact: [<p>When we realized that the creation of a nickase from the endonucleases in use was a desired outcome, it became necessary to talk to an expert in the field of enzyme engineering. Our first contact was Kai Schülke, a former iGEMer and PhD student under the guidance of Prof. Dr. Hammer[Link Hammer], who is the leader of the working group organic chemistry and bioanalytics at Bielefeld University.</p>],
+ insights: [<p>In the process of our interaction with Kai, we learned about the various methods employed in enzyme engineering. He demonstrated the complexity of this field of research and emphasized the importance of choosing the right approach. As a former iGEMer, Kai, inspired by his past experiences, is highly motivated and determined to develop an outstanding project. He pointed out that we cannot rely on classical methods such as directed evolution, but instead should use a rational approach to select mutation candidates. His insights and enthusiasm have encouraged us to think critically and pursue innovative solutions in our work. </p>],
+ implementation: [<p>We incorporated Kai's insights into our project by shifting our approach to enzyme engineering. By focusing on a more targeted approach, we were able to refine our enzyme optimization process, ensuring that the modifications we made were based on informed, calculated decisions. This not only streamlined our research but also improved the chances of success by reducing the trial-and-error inherent in traditional methods. </p>],
+ summary: "The team reached out to Kai Schülke, a former iGEM participant and enzyme engineering expert, for guidance on developing a nickase from the endonucleases in use. Kai emphasized the need for a rational, targeted approach rather than traditional methods like directed evolution. His insights helped the team refine their enzyme optimization process, making it more strategic and efficient. This shift reduced trial-and-error efforts and improved the chances of success, driving innovation in their project.",
+ months: "July"
+ },
+ {
+ title: "M.Sc.",
+ vorname: "Nils",
+ nachnname: "Berelsmann",
+ job: "PhD Working group: Prof. Dr. Gabriele Fischer von Mollard ",
+ affiliation: "University of Bielefeld",
+ pictureurl: pics['nilshefe'],
+ language: "de",
+ tag: "Academia",
+ heading: "Adapting expression strategies for Fanzor nickases and exploring the potential of Pichia pastoris for SpuFz1 nickase variants ",
+ interviewtabid: "nberelsmann",
+ cardtext: "",
+ quote: "X",
+ aimofcontact: [<p>During our interview with Makoto Saito[Linkinterview] about fanzor[link fanzor], it became evident that the expression of our fanzor nickases in yeast is very promising. We then refined our expression strategy for the nickases and approached Nils Berelsmann, who is currently working on his PhD thesis with the yeast strain Pichia pastoris (SMD1163). This particular strain could be ideal for expressing the SpuFz1 nickase variants. Our main aim in contacting Nils was to gain insight and advice on yeast expression and he generously shared his expertise with us. Not only did he give us valuable advice, but he also provided us with the yeast strain itself, along with a corresponding expression vector for possible experiments. He also provided us with detailed protocols and the plasmid map of the vector and gave us practical tips on how to optimize the expression process. His support was invaluable in moving our work forward. </p>],
+ insights: [<p>Pichia pastoris (SMD1163) is a promising option for expressing SpuFz1 nickase variants. Refining expression strategies based on expert insights is crucil for success. Nils provided practical tips on yeast expression, including optimizing growth conditions and fine-tuning induction protocols.</p>],
+ implementation: [<p>We adapted our expression strategy for Fanzor nickases in yeast by incorporating the Pichia pastoris strain (SMD1163) and the provided expression vector into our experiments. Following Nils' detailed protocols and plasmid map, we optimized key steps, enhancing expression efficiency and protein yield.</p>],
+ summary: "The team sought expert advice from Nils to optimize yeast expression for Fanzor nickases. Nils provided invaluable guidance on addressing potential challenges and troubleshooting the process. He supplied the Pichia pastoris (SMD1163) strain along with a suitable expression vector, crucial for expressing SpuFz1 nickase variants. Additionally, he shared detailed protocols for yeast transformation and growth optimization, enabling the team to replicate his methods effectively for their experiments.",
+ months: "July",
+ },
+ {
+ title: "Dr.",
+ vorname: "Timm",
+ nachnname: "Weber",
+ job: "Staff Scientist, Project- and Quality Manager",
+ affiliation: "Central Biobank of the University of Bielefeld",
+ pictureurl: pics['biobank'],
+ tag: "Academia",
+ heading: "Discussed the processes involved in the storage, processing, and security of patient samples.",
+ interviewtabid: "timm",
+ cardtext: "",
+ quote: "A biobank is not just a collection of samples; it's a bridge between patient trust and scientific discovery, ensuring that valuable biological data is safeguarded while contributing to future research.",
+ aimofcontact: "Contact was established with Timm for the purpose of gaining deeper insights into the functioning of the biobank and of deepening our understanding of the processing of patient samples.",
+ insights: "We were provided with invaluable insights into the quality and project management of the biobank and storage of patient samples. It was of particular interest to note that Biobank OWL occupies a distinctive position in this context, insofar as a trustee is not a mandatory figure within its system and is therefore not provided for as a standard component. However, Biobank OWL has elected to integrate a trustee in order to enhance the security standards for the safeguarding of patient data. This illustrates the biobank's dedication to ensuring the optimal protection and security of sensitive patient data.",
+ implementation: "The insights gained have facilitated a deeper comprehension of the significance of quality management in the processing of patient samples. This understanding has been integrated into our project processes, thereby enhancing the accuracy and reliability of our procedures. ",
+ summary: "The interview focused on understanding the operations of the Biobank OWL, particularly in the areas of quality management and sample processing. Provided a detailed overview of biobank activities, including sample collection, storage conditions, and data protection measures",
+ language: "de",
+ interview: <>
+ <QaBox q="Can you briefly explain to us what exactly a biobank is and what its main tasks are?"
+ a="A biobank is a specialized facility that collects, stores, and manages biological samples and associated data for research purposes. Each biobank is unique in its operations and functions. In Bielefeld and Lippe, the Biobank BOWL (Biobank OWL) is responsible for the storage of patient samples. The Data Integration Centre (DIZ) stores data pertaining to these samples. A trustee oversees the pseudonymisation of data, acting as an interface between BOWL and DIZ, ensuring that patient data cannot be directly linked to patient samples." />
+ <QaBox q="What types of samples are collected in your biobank and for what research purposes are they used?"
+ a="The biobank collects a wide variety of samples, including blood, stool, and soil. Samples may be gathered for specific research projects or for establishing a general repository under 'broad consent.' Researchers wishing to use these samples must apply to the 'use access committee,' which evaluates whether the requested samples and data can be released for their research." />
+ <QaBox q="How large is your biobank? How many samples do you currently store and how many new samples are added on average?"
+ a="The biobank is still in the process of establishing itself and has not yet reached its full sample capacity. However, it is anticipated to accumulate a significant number of samples in the near future, with several thousand samples expected to be analyzed in dedicated sessions." />
+ <QaBox q="What requirements and criteria must be met for a sample to be included in your biobank?" a="Samples must be processed according to highly detailed protocols, and regular audits are conducted to ensure compliance with all standards." />
+ <QaBox q="Which other research institutions or biobanks do you cooperate with and what form does this cooperation take?"
+ a="Biobank OWL has a second location in Lippe, in addition to Bielefeld. Collaborations exist with the DIZ, the Treuhand, and three university hospitals. It is anticipated that cooperation with other working groups will increase in the future." />
+ <QaBox q="What specific storage conditions (e.g. temperature, humidity) must be observed for different sample types?"
+ a="Samples are stored under various temperature conditions, including -20°C, -80°C, and -150°C, along with the use of liquid nitrogen." />
+ <QaBox q="How do you ensure that the samples remain stable and usable over longer periods of time?"
+ a="Samples are stored in nitrogen for long-term stability." />
+ <QaBox q="What encryption techniques or data protection measures are used in your biobank to prevent unauthorized access to patient data? Are there special regulations for the anonymisation of data and how is it ensured that patients cannot be traced?"
+ a="Pseudonyms are created using specialized software such as CentraXX or REDcap to protect patient data." />
+ <QaBox q="What rights do patients have in relation to their samples, and how are these rights safeguarded in your biobank?"
+ a="Patients have the right to revoke their consent at any time, which can be done at the clinic or biobank. The trustee, acting as an intermediary, will notify BOWL and DIZ to destroy the corresponding samples or data." />
+ </>,
+ months: "august"
+ },
+ {
+ vorname: "'Schüler*innen Akademie'",
+ nachnname: "",
+ pictureurl: pics['schueler'],
+ tag: "Education",
+ heading: "Empowering students through synthetic biology",
+ interviewtabid: "schueler",
+ type: "meta",
+ cardtext: "",
+ quote: "x",
summary: "",
- months: ""
+ months: "August"
},
{
vorname: "Benjamin",
title: "Dr.",
nachnname: "Winkeljann",
- job: "Co-Founder and CEO at RNhale",
+ job: "Co-Founder and CEO at ",
affiliation: "RNhale",
pictureurl: pics['winkeljann'],
tag: "Industry",
@@ -1020,87 +1270,71 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="Would you be willing to support us in our project? Would you dry our LNPs?" a="The spray dryer requires 5 mL of a solution with 5% lipid solids and 0.02% RNA. We’ve published recovery rates of 70%. You can send us the mRNA and LNP components to encapsulate and dry."/>
</>,
summary: "The conversation focused on spray-drying LNPs, emphasizing the shelf life of RNA-based formulations, optimal storage conditions, and technical requirements for the drying process. Corden Pharma shared insights on the protocol, highlighting the need for testing each LNP formulation individually for stability. AI technology is used to optimize LNP formulations, and potential collaborations were discussed, including support for drying LNPs.",
- months: "juli"
+ months: "august"
},
{
- title: "XXX",
- vorname: "David",
- nachnname: "Liu",
- job: "",
- affiliation: "",
- pictureurl: pics['placeholder'],
- tag: "Academia",
- heading: "Influence of research by David Liu on our design decisions ",
- interviewtabid: "liu",
+ vorname: "Max",
+ nachnname: "Beckmann",
+ job: "Patient and Student",
+ affiliation: "University Bielefeld",
+ pictureurl: pics['max'],
+ tag: "Patient",
+ heading: "Consultation on University Hygiene Risks and Improvement for Hygiene Concept",
+ interviewtabid: "maxhygiene",
cardtext: "",
- quote: "",
+ language: "de",
+ quote: "x",
aimofcontact: "",
insights: "",
implementation: "",
summary: "",
- months: ""
+ months: "August"
},
{
- vorname: "Steffen Bira and",
- nachnname: "Serra Gürcan from Corden Pharma",
- job: "Associate director",
- affiliation: "Corden Pharma",
- pictureurl: pics['corden'],
- tag: "Industry",
- heading: "Lipid Nanoparticles in Gene Therapy: perspectives from Corden Pharma ",
- interviewtabid: "corden",
+ title: "Dr.",
+ vorname: "Katharina",
+ nachnname: "Kolonko",
+ job: "Expert for nanocapsules",
+ affiliation: "Biologist",
+ pictureurl: pics['kolonko'],
+ tag: "Academia",
+ heading: "Optimizing our mRNA Delivery Systems",
+ interviewtabid: "kolonkofirst",
cardtext: "",
- language: "en",
- quote: "The stability of LNPs depends on the specific lipid and RNA components used, but ensuring the overall stability of a new formulation requires rigorous empirical testing under various conditions.",
- aimofcontact: [<p>The primary aim of the communication with Steffen Bira and Serra Gürcan from Corden Pharma was to explore the technical aspects and practical applications of Lipid Nanoparticles (LNPs) in advanced medical therapies, including gene therapy and inhalation treatments. The conversation focused on the possibility of using Corden Pharma’s LNP starter kits, understanding the factors affecting the stability of LNPs, and exploring options for incorporating antibodies into LNPs to target specific cells. </p>],
- insights: [<p>The discussion with <a href="https://cordenpharma.com/">Corden Pharma</a>, led by Steffen Bira and Serra Gürcan, offered key insights into LNPs and their applications. While Corden Pharma hasn't extensively explored spray drying for LNPs, they recommended consulting specialists to evaluate its feasibility, especially concerning lipid stability during the process. Stability was highlighted as crucial for inhalation therapies, requiring thorough testing of entire LNP formulations, possibly aided by cryoprotectants and controlled temperatures.
- Corden Pharma's LNP starter kits are based on well-researched lipid combinations designed for stability and encapsulation efficiency, making them suitable for multiple experiments. They suggested that modifying lipid components, such as incorporating cholesterol derivatives, could enhance cellular uptake and overall efficacy. Additionally, they confirmed the possibility of incorporating antibodies into LNPs and emphasized the importance of considering intellectual property when selecting lipids for commercial use. They also showed openness to collaboration,
- including offering discounts in exchange for recognition in publications.
- The interaction with Corden Pharma provided several key insights:</p>,
+ language: "de",
+ quote: "Wow, you’re already further along than I was! That’s a really good approach, especially since dry powder formulations can help with stability.",
+ aimofcontact: [<p>Our goal in reaching out to Dr. Katharina Kolonko, who earned her PhD working on chitosan-based nanoparticles for delivering nucleic acids to human respiratory cells in the context of cystic fibrosis, was to seek her advice on the design, stability, and application of nanoparticles. We wanted to learn from her experience with chitosan-capsaicin nanoparticles, especially the challenges she encountered, and apply her insights to improve our own project. Specifically, we aimed to better understand nanoparticle stability, transfection methods, and how to effectively design our experiments.
+ </p>],
+ insights: [<p>Dr. Kolonko provided us with valuable insights into working with nanoparticles, particularly emphasizing the advantages of nanocapsules. She highlighted that nanocapsules are more stable than nano-complexes, which is crucial for experiments involving complex environments and high shear forces. Additionally, her use of capsaicin wasn’t aimed at improving transfection efficiency but was part of a broader strategy targeting specific channels. She also shared practical methods for measuring particle stability and cytotoxicity, giving us clear guidance on tools and techniques that we can apply to our project.
+ Furthermore, Dr. Kolonko discussed the use of chitosan as a component in nanoparticle formulations. Chitosan, with its positive charge, can interact with mRNA, potentially enhancing the stability of the cargo. As an outlook, we plan to explore modifications using chitosan to improve the stability and performance of our mRNA delivery system. This approach may provide a more robust solution for optimizing nanoparticle formulations in future experiments. </p>],
+ implementation:[<p>We directly applied Katharina’s insights to improve our nanoparticle design and testing methods. Her recommendations on using nano-capsules and OptiMEM as the transfection medium helped refine our experimental approach. She emphasized using a medium with fewer additives, like OptiMEM, and suggested removing antibiotics 24 hours before transfection to avoid interference, ensuring more controlled and effective conditions. We also explored new stability testing ideas, including nasal spray solutions and dry powder formulations.
+ We incorporated her insights as follows: </p>,
<ul>
- <li><strong>Spray Drying Feasibility:</strong> Corden Pharma hasn’t explored spray drying extensively; consultation with specialists is recommended for assessing feasibility and lipid stability.</li>
- <li><strong>LNP Stability:</strong> Stability of LNPs, particularly for inhalation therapies, needs empirical testing, considering shear forces and the potential use of cryoprotectants or temperature control.</li>
- <li><strong>Lipid Selection in Kits:</strong> Starter kits use well-researched lipid combinations, tested for stability, encapsulation efficiency, and potency. They provide materials for multiple experimental batches.</li>
- <li><strong>Lipid Modifications:</strong> Exploring alternative lipids (e.g., cholesterol derivatives) could enhance stability and cellular uptake, tailored to project needs.</li>
- <li><strong>Antibody Incorporation:</strong> Antibodies can be incorporated into LNPs during preparation or afterward, depending on targeting requirements.</li>
- <li><strong>Intellectual Property:</strong> IP considerations are crucial when selecting lipids for LNP formulations, as many lipids are patented.</li>
- <li><strong>Collaboration Opportunities:</strong> Corden Pharma is open to offering discounts or forming partnerships, with recognition in publications or acknowledgments.</li>
+ <li><strong>Nano-Capsules Focus:</strong> Based on Katharina’s advice, we prioritized nano-capsules for their enhanced stability over nano-complexes.</li>
+ <li><strong>Chitosan for Stability:</strong> We're exploring chitosan to improve mRNA delivery system stability due to its positive charge, which binds mRNA to the nanoparticle surface.</li>
+ <li><strong>OptiMEM for Transfection:</strong> OptiMEM is now our chosen transfection medium, with the suggestion to remove antibiotics 24 hours prior.</li>
+ <li><strong>MTT Test for Cytotoxicity:</strong> We adopted the MTT test for cytotoxicity due to its simplicity and reliability.</li>
+ <li><strong>Nasal Spray and Dry Powder Testing:</strong> We are considering testing nanoparticle stability using nasal spray solutions and exploring dry powder formulations.</li>
+ <li><strong>Capsaicin Exclusion:</strong> As capsaicin did not significantly impact transfection efficiency in Katharina’s research, we decided not to include it in our project.</li>
</ul>
- ],
- implementation: [<p>The insights from Corden Pharma had a major impact on our project, especially in selecting lipids critical for LNP stability and optimizing gene therapy applications. Initially, we used the Cayman kit, but it was suboptimal for delivering our Primeguide. After receiving feedback, we switched to Corden Pharma’s kit #2, which includes advanced lipid components like cationic lipids that improve cellular uptake and enhance LNP stability. This shift has significantly boosted the efficiency and robustness of our formulations.
- Additionally, Corden Pharma's guidance on lipid modifications and antibody incorporation opened new possibilities for targeted therapies. These insights not only improved our technical approach but also paved the way for potential collaborations, offering cost benefits and increased scientific recognition. The feedback will continue to shape our testing process and improve therapeutic delivery. </p>],
- interview: <>
- <QaBox q="Is it possible to dry the LNPs designed by Corden Pharma, such as through spray drying?" a="It has not been confirmed whether LNPs have been successfully dried using spray drying. Further investigation or consultation with a specialized company would be required to determine feasibility."/>
- <QaBox q="How is stability ensured in LNPs, particularly for use in inhalation therapy?" a="The stability largely depends on the specific lipid and RNA components used in the formulation. While the stability of individual lipids can be assessed, the overall stability of a new LNP formulation requires empirical testing under various conditions."/>
- <QaBox q="How are lipid combinations selected for inclusion in the LNP starter kits, and what testing is conducted?" a="Lipid combinations in the LNP starter kits are selected based on known interactions, particularly in formulations containing RNA. Testing is conducted to assess physical-chemical properties, encapsulation efficiency, and overall potency. The kits are designed to provide sufficient material for multiple experimental batches."/>
- <QaBox q="Is it advisable to modify the lipid components in an existing LNP formulation?" a="It is generally advisable to consider alternative lipid components, as different lipids may offer improved stability or efficacy. However, the specific needs of the project will dictate whether changes are necessary."/>
- <QaBox q="Is it possible to incorporate antibodies into LNPs, and what is the recommended approach?" a="Yes. Incorporation of antibodies into LNPs is possible. This can be achieved either during the initial preparation phase or by incubating antibodies with LNPs after their formation, depending on whether surface or internal localization of antibodies is desired. Reference to specific studies may provide further guidance."/>
- <QaBox q="What is Corden Pharma's position on projects involving gene therapy?" a="Corden Pharma operates as a service provider, focusing on the manufacturing of active pharmaceutical ingredients (APIs) and excipients rather than developing therapeutic products. Consideration should be given to the intellectual property status of the lipids used in LNP formulations, particularly for commercial applications."/>
- <QaBox q="Is it possible to obtain a discount on LNP starter kits or establish a collaborative relationship with Corden Pharma?" a="We would need to discuss this internally but generally we would be open to potential collaborations that could involve recognition in publications or other forms of acknowledgment, pending approval from relevant management."/>
+ ] ,
+ interview:<>
+ <QaBox q="How did you approach the design of Lipid Nanoparticles (LNPs)? What were the first steps you took at that time? Were you already familiar with LNPs, or was that a completely new experience for you?" a="I started working with nanoparticles during my bachelor’s thesis. I continued with nanoparticles into my master’s thesis, working on a project related to cystic fibrosis. Initially, I worked with nano-complexes, but later switched to nano-capsules due to their stability, especially in cell culture media." />
+ <QaBox q="Since you’re focused on stability and applying high shear forces, could you explain why nano-capsules are more stable than complexes in this context?" a="Nano-capsules are generally more stable in cell culture media compared to nano-complexes, which often react with additives and proteins. However, I didn’t explore shear forces much further. My main goal was to stabilize the particles in cell culture media for testing on cells." />
+ <QaBox q="What kind of cell culture medium did you use for these experiments?" a="For transfection, we used Optimem as the medium, after removing antibiotics from the culture medium 24 hours prior to transfection." />
+ <QaBox q="How long did it take you to get to the point where you used nano-capsules?" a="I only started working with nano-capsules towards the end of my PhD. I spent much of my time with nano-complexes, but when I visited a lab in Leeds, I shifted to nano-capsules. This transition happened quite late, just months before I finished my thesis." />
+ <QaBox q="You mentioned capsaicin in your recent paper. Does it significantly affect transfection efficiency, and is it worth including in our experiments?" a="No, capsaicin didn’t affect transfection efficiency in our experiments. It was included to inhibit the Ina-channel as part of a dual strategy targeting both CFTR and Ina-channels, but it might not be necessary for your project." />
+ <QaBox q="Were there any critical components in the formulation of your nanoparticles that you couldn’t do without?" a="No, the main comparison was between nano-complexes and nano-capsules. Nano-complexes were inconsistent in size and stability, while nano-capsules were stable and smaller, which I believed would work better in later experiments." />
+ <QaBox q="Is there a way to check if the mRNA sticks to the outside of the nanoparticle or ends up inside?" a="I believe the mRNA sticks to the outside. The process involved forming nano-capsules using lecithin and oil, and after the ethanol was evaporated, mRNA was added last. The mRNA likely adhered to the positively charged chitosan on the outside of the capsule." />
+ <QaBox q="Do you remember the ratio of mRNA to nano-capsule?" a="I don’t remember the exact ratio offhand, but it’s documented in my dissertation. I optimized the amount of mRNA that needed to enter the cell for effective results, but didn’t do extensive testing with nano-capsules." />
+ <QaBox q="Was determining the optimal amount of nanoparticles trial and error?" a="Yes, definitely. It involved a lot of optimization." />
+ <QaBox q="You used the MTT test for cytotoxicity. Would you recommend it for us, or are there better alternatives?" a="Yes, the MTT test is simple and reliable. You just need to pipette accurately. We used it frequently, and it worked well." />
+ <QaBox q="How did you assess the stability of the nano-capsules? Did you use microscopy or another method?" a="We used a device called a Zetasizer, which measures size, zeta potential, and polydispersity index (PDI). We used it to assess stability in cell culture medium over time, from half an hour to 24 hours." />
+ <QaBox q="Do you have any advice for our project or anything we might have missed?" a="If you’re planning to use a diffuser for nasal administration, you might want to test the stability of the nanoparticles in a saline solution or standard nasal spray solution. It could be useful to see how they behave in such a medium. Otherwise, it seems like you’re well ahead of where I was!" />
+ <QaBox q="Thank you so much for your time and insights!" a="You’re welcome! I’m glad I could help." />
</>,
- summary: "The primary goal of the communication with Steffen Bira and Serra Gürcan from Corden Pharma was to explore the use of Lipid Nanoparticles (LNPs) in gene therapy and inhalation treatments. Corden Pharma recommended consulting specialists for assessing spray drying feasibility, while highlighting the importance of testing LNP stability under various conditions. Their LNP starter kits are optimized for stability and encapsulation efficiency, with potential for lipid modifications to enhance cellular uptake. They also confirmed that antibodies can be incorporated into LNPs and emphasized considering intellectual property when selecting lipids. Based on Corden Pharma’s insights, we switched to their kit #2 for our project, hoping to improve the stability and efficiency of our LNP formulations. Their guidance also opened opportunities for targeted therapies and collaborations. This feedback will continue to enhance our testing and therapeutic approaches.",
- months: "august",
- pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/hp/hp-corden-zoom.png"
-
- },
- {
- vorname: "Mattijs",
- nachnname: "Bulcaen",
- job: "PhD Researcher at Laboratory for Molecular Virology & Gene Therapy",
- affiliation: "KU Leuven",
- pictureurl: pics['mattijs'],
- tag: "Academia",
- heading: "",
- interviewtabid: "mattijsvisit",
- cardtext: "",
- language: "en",
- quote: "",
- aimofcontact: "",
-
- insights: "",
- implementation: "",
- summary: "",
- months: ""
+ summary: "We identified several crucial insights to guide our project development. Nano-capsules were found to be more stable than nano-complexes, making them our preferred formulation choice. We will utilize chitosan to enhance mRNA stability due to its positive charge, while capsaicin was deemed irrelevant for our purposes. For transfection, we will use OptiMEM as the medium, removing antibiotics 24 hours prior to the procedure. We will assess stability with a Zetasizer and evaluate cytotoxicity using the MTT test. Additionally, we are exploring nasal spray and dry powder formulations to improve nanoparticle delivery. These insights will significantly shape our approach to optimizing mRNA delivery systems.",
+ months: "August"
},
{
title: "Dr.",
@@ -1110,45 +1344,58 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
affiliation: "Research Group Cellular Neurophysiology",
pictureurl: pics['draeger'],
tag: "Academia",
- heading: "",
+ heading: "New approaches in electrophysiology and their application in our project ",
interviewtabid: "patchclamp",
cardtext: "",
language: "de",
quote: "",
- aimofcontact: "",
-
- insights: "",
- implementation: "",
+ aimofcontact:[<p></p>],
+ insights: [<p></p>],
+ implementation: [<p></p>],
summary: "",
- months: ""
+ months: "August",
},
{
- title: "",
- vorname: "Nils",
- nachnname: "Berelsmann",
- job: "",
- affiliation: "University of Bielefeld",
- pictureurl: pics['nilshefe'],
- tag: "Academia",
- heading: "Focus on adapting expression strategies for Fanzor nickases and exploring the potential of Pichia pastoris (SMD1163) for SpuFz1 nickase variants ",
- interviewtabid: "nberelsmann",
+ vorname: "GxP course",
+ nachnname:"",
+ pictureurl: pics['gxpcourse'],
+ tag: "Industry",
+ heading: "Successful participation of a team member in a 5 day GxP course",
+ interviewtabid: "gxpcourse",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
- },
- {
- title: "",
- vorname: "Michael",
- nachnname: "Johannfunke",
- job: "Representative body for severely disabled persons",
- affiliation: "University Bielefeld",
- pictureurl: pics['johannfunke'],
- tag: "Academia",
- heading: "urgent requirement for a hygiene concept for students with disabilities and immunocompromised employees ",
+ quote: "The GXP course was extremely useful as it provided us with important knowledge that supports our entire team in complying with quality standards. This knowledge will help us to organise our processes efficiently and in accordance with regulations in the future.",
+ quoteVorname:"Kaya",
+ quoteNachname:"Lange",
+ text: [<p>I, Kaya, Team Member of iGEM Bielefeld 2024, recently participated in an intensive one-week GXP (Good Practice) training course, which was pivotal experience for both me and our project. The course covered essential regulatory frameworks, including</p>,
+ <ul>
+ <li>Good Laboratory Practice (GLP)</li>
+ <li>Good Clinical Practice (GCP)</li>
+ <li>Good Manufacturing Practice (GMP)</li>
+ </ul>,
+ <p>
+ which are all designed to ensure quality, safety, and compliance across every phase of scientific research and development.
+ As the head of Integrated Human Practices, I found this training particularly valuable. It provided me with a deeper understanding of the rigorous standards that need to be maintained in research, especially concerning ethics, data integrity, and patient safety. I learned how to properly document research processes, ensure the reproducibility of results, and assess and mitigate risks, all while keeping the ethical considerations of our project at the forefront.
+ I have acquired the ability to create standard operating procedures (SOPs) that guarantee the transparent and traceable documentation of each stage of the research process. This not only facilitates internal organisation but is also crucial for subsequent approvals and audits by regulatory authorities.
+ It is of paramount importance to ensure the reproducibility of our experiments by maintaining accurate protocols and meticulously documenting all variables. This is of particular importance should the intention be to pursue clinical research at a later stage, as the reproducibility of experiments is a crucial factor in the validity of the results.
+ I acquired knowledge of techniques for risk assessment, including Failure Mode and Effects Analysis (FMEA). This process enables the identification of potential risks in a project at an early stage, thus facilitating the development of strategies to minimise them. This approach allows us to identify and address potential sources of error before they lead to significant issues.
+ This knowledge is crucial as we think about the future of our project, particularly if we aim to move our gene therapy approach for cystic fibrosis closer to clinical trials and real-world applications. My participation in the GXP training has equipped me with the necessary tools to potentially guide our team through the complex regulatory landscape, ensuring our work remains aligned with industry standards and ready for the next steps in development.
+ One of the key speakers during the GXP course was Dr. Marcus Berger [LINK INtreview Beerger], whose expertise was invaluable to me and the entire team. After the course, I had the opportunity to ask Dr. Berger some questions, further deepening my understanding of the practical applications of GXP in research. The connection with Dr. Berger has been highly beneficial, as his insights helped shape key aspects of our project’s development and compliance with industry standards. His guidance will continue to be a valuable resource for our team moving forward.
+ Through this training, I feel better positioned to contribute to the team’s efforts, ensuring our project adheres to global safety and ethical guidelines. This experience has strengthened our approach and set a solid foundation for future progress, ensuring that our research, public engagement, and potential clinical applications continue to meet the highest regulatory standards. </p>],
+ type: "meta",
+ summary: "Kaya, a member of the iGEM Bielefeld 2024 team, completed an intensive one-week GXP (Good Practice) training, which covered Good Laboratory Practice (GLP), Good Clinical Practice (GCP), and Good Manufacturing Practice (GMP). The training provided valuable insights into maintaining high standards of quality, safety, and ethics throughout the research process. Kaya learned crucial skills, such as documenting research processes for reproducibility, creating standard operating procedures (SOPs), and conducting risk assessments using techniques like Failure Mode and Effects Analysis (FMEA). This knowledge is essential for advancing their cystic fibrosis gene therapy project toward clinical trials and ensuring compliance with regulatory standards. Dr. Marcus Berger, a key speaker in the course, provided additional guidance, offering valuable insights that will continue to benefit the team.",
+ months: "august",
+ pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/for-wiki-texts/gxp/gxp-course-kaya.webp",
+ pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/for-wiki-texts/gxp/gxp-course-group.webp",
+ },
+ {
+ title: "",
+ vorname: "Michael",
+ nachnname: "Johannfunke",
+ job: "Representative body for severely disabled persons",
+ affiliation: "University Bielefeld",
+ pictureurl: pics['johannfunke'],
+ tag: "Academia",
+ heading: "Hygiene concept for students with disabilities and immunocompromised employees",
interviewtabid: "johannfunke",
cardtext: "",
quote: "The implementation of the hygiene concept is proving more difficult than expected due to the bureaucracy at the university. Nevertheless, the interview gave us a good insight into this labyrinth of regulations and we got started the prozess of implementation.",
@@ -1165,7 +1412,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="What are the next steps in implementing the hygiene concept?" a="It is essential that the concept be implemented in small, strategic steps. At the same time, it is vital that the rectorate and other decision-makers be consulted on a regular basis to ensure that this matter remains at the forefront of discussions. Furthermore, it is of great importance to engage in political lobbying to secure additional support for this issue." />
</>,
summary: "We got in touch because there was an acute hygiene problem for particularly vulnerable groups like immunocomprised persons at the university. We learnt from the exchange that despite a well-developed hygiene policy, strategic steps are still needed, especially to overcome bureaucratic hurdles. We have integrated these lessons into our project by focusing on continuous collaboration with the Central contact point Barrier-free and other decision-makers.",
- months: ""
+ months: "September"
},
{
title: "Dr.",
@@ -1175,7 +1422,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
affiliation: "Nationwide Children’s Hospital",
pictureurl: pics['placeholder'],
tag: "Academia",
- heading: "F508del mutation confirmed as the most common CFTR mutation worldwide, including Asia, supporting the efficacy of existing therapies for the majority of patients. ",
+ heading: "F508del mutation confirmed as the most common CFTR mutation worldwide, including Asia",
interviewtabid: "sriram",
language: "en",
cardtext: "",
@@ -1186,17 +1433,37 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
implementation: [<p>The data were incorporated by confirming that the F508del mutation is not only the most common in Europe but also globally, including in Asia, highlighting a broader perspective and contributing to a significant horizon expansion in understanding the mutation's worldwide prevalence. This finding lends support to the idea that existing therapies targeting the F508del mutation will be effective for many patients worldwide, thereby providing a solid foundation for treatment. As a starting point, this is promising, but future efforts will focus on adapting therapies to address other, rarer mutations found in specific populations.
</p>],
summary: "The contact provided valuable insights into cystic fibrosis (CF) in Asia and confirmed that the F508del mutation is the most common, as it is globally. However, the genetic diversity observed in Asia, together with the exacerbation of symptoms by environmental factors such as air pollution, highlights the need for more region-specific research. Future efforts will focus on refining treatments for rarer mutations and improving diagnostic accuracy in Asian populations.",
- months: ""
+ months: "September",
+ interview:<>
+ <QaBox q="Should we explain our project a little bit in the beginning?" a="That would be great."/>
+ <QaBox q="Okay. As I wrote to you in the email, we are part of the Bielefeld-CeBiTec item team in Germany this year, and we have a project called Precyse, where we want to develop a platform for next-generation prime editing. With that, we aim to tackle CF mutations, starting with the F508del mutation. The complex should be adaptable for different mutations with minor changes. Essentially, we want to operate like Cas-based projects using prime editing tools but may transition to using a fanzor nickase in the future. Currently, we are working on a delivery system utilizing lipid nanoparticles to transport our complex into the lungs. These nanoparticles will be loaded with mRNA and designed for inhalation, similar to how asthma medications are delivered. Our goal is to advance personalized medicine and improve the standard of living for those affected by the disease, especially since a friend of one of our team members has cystic fibrosis and shares insights on the challenges faced by those with the condition." a="That's a very nice summary. I have a couple of clarifying questions. Are you undergraduates?" />
+ <QaBox q="No, we are overgraduates at the University of Bielefeld." a="Oh, great! So, how are you optimizing prime editing, and who is helping you with this process?" />
+ <QaBox q="We tried to reach out to Dr. Liu, but he hasn't responded yet." a="You should also contact Marianne Carlon in Leuven, Belgium. They didn't work on Delta F508, but Mattijs is a graduate student there and could be helpful." />
+ <QaBox q="We’ve already been in touch with Mattijs; he’s been very nice." a="Perfect! You're reaching out to the right people. Are you aware of the Liu lab's Nature Biomedical Engineering paper that optimizes prime editing?" />
+ <QaBox q="Yes, we are aware of it." a="Great! How can I help you further?" />
+ <QaBox q="We have some questions about cystic fibrosis (CF) in Asia, specifically about its perceived rarity and its impact on diagnosis and research efforts." a="I can share some insights. While CF is often considered rare in Asia, it's essential to recognize that it affects various populations. In my lab, we analyze CF-causing mutations across different populations using data from Nomad, which focuses on genetic ancestry rather than self-identified race. CF is most prevalent in individuals of European origin, but it remains common across many populations worldwide, particularly in South Asia and other regions, although East Asia has lower prevalence rates." />
+ <QaBox q="That’s helpful. Can you explain how the perceived rarity of CF impacts diagnosis and research in Asia?" a="The perception of CF as a 'white people disease' leads to underdiagnosis and fewer research efforts in Asian populations. For instance, in India, physicians are more familiar with conditions like sickle cell disease and thalassemia, leading to limited awareness of CF. Early diagnosis is challenging due to the lack of screening programs for CF, and currently, many regions are only beginning to implement sweat chloride testing." />
+ <QaBox q="Thank you! What mutations are more common in Asia?" a="Delta F508 is still the most frequent variant across different populations, but its frequency decreases in non-European groups. While there are several other common variants, they do not respond to modulator therapeutics, which complicates treatment options. Focusing on Delta F508 is sensible, but you might face challenges in finding individuals with other variants due to the limited awareness and screening for CF in those populations." />
+ <QaBox q="Okay. But you would say that the other mutations are a bit under-researched, right?" a="They are completely under-researched, right? Like I have not seen anybody work on L218X. I didn't even know L218X existed until I started looking into that group. And it seems like L218X, I've not seen it in any other population that frequently. So a lot of these variants seem to be unique. I don't know if they're completely unique to South Asians or if you go to Iran or something, whether you're going to start finding these people or whether if you go to Southeast Asia, then you're going to start finding some of these people, right? So we don't know that, but I think it will be really hard for you to find cells with these variants."/>
+ <QaBox q="What do you believe, what steps should be done in the scientific community to ensure better representation of Asian populations, or in general, not only Western populations, in the genetic studies for cystic fibrosis?" a="Ah, it's a very good question; it's also a question that doesn't have a very straightforward answer, right? In the sense that one, you know, you have to raise awareness and ensure that people are getting diagnosed, that's step number one, right. A lot of the numbers they showed you are based on prediction; they're not based on a registry in any country with some of these populations, right. India doesn't have a registry, China doesn't have a registry. There are efforts to make a registry, but you know it's a little hard to get started when you know if you go and argue that there are 6,000 children with CF that are being born in a year in India, and then the response that I've received before is well, there are about 60,000 kids dying of diarrhea, and you know so the priority from public health is often different. It's starting to change. So one focus would be diagnosis and raising awareness, and if you come across groups that are trying to improve their ability to diagnose then you try to help them, right. What's your other question in terms of treatment and research? I think being aware of it if you do end up finding samples that have some of these unique variants. Right then, trying to form a strategy around it is not bad; I've seen people identify variants of CF that are unique to different populations. I think even Marianne Carlon's group picked up variants that are unique to Belgium and then tried prime editing on that right, so those sort of strategies help quite a bit, right? And this is partially the reason you know I continue to focus on mutation-agnostic strategies; I've not really branched out into the Prime Editing world or the Base Editing world primarily because this seems to be a pretty challenging issue, but that said, you want as many shots at goal as possible. So, you know, if you're able to use prime editing to fix Delta 508, that still helps the most number of people with CF. And then maybe, you know, you could use that platform that's really well validated at that point, then go start targeting individual variants. The other idea with prime editing is that there's hope that you can probably at least try to replace one exon at a time. And that sort of makes things a little bit more manageable, right? So if you, you know, it's a little bit more, like if you have to target every single variant, then you're thinking about hundreds of different medical products. But if you're thinking of replacing every exon, that's 27, that's a countable number. And I would argue that you don't even have to do all 27. You start with the hotspots, right? You start with exon 11, you start with exon 12. But then as part of the conversation, I think we should just be thinking about exons that might be hotspots for variants in other populations and at least, you know, keeping them somewhere in the packing order so that as you go through the most frequent variants, right? And you're starting to develop strategies for the most frequent variants. Those are on the list so that we eventually get to them."/>
+ <QaBox q="Yeah. Okay. And what do you think? Are there any healthcare challenges managing cystic fibrosis in Asian countries?" a="Oh, absolutely. Right. I think, you know, if you're in a high-income country like Japan, I think, you know, the biggest challenge is awareness and getting diagnosed. If you're in low- and middle-income countries, right, then the challenge becomes access to drugs. So modulators are not available in most of Asia, or at least in the lower and middle-income countries in Asia, that's not available, like, it's not available for sale. Primarily, partly because they think that those people, like people at CF, don't exist in enough numbers in those countries. And partly because then it creates problems in terms of monetary, like factors, right. In terms of how much they charge for the modulators. Right. So those don't exist. I don't know, like they have access to pancreatic enzymes and things like that, but, you know, regular access to regular care is still a challenge. The base of physicians that are family with treating CF is only growing in some of those places at least from my personal experience but it’s not by where we are. The children there are still mostly passing away when they are in their teenage years or early twenties. The sort of life span and life expectancy is no where close to what you would see in high-income countries. So, I think that is the biggest challenge, access to resources both, publicly and also from a private point."/>
+ <QaBox q="Okay. And do you think that the environmental or like pollution is doing, like, I don't know how to say it correctly. So that there are like respiratory challenges because, yeah, right." a="Absolutely! Right. It's, you know, your exposure to pathogens is higher. Your exposure to pollution is significantly higher in a lot of these countries, right? Even China, which is, you know, much better off than a country like India still has a lot of pollution, right? And so, or I've never been to China, but at least that's what I read, right? But India, I grew up there and I can firsthand tell you that there's a lot of pollution there, right? And that I'm sure it affects it because it's been documented to affect people, just people without CF, right? On an average, it's supposed to like, the reports that I've seen are like, it takes a few years off of life expectancy for even people that have not been diagnosed with anything else, right? So I'm sure if there's a person with CF, then that's going to affect them even more negatively in places like India. And then I think that the exposure to pathogens might be slight, it might be higher and the lower down in the economic scale you go, the more, the exposure to pathogens are going to have, right? I think the third factor would also might be nutrition, right? Like, so if you have CF, you, you know, here, one way they were managing it was through high fat calorie rich diet and access to those sort of high fat calorie rich diets may not be that prevalent in some of these other places. And so that might become a factor."/>
+ <QaBox q="Okay. And then one last question. So it's more a bit about you and your research. So what drew your attention to the issue of CF in Asia? So why did you start researching this topic?" a="Oh, so that was a very okay. So the way it happened was I was at a conference and I was a postdoc at Stanford previously, as you know. So I was at a conference and I was, we were just, I think had a sort of a, you know, a dinner for everybody from our institution. And I was chatting with the nurse in the table and sitting in the table and so the San Francisco area has a lot of people from Asia. And so the nurse particularly said that they had a lot of children with CF who happened to originate in India, which really surprised me because that's not a disease that I'd heard growing up as being some, a common genetic disease there. Right. And so that kind of drew my attention. And then in subsequent conversations, when I spoke with physicians who are practicing in India, nobody said that they hadn't seen people with CF, right? Whenever I had the conversation, people would be like, oh yeah, I've seen a patient with CF or I know of somebody that has a child with CF, right? Which was surprising. And then when I went to India, I went to a hospital in my hometown and I was talking to the pulmonologist there. And he said he had 30 or 40 patients with CF in that, just that hospital. And that's, you know, it's not even, you know, it's probably one of the bigger cities in India, but I wouldn't even know if it's in the top 10 biggest cities in India. And this is not even the biggest center in that city necessarily. Right. So I was just really surprised. And he said he didn't have to really put in any effort to go seek out any people with CF. This was just in the people that walked into that hospital that got diagnosed with CF, right? So that kind of solidified the perception that perhaps it was underdiagnosed. And then the reason it happened was because the pandemic sort of forced most of us out of the lab. And so this ended up being something you can actually work on without going to lab."/>
+ <QaBox q="Yeah, sounds really interesting the way you got there. at the beginning, you said you could tell us something, what came to you in mind when you heard about Project First, and you already prepared some slides. So I just want to ask if you maybe have something more to tell us that you haven't thought about yet, because the interview until now is highly interesting and I think we got a lot of new information until now. So maybe you have something. Maybe you have more interesting information for us." a="I think you guys covered a lot of what I had in mind, right? In terms of, I think if I were to start with prime editing and Delta 508, this is exactly the argument I would have made. I would have talked to all of the exact people that you have already spoken with, right? So I think that I don't have too much to add there. The only thing you could do is if you are looking at variants, right? Maybe I would try to come up with a plan for what variants you might go after, after Delta 508, right? And that could become part of your future research or something like that. Or if you can, I guess the one question I had for you is, so the correction of Delta 508 is sort of published, right? People have reported using lipid nanoparticles, at least to deliver base editing reagents, right? So how are you thinking about, like when you pitch the innovation in your project, how are you thinking about pitching your innovation? "/>
+ <QaBox q="Okay. So I think, our idea was that we want to do like the, the prime editor is more efficient. Our idea is to use a smaller reverse transcript case and maybe when we will when the time is letting us do it, maybe not, we don't know if we will get in time there and everything will be ready. But in the Liu paper, I think was something about the La motive and some other stuff that we want to add to the prime editor so that it's even more efficient. And even then the idea of the LNP to spray dry because we thought then when there is not the that it has to be cool, like other medication, it's easier to bring it like to Asia or other places because they can just get the inhalator. " a="Right. So the one question I had for you on that is, have you looked at Daniel Siegwarts work on LNP? "/>
+ <QaBox q="No,not yet." a="Because they had a paper in science where they did intravenous delivery of lipid nanoparticles and they, they delivered a base editor. So when you present this, this might come up. So, you know, so I'm not saying you should change your strategy, but, you know, if you're trying to do a lot of work on lipid nanoparticles, you're going to have to do a lot of work on So you want to have thought about a response. And it's, it's a really interesting strategy. Um, trying to see if I can find it. Yeah. Here. It's a really interesting sort of nanoparticle strategy that they have presented. Uh, the nice thing is it is an IV delivery, which gets you to basal cells and they were able to get about 20% correction, right? So you could almost think of it as an alternative strategy. So if, you know, if you get the pushback or I don't know how you have to write to your proposal, but if the, the, the spray drying or the nebulization process is not going well for you, this might be something that you could propose as a backup because here they've shown that you can deliver lipid nanoparticles intravenously and they get to the basal cell population. Okay. And they get to, yeah. So that might be the only other direction I would point to as you're thinking about your delivery strategy. Right. Uh, the other innovation could be that if you can even optimize your prime editing to go a little bit beyond just Delta, if I wait, if you can show that you even replace the whole Exxon, you might have something that is a little bit more unique than what's out there. And then you don't have to even worry about delivery necessarily. "/>
+ <QaBox q="Okay. Thank you so much. We will look at the paper and we'll check it out. All right. Okay. Then, yeah. Thank you very much for the interview. " a="Yeah, that's perfectly fine. Good luck with your project. It's a really interesting project."/>
+ </>
},
{
- title: "",
+ title: "M.Sc.",
vorname: "Philipp",
nachnname: "Kühnel",
job: "PhD student in the Otorhinolaryngology working group at Bielefeld University",
affiliation: "Universität Bielefeld",
pictureurl: pics['kühnel'],
tag: "Academia",
- heading: "Philipp Kühnel’s guidance significantly improved our culture protocols and experimental outcomes, particularly in maintaining ALI cultures and addressing fungal contamination issues.",
+ heading: "Improvement of our culture protocols and experimental outcomes, particularly in maintaining ALI cultures",
interviewtabid: "pkuehnel",
cardtext: "",
quote: "x",
@@ -1205,86 +1472,12 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
implementation: [<p>We incorporated Philipp’s advice by refining our culture protocols, particularly adjusting the conditions for ALI cultures to improve cell differentiation and overall culture health. This directly enhanced the reliability of our experimental results, ensuring that our work with primary cultures was both accurate and reproducible. </p>],
language: "de",
summary: "The contact aimed to leverage Philipp’s expertise in ALI cultures to improve our experimental protocols Gained insights into optimizing conditions for primary cell cultures and managing common challenges like fungal contamination",
- months: ""
- },
- {
- title: "",
- vorname: "Timm",
- nachnname: "Weber",
- job: "Quality Manager | Immunologist",
- affiliation: " Biobank OWL (Bielefeld and Lippe)",
- pictureurl: pics['placeholder'],
- tag: "Academia",
- heading: "Discussed the processes involved in the storage, processing, and security of patient samples.",
- interviewtabid: "timm",
- cardtext: "",
- quote: "A biobank is not just a collection of samples; it's a bridge between patient trust and scientific discovery, ensuring that valuable biological data is safeguarded while contributing to future research.",
- aimofcontact: "Contact was established with Timm for the purpose of gaining deeper insights into the functioning of the biobank and of deepening our understanding of the processing of patient samples.",
- insights: "We were provided with invaluable insights into the quality and project management of the biobank and storage of patient samples. It was of particular interest to note that Biobank OWL occupies a distinctive position in this context, insofar as a trustee is not a mandatory figure within its system and is therefore not provided for as a standard component. However, Biobank OWL has elected to integrate a trustee in order to enhance the security standards for the safeguarding of patient data. This illustrates the biobank's dedication to ensuring the optimal protection and security of sensitive patient data.",
- implementation: "The insights gained have facilitated a deeper comprehension of the significance of quality management in the processing of patient samples. This understanding has been integrated into our project processes, thereby enhancing the accuracy and reliability of our procedures. ",
- summary: "The interview focused on understanding the operations of the Biobank OWL, particularly in the areas of quality management and sample processing. Provided a detailed overview of biobank activities, including sample collection, storage conditions, and data protection measures",
- language: "de",
- interview: <>
- <QaBox q="Can you briefly explain to us what exactly a biobank is and what its main tasks are?"
- a="A biobank is a specialized facility that collects, stores, and manages biological samples and associated data for research purposes. Each biobank is unique in its operations and functions. In Bielefeld and Lippe, the Biobank BOWL (Biobank OWL) is responsible for the storage of patient samples. The Data Integration Centre (DIZ) stores data pertaining to these samples. A trustee oversees the pseudonymisation of data, acting as an interface between BOWL and DIZ, ensuring that patient data cannot be directly linked to patient samples." />
- <QaBox q="What types of samples are collected in your biobank and for what research purposes are they used?"
- a="The biobank collects a wide variety of samples, including blood, stool, and soil. Samples may be gathered for specific research projects or for establishing a general repository under 'broad consent.' Researchers wishing to use these samples must apply to the 'use access committee,' which evaluates whether the requested samples and data can be released for their research." />
- <QaBox q="How large is your biobank? How many samples do you currently store and how many new samples are added on average?"
- a="The biobank is still in the process of establishing itself and has not yet reached its full sample capacity. However, it is anticipated to accumulate a significant number of samples in the near future, with several thousand samples expected to be analyzed in dedicated sessions." />
- <QaBox q="What requirements and criteria must be met for a sample to be included in your biobank?" a="Samples must be processed according to highly detailed protocols, and regular audits are conducted to ensure compliance with all standards." />
- <QaBox q="Which other research institutions or biobanks do you cooperate with and what form does this cooperation take?"
- a="Biobank OWL has a second location in Lippe, in addition to Bielefeld. Collaborations exist with the DIZ, the Treuhand, and three university hospitals. It is anticipated that cooperation with other working groups will increase in the future." />
- <QaBox q="What specific storage conditions (e.g. temperature, humidity) must be observed for different sample types?"
- a="Samples are stored under various temperature conditions, including -20°C, -80°C, and -150°C, along with the use of liquid nitrogen." />
- <QaBox q="How do you ensure that the samples remain stable and usable over longer periods of time?"
- a="Samples are stored in nitrogen for long-term stability." />
- <QaBox q="What encryption techniques or data protection measures are used in your biobank to prevent unauthorized access to patient data? Are there special regulations for the anonymisation of data and how is it ensured that patients cannot be traced?"
- a="Pseudonyms are created using specialized software such as CentraXX or REDcap to protect patient data." />
- <QaBox q="What rights do patients have in relation to their samples, and how are these rights safeguarded in your biobank?"
- a="Patients have the right to revoke their consent at any time, which can be done at the clinic or biobank. The trustee, acting as an intermediary, will notify BOWL and DIZ to destroy the corresponding samples or data." />
- </>,
- months: ""
- },
- {
- title: "M.Sc.",
- vorname: "Nils",
- nachnname: "Berelsmann",
- job: "PhD Working group: Prof. Dr. Gabriele Fischer von Mollard ",
- affiliation: "University of Bielefeld",
- pictureurl: pics['placeholder'],
- tag: "Academia",
- heading: "",
- interviewtabid: "nberelsmann",
- cardtext: "",
- quote: "X",
- aimofcontact: [<p>During our interview with Makoto Saito[Linkinterview] about fanzor[link fanzor], it became evident that the expression of our fanzor nickases in yeast is very promising. We then refined our expression strategy for the nickases and approached Nils Berelsmann, who is currently working on his PhD thesis with the yeast strain Pichia pastoris (SMD1163). This particular strain could be ideal for expressing the SpuFz1 nickase variants. Our main aim in contacting Nils was to gain insight and advice on yeast expression and he generously shared his expertise with us. Not only did he give us valuable advice, but he also provided us with the yeast strain itself, along with a corresponding expression vector for possible experiments. He also provided us with detailed protocols and the plasmid map of the vector and gave us practical tips on how to optimize the expression process. His support was invaluable in moving our work forward. </p>],
- insights: [<p>Pichia pastoris (SMD1163) is a promising option for expressing SpuFz1 nickase variants. Refining expression strategies based on expert insights is crucil for success. Nils provided practical tips on yeast expression, including optimizing growth conditions and fine-tuning induction protocols.</p>],
- implementation: [<p>We adapted our expression strategy for Fanzor nickases in yeast by incorporating the Pichia pastoris strain (SMD1163) and the provided expression vector into our experiments. Following Nils' detailed protocols and plasmid map, we optimized key steps, enhancing expression efficiency and protein yield.</p>],
- summary: "The team sought expert advice from Nils to optimize yeast expression for Fanzor nickases. Nils provided invaluable guidance on addressing potential challenges and troubleshooting the process. He supplied the Pichia pastoris (SMD1163) strain along with a suitable expression vector, crucial for expressing SpuFz1 nickase variants. Additionally, he shared detailed protocols for yeast transformation and growth optimization, enabling the team to replicate his methods effectively for their experiments.",
- months: "July"
- },
- {
- title: "M.Sc.",
- vorname: "Kai",
- nachnname: "Schülke",
- job: "PhD student Working group: Organic chemistry and biocatalysis  ",
- affiliation: "University of Bielefeld",
- pictureurl: pics['kaihammer'],
- tag: "Academia",
- heading: "First insights of Enzym Engineering",
- interviewtabid: "hammerkai",
- cardtext: "",
- quote: "x",
- aimofcontact: [<p>When we realized that the creation of a nickase from the endonucleases in use was a desired outcome, it became necessary to talk to an expert in the field of enzyme engineering. Our first contact was Kai Schülke, a former iGEMer and PhD student under the guidance of Prof. Dr. Hammer[Link Hammer], who is the leader of the working group organic chemistry and bioanalytics at Bielefeld University.</p>],
- insights: [<p>In the process of our interaction with Kai, we learned about the various methods employed in enzyme engineering. He demonstrated the complexity of this field of research and emphasized the importance of choosing the right approach. As a former iGEMer, Kai, inspired by his past experiences, is highly motivated and determined to develop an outstanding project. He pointed out that we cannot rely on classical methods such as directed evolution, but instead should use a rational approach to select mutation candidates. His insights and enthusiasm have encouraged us to think critically and pursue innovative solutions in our work. </p>],
- implementation: [<p>We incorporated Kai's insights into our project by shifting our approach to enzyme engineering. By focusing on a more targeted approach, we were able to refine our enzyme optimization process, ensuring that the modifications we made were based on informed, calculated decisions. This not only streamlined our research but also improved the chances of success by reducing the trial-and-error inherent in traditional methods. </p>],
- summary: "The team reached out to Kai Schülke, a former iGEM participant and enzyme engineering expert, for guidance on developing a nickase from the endonucleases in use. Kai emphasized the need for a rational, targeted approach rather than traditional methods like directed evolution. His insights helped the team refine their enzyme optimization process, making it more strategic and efficient. This shift reduced trial-and-error efforts and improved the chances of success, driving innovation in their project.",
- months: "July"
+ months: "several times"
},
{
title: "Prof.Dr.",
vorname: "Zoya",
- nachnname: "Ingatova",
+ nachnname: "Ingatova and Dr. Nikhil Bharti",
job: "Group Leader",
affiliation: "Institute of Biochemistry and Molecular Biology at Hamburg University",
pictureurl: pics['zoya'],
@@ -1294,9 +1487,9 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
cardtext: "",
quote: "Precision is key to minimizing side effects and ensuring the safety of your therapy.",
aimofcontact: "We conducted the interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti from the Institute of Biochemistry and Molecular Biology at the University of Hamburg, seeking to deepen our understanding of their research on cystic fibrosis (CF) and explore additional CF mutations, as well as to learn more about cell culture techniques specific to CF research, since they send us the CFBE41o- cell line. Our aim was also to gather more information about their approaches in CF research, particularly their focus on treating genetic mutations like nonsense mutations, which are highly prevalent in CF.",
- insights: [<p>We were struck by Ignatova’s story about founding the iGEM team in Hamburg. Her passion for fostering creativity and innovation in science was inspiring. On a technical level, their advice on cell culture was incredibly practical and immediately useful. Dr. Nikhil Bharti explained how they handle CFBE41o- cells and ALI (air-liquid interface) cell culture. This advice directly addressed the challenges we’ve faced in our own lab, giving us a method to improve our cell culture success rates. During our interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti, their innovative approach to cystic fibrosis (CF) therapy, particularly "read-through" and tRNA-based therapies, stood out. "Read-through" therapies aim to bypass premature stop codons that prevent full protein production, offering a way to restore the function of critical proteins like CFTR in CF. This approach has the potential to treat a broad range of genetic diseases caused by similar mutations. The tRNA-based therapy is even more precise, targeting mRNA to correct faulty codons without altering the DNA, making it safer for long-term use. This flexibility, along with the ability to apply these therapies beyond CF, broadened our understanding of how such strategies can revolutionize treatments for genetic disorders. A key focus throughout the discussion was safety. Prof. Ignatova emphasized the importance of ensuring that the therapies are highly specific, targeting only the defective codons while avoiding natural stop codons to prevent uncontrolled protein production. Moreover, their careful consideration of delivery systems further demonstrated their commitment to minimizing risks like toxicity in unintended organs. Their meticulous approach to safety has influenced how we think about developing and applying these therapies, making it clear that ensuring patient safety is as critical as achieving therapeutic success.</p>],
+ insights: [<p>We were struck by Ignatova’s story about founding the iGEM team in Hamburg. Her passion for fostering creativity and innovation in science was inspiring. On a technical level, their advice on cell culture was incredibly practical and immediately useful. Dr. Nikhil Bharti explained how they handle CFBE41o- cells and ALI (air-liquid interface) cell culture. This advice directly addressed the challenges we’ve faced in our own lab, giving us a method to improve our cell culture success rates. During our interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti, their innovative approach to cystic fibrosis (CF) therapy, particularly "read-through" and tRNA-based therapies, stood out. "Read-through" therapies aim to bypass premature stop codons that prevent full protein production, offering a way to restore the function of critical proteins like CFTR in CF. This approach has the potential to treat a broad range of genetic diseases caused by similar mutations. The tRNA-based therapy is even more precise, targeting mRNA to correct faulty codons without altering the DNA, making it safer for long-term use. This flexibility, along with the ability to apply these therapies beyond CF, broadened our understanding of how such strategies can revolutionize treatments for genetic disorders.Ignatova highlighted, that using cell models closely resembling properties of the cell types targeted by our therapy is important for ensuring applicability of our approach to patient cells and its safety. A key focus throughout the discussion was safety. Prof. Ignatova emphasized the importance of ensuring that the therapies are highly specific, targeting only the defective codons while avoiding natural stop codons to prevent uncontrolled protein production. Moreover, their careful consideration of delivery systems further demonstrated their commitment to minimizing risks like toxicity in unintended organs. Their meticulous approach to safety has influenced how we think about developing and applying these therapies, making it clear that ensuring patient safety is as critical as achieving therapeutic success.</p>],
implementation: "Prof. Ignatova's practical advice on cell culture had a transformative impact on our project. By adopting her method for CFBE41o- cells and improving our lab's sterilization protocols, we successfully established the cell line and significantly reduced the risk of contamination. In addition, her emphasis on safety in gene therapy guided us to review our Prime Editing construct and lipid nanoparticle (LNP) design. We focused on minimizing toxicity and off-target effects while ensuring precise targeting of lung tissues and the F508del mutation of the CFTR gene, making our approach safer and more efficient",
- summary: "Test",
+ summary: "In our interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti from the Institute of Biochemistry and Molecular Biology at the University of Hamburg, we aimed to deepen our understanding of their research on cystic fibrosis (CF) and explore additional mutations. Their innovative approaches, particularly tRNA-based therapies, aim to restore the function of proteins like CFTR by bypassing premature stop codons, presenting potential treatments for various genetic diseases. They highlighted the importance of precision in therapy to minimize side effects and enhance safety, emphasizing careful delivery systems to avoid toxicity. Prof. Ignatova's practical insights into cell culture techniques significantly improved our lab's success with CFBE41o- cells, guiding us to enhance sterilization protocols and refine our Prime Editing constructs. This transformative exchange underscored the critical balance between achieving therapeutic success and ensuring patient safety.",
language: "en",
interview: <>
<QaBox q="We have heard you are passionate about iGEM. What inspired you to get involved, and what has your experience been like with the competition?" a="My journey with iGEM began when I moved to Hamburg in 2014. Back then, Hamburg did not have its own iGEM team. Despite Hamburg lacking an iGEM presence, there were motivated students who were eager to establish a team. We started quite late with me as a principal instructor, around April, with the competition scheduled for October, so we had limited time. However, we managed to form a team and participate. Fortunately, we were successful in convincing the university administration to establish a steady support for the initiative, which ensured stable funding, including covering registration fees early on. This financial and logistical support gave the team the security to focus on their projects. Over the years, the Hamburg iGEM team has become a well-known and respected group at the university. It is a creative environment where students can push the boundaries of science through interesting and impactful projects. I moved on to other duties after several years of supervision, but I am proud to have played a role in its foundation. The university has recognized iGEM within the curriculum of Molecular Life Science, allowing students to earn credit points and have their work reflected on their transcripts. This acknowledgment further incentivizes students and ensures that their efforts are formally recognized." />
@@ -1312,190 +1505,157 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="What are your thoughts on using lipid nanoparticles (LNPs) versus other delivery systems, like AAV vectors?" a="Lipid nanoparticles (LNPs) are a promising delivery system for many genetic therapies, but they have limitations. While LNPs can effectively target certain organs, such as the lungs and liver, they cannot cross the blood-brain barrier and thus unsuitable (for now) to target neuronal pathologies. For these conditions, adeno-associated viral (AAV) vectors may be more effective, as they exhibit an inherent ability to cross the blood-brain barrier. For CF specifically, we have used LNPs to deliver sup-tRNAs directly to the lungs. We teamed up with an US company that develops safe LNPs used also for vaccines. Delivery methods like intratracheal instillation—where the LNPs are introduced into the trachea—allow for targeting the lung tissue more directly, which is critical for treating CF." />
<QaBox q="How do you view prime editing compared to other gene editing technologies?" a="Prime editing is an exciting development in the field of gene editing, but it is important to recognize that no single approach is universally superior. Technologies like prime editing, CRISPR-Cas, and our own tRNA-based therapies each have their strengths and limitations. For instance, prime editing offers a highly precise method for correcting mutations directly at the DNA level, potentially providing a one-time, lifelong cure. However, our approach, which focuses on restoring mRNA translation, does not introduce permanent changes to the genome and unforeseen, also individuum-specific side effects, can be counteracted by immediate termination of the therapy. In turn, it requires continuous re-administration over time. Ultimately, the safety and efficacy of any approach must be carefully weighed. We are not yet at a point where we can definitively rank these technologies because the field is still evolving. Each approach has potential, and the choice of which to use will likely depend on the specific disease and mutation being targeted." />
</>,
- months: "September"
+ months: "September",
+ pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/for-wiki-texts/interview-ignatova/interview-ingatova.webp ",
},
{
- title: "",
- vorname: "Muko",
- nachnname: "Dino",
- job: "",
- affiliation: "",
+ vorname: "'MukoDino'",
+ nachnname: "Thomas Malenke ",
+ job: "Patient",
+ affiliation: "and CF Activist",
pictureurl: pics['placeholder'],
- tag: "Academia",
- heading: "",
+ tag: "Patient",
+ heading: "Shaping CF Therapies: Lessons Learned from Patients' Real-World Experiences",
interviewtabid: "dino",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
- },
- {
- title: "Dr.",
- vorname: "Marco",
- nachnname: "Radukic",
- job: "Postdoc at AG Cellular and Molecular Biotechnology",
- affiliation: "University Bielefeld",
- pictureurl: pics['marco'],
- tag: "Academia",
- heading: "Optimizing LNP Transfection: Insights into working with LNP Kits",
- interviewtabid: "radukic",
- language:"de",
- cardtext: "",
- quote: "x",
- aimofcontact: [<p>The primary objective was to tackle challenges in LNP transfection related to manufacturing and cell transfection methods. The focus was on improving LNP formulation and application protocols to enhance gene delivery effectiveness,
- and on acquiring specialized expertise to optimize these processes. Dr. Radukic from Bielefeld University provided crucial insights for troubleshooting and protocol optimization to enhance LNP efficacy. </p>],
- insights: [<p> Dr. Radukic told us that the efficiency of LNPs is significantly affected by lipid-to-nucleic acid ratios and that optimizing ratios like 22:1 versus 10:1 can improve transfection.
- pH adjustments and buffer composition (e.g., modifying sodium acetate solutions) are also crucial for LNP performance. Proper storage at 4 °C, precise pipetting, and thorough mixing are essential to maintain LNP functionality.
- In addition, he suggested quality control measures such as fluorescence testing, zeta potential, and light scattering analyses help ensure our LNP is stable. Additionally, spray drying was evaluated for potential use in long-term LNP stabilization. </p>],
- implementation: [<p>Incorporating the insights Dr. Radukic and advice from recent consultations, we adjusted the lipid-to-nucleic acid ratio from 22:1 to 10:1 to enhance efficiency and modified the pH and concentration of sodium acetate solutions for better packaging.
- Storage conditions were strictly managed at 4 °C, and pipetting/mixing techniques were refined to ensure quality consistency. Quality control was expanded to include fluorescence testing, zeta potential measurements, and light scattering, alongside cytotoxicity tests.
- These improvements not only address transfection challenges but also strengthen our foundation for future LNP applications. </p>],
- summary: "The aim of the contact was to address challenges in LNP transfection and improve formulation protocols for enhanced gene delivery. Dr. Radukic highlighted the importance of lipid-to-nucleic acid ratios, recommending adjustments like 10:1 for better transfection efficiency. He also emphasized optimizing pH and buffer composition, as well as strict storage and mixing practices. Additionally, quality control measures such as fluorescence testing and zeta potential analysis were suggested to ensure LNP stability. These insights were implemented into the project, improving transfection efficiency and paving the way for future LNP applications.",
- months: "september"
+ quote: "Living with cystic fibrosis has been a journey of perseverance and adaptation. Despite the challenges, I've learned that taking personal responsibility for my health, staying informed, and embracing the advancements in research can lead to a much-improved quality of life.",
+ aimofcontact: [<p>Our goal in reaching out to cystic fibrosis (CF) patients was to gain a firsthand understanding of their day-to-day experiences living with the condition. We aimed to explore how recent advancements in treatments have impacted their lives and transformed their approach to managing the disease. This initiative is vital for our project, as it helps us comprehend the significance of these treatments and their broader implications. A key aspect of our outreach was partnering with the MukoDino[Link https://www.instagram.com/accounts/login/?next=https%3A%2F%2Fwww.instagram.com%2Fmukodino%2F&is_from_rle] who has extensive reach within the CF community. This collaboration enabled us to distribute our survey widely among those affected, as we recognized that we could not conduct as many interviews as we could reach through the survey. We also sought to gather insights on research priorities and the future direction of CF therapies. </p>],
+ insights: [<p>From talking with the CF patient, we gained some valuable insights that have shaped how we move forward with our project:
+ Firstly, hearing about the day-to-day challenges with current CF treatments was eye-opening. It highlighted just how crucial it is for us to develop therapies that are not only effective but also make life easier for patients. We’ve taken this to heart and are working to make our treatment process as efficient and user-friendly as possible.
+ The patient also talked about the heavy toll that existing treatments can take, both in terms of time and physical strain. This feedback has pushed us to focus on streamlining our approach, especially with our prime editing and inhalation therapy. We want to cut down on the complexity and frequency of treatments, making things simpler and less burdensome for patients.
+ We also learned about the ongoing need for more advanced therapies. The patient stressed the importance of continuing to push the boundaries with modulator therapies and gene editing. This has really driven us to prioritize our spray-dried lipid nanoparticles (LNPs) approach, which aims to deliver gene-editing tools straight to the lungs, tackling the root cause of CF.
+ The discussion also gave us a clearer picture of the global disparities in CF care. It became evident that access to advanced treatments varies a lot around the world. This has made us think about how we can scale our therapy to be more accessible, even in places with fewer resources.
+ The patient’s personal experiences underscored the need for treatments that are tailored to individual needs. We’re using this insight to make sure our therapy can be adapted to different genetic mutations and patient responses.
+ Finally, their emphasis on enzyme development and gene therapy has influenced our research focus. We’re now prioritizing these areas to ensure our work addresses both current needs and future possibilities in CF treatment.
+ Overall, these insights have really helped us align our project with the real-world needs of CF patients, making sure that what we’re developing is both practical and impactful. </p>],
+ implementation: [<p>We incorporated the CF patient’s insights into our project by making several key adjustments to better align with the real-world needs of CF patients. Understanding the challenges they face and the impact of current treatments helped us refine our approach significantly.
+ Firstly, we focused on developing prime editing via inhalation therapy with spray-dried lipid nanoparticles (LNPs). This method directly targets genetic mutations in the lungs, addressing the patient’s need for more effective and less invasive treatments. We’re also fine-tuning the formulation and delivery of our LNPs to ensure they reach lung tissue efficiently while minimizing any discomfort.
+ The feedback on global disparities in CF care highlighted the importance of creating a treatment that is not only effective but also affordable and accessible. We’re considering how to scale our therapy to make it available in regions with limited access to advanced treatments.
+ Additionally, the emphasis on the need for continued advancements in enzyme development and gene therapy has guided us to balance immediate therapeutic benefits with long-term research goals. This ensures that our project addresses both current needs and future possibilities in CF treatment.
+ By integrating these insights, we aim to ensure our project not only advances scientific understanding but also meets the practical needs of CF patients, ultimately leading to more effective and accessible treatments. </p>],
+ summary: "The aim of our outreach to cystic fibrosis (CF) patients was to gain insights into their daily experiences and how recent treatment advancements have affected their lives. Partnering with MukoDino allowed us to distribute a survey widely, as interviews were limited. Through discussions with patients, we learned about the challenges posed by current treatments, emphasizing the need for therapies that simplify management and reduce physical strain. This feedback has driven our focus on developing more efficient inhalation therapies using spray-dried lipid nanoparticles (LNPs) to deliver gene-editing tools directly to the lungs. We also recognized the global disparities in CF care, motivating us to consider scalability and accessibility for those in resource-limited regions. Patient insights underscored the importance of personalized treatments tailored to individual genetic mutations, prompting us to prioritize enzyme development and gene therapy in our research. Overall, these insights have shaped our project to align closely with the real-world needs of CF patients, aiming for impactful and accessible solutions.",
+ months: "September",
+ interview: <>
+ <QaBox q="How did you come up with the name ‘Muko-Dino’?" a="The name ‘Muko-Dino’ has a personal history. At a cystic fibrosis meeting, another patient joked that I, as a 58-year-old cystic fibrosis patient, was a ‘Muko-Dino’. The name alludes to my age, because in the past, cystic fibrosis patients had a much shorter life expectancy. So it was unusual for someone with the disease to still be so active at my age. The name stuck - initially as a joke, but I have since adopted it as a kind of nickname." />
+ <QaBox q="When were you diagnosed with cystic fibrosis?" a="I was diagnosed at the age of six after a sweat test. I had lost a lot of weight in the first year of my life and cystic fibrosis was already suspected, but it took a while before the diagnosis was actually confirmed." />
+ <QaBox q="What were your thoughts and those of your family after the diagnosis?" a="My mum was naturally shocked. When you have a child, you are happy, and then to receive a diagnosis like this is a huge shock. For parents of cystic fibrosis patients, there is always a basic fear because the disease is life-threatening. Today, many parents hope that their child will live to be at least two years old so that they can receive modulators that can greatly improve the symptoms." />
+ <QaBox q="What mutation do you have?" a="I have the Delta 508 mutation in duplicate, i.e. homozygous. This is the most common form of cystic fibrosis mutation and causes the typical symptoms associated with the disease, such as digestive and lung problems." />
+ <QaBox q="What were the first steps after the diagnosis?" a="Back then, in the 1960s, there were hardly any specialised cystic fibrosis outpatient clinics. My paediatrician prescribed me Pankreon, an early enzyme preparation that was supposed to help me with digestion. Nowadays, the process is very different: A child is referred to a specialised outpatient clinic immediately after diagnosis, where they receive comprehensive care. This includes medical care, nutritional counselling, physiotherapy and support with socio-legal issues." />
+ <QaBox q="How has treatment changed over the years?" a="The treatment of cystic fibrosis has improved dramatically over the last few decades. In the past, low-fat diets had to be followed, even with enzymes, which were not as effective as today's preparations. Back then, if you ate something fatty like chocolate or chips, you got diarrhoea. Nowadays, enzymes are much more effective and patients have hardly any dietary restrictions. As far as antibiotics are concerned, resistance is a big issue. That's why doctors change antibiotics regularly to prevent resistance from developing. Modulators have revolutionised the lives of many patients, even if they can trigger psychological side effects in some. But the alternative, frequent pneumonia, is much worse." />
+ <QaBox q="How do you assess the progress made in cystic fibrosis research?" a="The progress is absolutely remarkable. When I was born, the average life expectancy of a cystic fibrosis patient was one to five years. Today we are talking about over 60 years. This development is not only due to medical innovations, but also to better adherence on the part of patients, who follow their therapies more regularly and efficiently." />
+ <QaBox q="Is there anything you would like to see in the future of therapy?" a="Personally, I am almost perfectly happy, as the current modulators have given me an enormously improved quality of life. My wish for the entire cystic fibrosis community is that gene therapy will be further developed. A treatment that addresses the genetic causes of the disease would be a huge breakthrough and it would be ideal if this could be achieved without serious side effects." />
+ <QaBox q="Which areas of research do you think should be strengthened?" a="There are a few areas that I consider to be particularly important. On the one hand, enzymes should be further developed in order to work even better and further minimise side effects. Another major topic is bacteriophage research, which could help patients with multi-resistant germs. Modulators should also be further optimised and, of course, gene therapy must be driven forward in order to find a sustainable solution." />
+ <QaBox q="What role does personal responsibility play in cystic fibrosis?" a="Personal responsibility is extremely important. Anyone living with cystic fibrosis has to deal intensively with their own disease and take responsibility for their health. This means regularly informing yourself about new treatment options and being disciplined about your own treatment. If you don't take care of yourself, you run the risk of not fully utilising the advances in medicine. The healthcare system gives you many options, but you have to actively utilise them. Luck definitely plays a role, but it is not enough on its own. I have been lucky in my medical history because I live in a country with good access to medical care and have been able to benefit from the modulators. But even the best luck doesn't help without initiative and commitment. You have to play an active role in making the most of the opportunities that life offers you." />
+ <QaBox q="What does your daily routine look like?" a="My daily therapy takes about two to three hours. This includes taking enzymes and antibiotics as well as inhalations with saline solution and antibiotics. I also do regular autogenic drainage and stretching exercises to support my lungs. It is important to stick to this routine consistently, as even a few days without therapy can lead to inflammation." />
+ <QaBox q="What is the most important part of your daily routine?" a="It's difficult to single out one part because all aspects work together. If I left out the enzymes, I would only be able to eat salad because my body wouldn't be able to absorb any nutrients. Without the modulators and antibiotics, I would quickly get pneumonia, which means I would have to take weeks off. So regularity is the key to staying healthy in the long term." />
+ <QaBox q="Do you do any sporting activities?" a="Yes, I cycle to work every day and take the stairs to the eighth floor where I have my office. I don't do intensive sports like going to the gym, but I do exercise regularly in my everyday life, which is very important for my health." />
+ <QaBox q="Do you use physiotherapy?" a="I do my own physiotherapy at home as I have learnt the techniques well over the years. I used to visit a physiotherapist regularly, but I no longer need to. Also, there are hardly any specialised physiotherapists for cystic fibrosis patients in my area." />
+ <QaBox q="Which organ affects you the most in your everyday life?" a="Thanks to the modulators, none actually. But if I don't take the enzymes, I can't eat anything, which leads to rapid weight loss. Without the antibiotics and modulators, I would be more susceptible to infections and would quickly develop pneumonia, which would require weeks of recovery." />
+ <QaBox q="Have you or anyone close to you taken part in clinical trials?" a="I personally have not participated in clinical trials, but I encourage other patients to do so, as trials are often the only way to test and develop new treatments." />
+ <QaBox q="What impact do advances in cystic fibrosis research have on patients' lives?" a="Advances in cystic fibrosis research have had an enormous positive impact on patients' lives. In the past, life expectancy at diagnosis was often only a few years. Today, many patients can live into old age. The introduction of modulators that specifically stabilise the disease-causing proteins and improve their function has significantly improved patients' quality of life. Research has also contributed to patients being better informed about their disease and having effective therapies available, leading to better overall disease management." />
+ <QaBox q="How do you assess the differences in the treatment and support of cystic fibrosis patients in Germany compared to other countries?" a="In Europe, especially in Germany, we live at a very high medical level, even if there are challenges such as the shortage of specialists. The difference in prosperity in the USA is greater, but at the same time it is also a driver of innovation. Compared to countries as some third-world countries, where cystic fibrosis patients have to wait in hospitals for appointments between infectious patients, we are complaining at a high level here. There is always room for improvement, but overall we have a brilliant healthcare system." />
+ <QaBox q="Do you feel sufficiently informed by the available sources of information?" a="Yes, the information situation is good. You can find sufficient information from organisations such as the CF Foundation, the CF Trust and Mukoviszidose e.V.. However, you have to get involved yourself and actively seek out the information." />
+ <QaBox q="Is there anything else you would like to tell us?" a="It is important to understand that CF patients are very different, including in terms of their cultural and family background. In addition, surveys and questions should be reviewed in advance by patients or parents to ensure that they are understandable and do not contain unfortunate wording. If you need any help or anything, I would love to help you. Send me the link as soon as the website is ready and I'll give you feedback. I can also circulate the survey in the community. I'm always available if you have any further questions or need support." />
+ </>,
+ pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/hp/zoom-mukodino.webp",
},
{
- title: "",
- vorname: "",
- nachnname: "Psychologinnen",
- job: "",
- affiliation: "",
- pictureurl: pics['placeholder'],
- tag: "Academia",
- heading: "",
+ title: "Dr. Kerstin Landwehr",
+ vorname: "Senior Physician for Pediatrics & Pneumology and",
+ nachnname: "Elena Wiesler",
+ job: "Psychologists in child & adolescent medicine ",
+ affiliation: "at Bethel Hospital in Bielefeld",
+ pictureurl: pics['bethel'],
+ tag: "Medical Professional",
+ heading: "Holistic Care for Cystic Fibrosis: Exploring the Psychological Impact and Role of Gene Therapy",
interviewtabid: "psychol",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
+ language:"de",
+ quote: "Many families are confronted with extremely high therapy and treatment costs. These are often expensive and unavailable everywhere.",
+ quoteVorname: "Elena",
+ quoteNachname: "Wiesler",
+ aimofcontact: [<p>In previous interviews, Max[link] and Thomas[link] shared how the psychological burden of living with cystic fibrosis weighs heavily on patients. Parents Joshua[link] and Julia[link] also emphasized that mental health challenges are a major issue for both patients and their families. This prompted us to delve deeper into the psychological, social, and medical difficulties faced by cystic fibrosis (CF) patients and their support systems. A key goal was to understand how gene therapies are perceived and how they may affect the quality of life for CF patients. We aimed to gather insights from various perspectives—patients, caregivers, and healthcare professionals—to ensure our project aligns with their needs and addresses the most pressing challenges.
+ Given the complexity of these psychological aspects, it was crucial for us to engage with psychologists to gain a professional, expert opinion. We visited the medical professionals at the klinikum bethel. Their input helped us better understand the mental health impacts of CF and the potential emotional adjustments required when integrating gene therapies into treatment plans. This guidance was invaluable in shaping our approach to developing a holistic solution that addresses not only the medical needs but also the emotional well-being of patients and their families. It informed our Integrated Health Program (IHP) strategy, emphasizing the importance of interpersonal relationships, effective communication, and community engagement, extending beyond purely scientific considerations. </p>],
+ insights: [<p>Through our discussions, several valuable insights emerged that have significantly deepened our understanding of the challenges faced by CF patients and their families:
+ CF patients and their families often endure immense psychological strain. Anxiety, depression, and frustration are common, exacerbated by the constant uncertainty about the disease’s progression and the effectiveness of new treatments. The emotional toll is profound—not just due to the physical burden of the illness, but also because of the hope and fear that come with emerging therapies. While new treatments bring promise, they also raise concerns about their potential success and the unknowns that accompany them.
+ There’s a strong sense of optimism regarding gene therapies, as they hold the potential to significantly improve both life expectancy and quality of life for CF patients. Many are eager to embrace these innovations, seeing them as a long-awaited breakthrough. However, this excitement is often mixed with concerns about side effects, the accessibility of these therapies, and their long-term effectiveness. The prospect of such treatments brings hope, but also a degree of scepticism, particularly around whether they will be accessible to all who need them.
+ Psychological support, family counselling, and the involvement of patient communities are essential in helping patients and their families cope with the emotional and mental challenges of both the disease and its treatments. The strength of these support systems can make a profound difference in how well patients navigate the challenges of living with CF, particularly when adapting to new therapies.
+ We also learned that while new therapies are exciting, their success often depends on their practicality. Treatments that are complex, invasive, or burdensome are less likely to be adopted, even if they promise significant benefits. Simplicity, ease of use, and reducing the treatment burden are critical factors in ensuring patients fully engage with and benefit from these therapies.
+ These insights have shaped our approach, reinforcing the importance of considering both the psychological and practical aspects of new gene therapies, to better address the needs of CF patients and their families. </p>],
+ implementation: [<p>These findings directly influenced several key areas of the project. We adapted the project to emphasise ease of use and minimal disruption to patients' daily lives. For example, we focused on developing a therapy delivery system that was as non-invasive as possible. Recognising the mental health challenges, we integrated our project with a simple therapeutic method to reduce the mental burden on patients. We have emphasised transparency in communicating the benefits, risks and expectations of gene therapy to ensure that patients have a realistic understanding of the potential outcomes. This includes working closely with patient organisations to disseminate clear and accurate information. We are actively engaging with CF patient communities and healthcare professionals to gather ongoing feedback and ensure that the project evolves based on real patient experiences and challenges. Therefore we used our survey to gather feedback from patients and their families.
+ By integrating these insights, we aim to create a gene therapy project that addresses not only the medical needs, but also the emotional and practical concerns of CF patients and their families. </p>],
+ summary: "Our project aims to address the psychological and medical challenges faced by cystic fibrosis (CF) patients and their families, particularly regarding gene therapies. We engaged with psychologists and gathered insights from patients and caregivers, revealing significant emotional strain and a mix of optimism and concern about new treatments. Key findings highlighted the importance of psychological support and the practicality of therapies in ensuring patient engagement. In response, we are developing a user-friendly therapy delivery system that minimizes disruption to daily life while emphasizing transparent communication about treatment risks and benefits. Our goal is to create a comprehensive gene therapy solution that meets the medical and emotional needs of CF patients.",
+ months: "September",
+ interview:<>
+ <QaBox q="Which psychological challenges are particularly relevant for cystic fibrosis patients?" a="Psychological problems are often a major issue for cystic fibrosis patients. Many patients experience anxiety and depression, and their parents are also often affected. This is exacerbated by the constant strain and stress associated with the disease. Special attention is therefore paid to psychological support during diagnosis and ongoing treatment. Regular screenings for anxiety and depression as well as the early involvement of parents in the treatment process are central components of care."/>
+ <QaBox q="What significance do the new therapies have for cystic fibrosis patients?" a="New therapies are ‘game changers’ for cystic fibrosis patients, as they significantly improve life expectancy and quality of life. In the past, cystic fibrosis was mainly a paediatric disease with a short life expectancy. Today, new therapies make it possible to significantly extend life expectancy and improve quality of life. Nevertheless, the disease persists, and patients still require comprehensive treatment. Improving quality of life through early and continuous therapy therefore remains of great importance."/>
+ <QaBox q="How is psychological support integrated into regular treatment?" a="Psychological support is an integral part of the treatment of cystic fibrosis. Care is taken to ensure that both patients and their families are supported at an early stage. This includes regular screenings for anxiety and depression, psycho-educational measures and, if necessary, further psychotherapeutic support. The team works on an interdisciplinary basis to ensure that all aspects of patient care are taken into account. If necessary, external help is also arranged."/>
+ <QaBox q="How is co-operation between medical specialists and psychologists improved?" a="The collaboration between medical specialists and psychologists is characterised by short communication channels and close cooperation. Specialists can exchange information quickly and make decisions together. This enables comprehensive and coordinated care for patients. Effective communication channels are already in place and this close co-operation is seen as very positive. Improvements could be achieved through additional time slots for dialogue or expanded resources."/>
+ <QaBox q="How do families react to the news of a serious diagnosis and how important is it that they receive support at an early stage?" a="Families are often shocked at first when they receive the diagnosis. They first have to come to terms with it and process it. Initially, many don't ask for psychological support straight away, although that would be helpful. It would be good if they were informed about all available resources at an early stage, even if they don't want to make use of them straight away."/>
+ <QaBox q="How does access to gene therapy affect the psychological distress of patients and families?" a="Access to gene therapy can have a significant impact on psychological distress. When therapy is effective, families often see great progress and feel relieved. But if there is no suitable therapy, many are stuck with older, less effective treatments, which can lead to frustration and a sense of disadvantage. The difference in quality of life and outlook is huge."/>
+ <QaBox q="How do patients and families feel about gene therapy compared to traditional therapies?" a="The willingness to participate in gene therapies is often high, especially if the existing therapies are not sufficient. There is a great openness to new approaches, even if they are new and possibly not yet fully tested. The hope for progress and improvement is strong, but there are also concerns and uncertainties about new therapies."/>
+ <QaBox q="What psychological challenges can arise following the introduction of new therapies?" a="New therapies not only bring relief, but also challenges. Patients and families have to adapt to a changed reality. Identity crises can occur, especially if the illness has been a big part of life for a long time. The process of adjustment and the possible feelings of alienation from the previous community can cause additional psychological stress."/>
+ <QaBox q="How important is comprehensive information and psychological support in connection with gene therapies?" a="It is extremely important that patients receive comprehensive information and psychological support. People should know what they can expect from the therapy and what adjustments might be necessary. Talking openly about possible disappointments and challenges can help them to cope better with the changes."/>
+ <QaBox q="How does the role of support groups and patient organisations influence confidence in new therapies?" a="Support groups and patient organisations are crucial for confidence in new therapies. If they are actively involved in research and provide transparent information, this strengthens patient confidence. The use of donations and the establishment of registries by such organisations creates trust and shows that there are serious efforts to improve the situation."/>
+ <QaBox q="What are the challenges in adapting therapies to different genetic mutations?" a="Adapting therapies to different genetic mutations is a major challenge. While there has been progress in the treatment of certain mutations such as Delta-F508, we are still at the beginning with others. In the long term, a modular gene therapy that is customised to the specific mutations would be ideal. It will take a lot of work to develop these therapies for all relevant mutations."/>
+ <QaBox q="How do families and patients deal with the rapid feedback on experimental therapies?" a="If you realise that a therapy is not working as expected, this is communicated very quickly. The feedback system is quite effective: either there is cause for euphoria because everything is going well, or there is bad news. This quick feedback is also reassuring because it means you don't have to be in the dark for long. You are simply grateful when you know how the therapy is going, even if it is not having the desired effect."/>
+ <QaBox q="How important is the community for cystic fibrosis sufferers?" a="The cystic fibrosis community is incredibly strong and well connected. That's really impressive. Those affected often have no other point of contact than this community to exchange information. It's a reliable source of valid information, and that's worth its weight in gold. The community is honest and realistic - there is no sugarcoating, the information is direct and well documented."/>
+ <QaBox q="What is the relationship between different specialist disciplines in the treatment of cystic fibrosis?" a="In cystic fibrosis treatment, the specialist disciplines work together as equals. At congresses, all disciplines such as physios, doctors, psychologists and nutritionists are equally represented. Everyone takes each other seriously and there is a strong interest in developing each other further. This is really exciting because it shows that everyone is working together to provide the best care."/>
+ <QaBox q="How do families deal with the challenges of therapy and the financial aspects?" a="Many families are confronted with extremely high therapy and treatment costs. There has been progress and many treatment options in Germany, but these are often expensive and not available everywhere. As a result, some families are forced to leave their home country in order to receive better medical care. This is an enormous burden and shows how unfair the distribution of resources is worldwide."/>
+ <QaBox q="How is medical care for refugees organised?" a="For refugees from countries such as Ukraine or other crisis areas, care is often a challenge. During emergency care, the children are treated as if they were German patients. But when the refugees have to return to their home countries, the therapy often ends, which is an enormous burden for the families. It is difficult for them to prepare for the future when their status is unclear, and they constantly live with the fear of being deported."/>
+ <QaBox q="How much psychological stress is caused by therapies and their implementation?" a="Therapies can be a major psychological burden, even if they have fundamentally positive effects. Regular inhalations, tablets and other treatments are often tedious and require a lot of discipline. Some patients find it extremely challenging to stick to a regular therapy schedule, especially if the therapy does not bring any immediately visible progress in the long term. It is important to be realistic about the burden of therapy, as it can have a major impact on daily life and well-being."/>
+ <QaBox q="How do patients react to new therapies and the associated challenges?" a="Many patients are open to new therapies but implementing them can be a major challenge. If a new therapy doesn't work immediately at first or even has side effects, this can be demotivating. This is particularly difficult if you have been undergoing treatment for a long time and are hoping to make great progress. The path to a better condition is often arduous and not every therapy brings the desired improvement. Nevertheless, it is important to keep going and persevere with the therapy, even if there are hard times."/>
+ <QaBox q="How much of the overall illness is psychological distress, in addition to the physical symptoms and distress from therapies?" a="The psychological part of the burden is difficult to quantify, as it varies greatly from individual to individual and is influenced by many factors. The interaction between psychological stability and physical health is considerable, as psychological stress can impair self-care and thus physical health. At different stages of life, the psychological component can vary. For example, it can increase during puberty and young adulthood. The psychological component is therefore not small and varies depending on the individual situation and phase of life."/>
+ <QaBox q="How is the visibility of the disease assessed through projects such as MukoMove or projects for children?" a="The visibility of the disease through such projects can be helpful in raising awareness. With rare diseases such as cystic fibrosis, the disease often remains abstract if there are no people directly affected nearby. Educational projects such as MukoMove can help children develop a better understanding of the disease, even if the impact is limited if there are no direct points of reference. However, it can be helpful if patients themselves explain their disease in schools or classes, as this provides direct and personal insights."/>
+ <QaBox q="What are important aspects of designing a gene therapy project so that it is viewed positively by cystic fibrosis patients?" a="When designing a gene therapy project, care should be taken to minimise the practical hurdles. The therapy"/>
+ <QaBox q="What tips can be given to improve the accessibility and acceptance of projects or therapies in cystic fibrosis patients? " a="It is important to ensure the accessibility of projects and that they are practical to implement. The burden on patients should be minimised. This includes ensuring that the therapy is not only effective but also as pleasant as possible. In addition, communication about the progress of the therapy should be transparent and understandable to build trust and make it clear to patients how they can benefit from the new developments. "/>
+ </>,
+ pictureurl_aim:"https://static.igem.wiki/teams/5247/integrated-human-practices/on-our-way-to-interview-psychologists.webp",
},
{
- title: "",
- vorname: "",
+ title: "Dr.",
+ vorname: "Makoto",
nachnname: "Saito",
- job: "",
- affiliation: "",
- pictureurl: pics['placeholder'],
+ job: "Postdoc",
+ affiliation: "Broad Institute of MIT and Harvard",
+ pictureurl: pics['saito'],
tag: "Academia",
- heading: "",
+ heading: "Insights and Recommendations from Interview on Protein Engineering",
interviewtabid: "saito",
cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
- },
- {
- title: "M.Sc.",
- vorname: "Benjamin",
- nachnname: "Moorlach",
- job:"",
- affiliation:"",
- pictureurl: pics['moorlach'],
- tag: "Academia",
- heading: "Gathering information about Chitosan coating for RNA protection",
- interviewtabid: "moorlach",
- cardtext: "",
- language: "de",
- quote: "x",
- aimofcontact: [<p>The aim of the contact with Benjamin Willem Moorlach, M.Sc., from the Department of Engineering and Mathematics, was to gain a deeper understanding of how Chitosan could be applied in lipid-based nanoparticles (LNPs) and to explore its potential role in our project. We had several questions focusing on the properties of Chitosan, its advantages and disadvantages, and how it could be integrated into LNPs. Benjamin Moorlach provided extensive insights into Chitosan’s interactions with RNA, its behavior, and how we might leverage it for our formulations. </p>],
- insights: [<p>From our discussion, we gained valuable insights into the unique properties of Chitosan, a cationic polymer with significant potential to stabilize RNA. Notably, Chitosan offers strong protection against RNases, making it highly beneficial for formulations like lipid-based nanoparticles (LNPs). Another key feature is its heat stability, withstanding temperatures up to 121°C, which makes it suitable for processing methods such as spray drying. However, at higher concentrations (0.5% or more), Chitosan can become toxic, suffocating cells and displaying antimicrobial properties. While it differs from PEG and cannot serve as a direct alternative, Chitosan can be a valuable complement, especially in stabilizing RNA within LNPs.
- A critical point Benjamin emphasized is that Chitosan must be in an acidic environment, typically with a pH range of 4 to 6, to remain positively charged. This positive charge is essential for its effective interaction with RNA and successful integration into the LNP system.
- One of the most important attributes of Chitosan is its ability to form complexes with RNA, offering a high degree of protection, which is crucial for the stability of LNP formulations. This characteristic makes Chitosan particularly advantageous in enhancing RNA stability during processes like spray drying. However, incorporating Chitosan directly into the lipid shell of LNPs poses challenges due to its hydrophilic nature and incompatible charge ratios, which prevent its use as an external coating on LNPs. Instead, it is more suitable for forming stable RNA-Chitosan complexes that can be encapsulated within the LNP structure, ensuring improved stability and protection.</p>],
- implementation: [<p>We have integrated the information by primarily using Chitosan as an RNA stabilizer, rather than embedding it directly into the LNP lipid shell. Benjamin suggested forming Chitosan-RNA complexes first and then encapsulating them within LNPs to ensure the RNA remains stable and functional. For this, Chitosan with a low molecular weight (around 5 kDa) is ideal, as it helps produce smaller particles that can be efficiently encapsulated.
- Additionally, Benjamin recommended starting with small-scale tests (about 100 µL) before moving to larger formulations. The ratio of RNA to Chitosan is key to creating negatively charged particles, and a 2:1 ratio should be maintained. We will verify successful encapsulation using microscopic analysis and gel electrophoresis.
- This knowledge has directly shaped our approach to using Chitosan. Our focus is now on forming stable RNA-Chitosan complexes, which can be encapsulated in LNPs. We’ve also learned the importance of optimizing concentrations to prevent aggregation or toxicity while ensuring the particles stay within the desired nanometer range. Microscopy and electrophoresis will now be key methods in our protocol to confirm complete RNA encapsulation within the LNPs. </p>],
- summary: "In summary, the insights from Benjamin’s expertise were crucial in shaping our understanding of how to integrate Chitosan into our LNP formulations. Chitosan’s protective abilities for RNA, along with its heat stability, make it a valuable component in our project. However, its hydrophilic and cationic nature presents challenges for direct integration into LNP lipid shells, so we are focusing on its use as an encapsulation for the RNA. Benjamin’s advice on concentrations, molecular weight, and complex formation gave us a clear path forward, which will be validated through experimental testing. ",
- months: ""
- },
- {
- title: "",
- vorname: "",
- nachnname: "",
- job: "",
- affiliation: "",
- pictureurl: pics['placeholder'],
- tag: "Academia",
- heading: "",
- interviewtabid: "biophysik",
- cardtext: "",
- quote: "",
- aimofcontact: "",
- insights: "",
- implementation: "",
- summary: "",
- months: ""
- },
- {
- vorname: "Collaborations",
- nachnname: "iGEM Team Linköping ",
- pictureurl: pics['placeholder'],
- tag: "Other",
- heading: "Cooperation to create a Lipid Delivery System Handbook",
- interviewtabid: "linköping",
- cardtext: "",
- quoteVorname: "Kaya",
- quoteNachname: "Lange",
- quote: "We were genuinely excited when Linköping University approached us for collaboration. From the very beginning, their ideas resonated with us, and our shared enthusiasm laid a strong foundation for a productive partnership. We're happy to work together, also with the other teams, and explore new possibilities.",
- aimofcontact: [<p>The initial contact for our collaboration came from the iGEM team 2024 of Linköping, Sweden, who approached us with a proposal to create a “Delivery-Based Handbookâ€[link Handbook]. Their goal was to reduce the steep learning curve associated with these technologies by sharing collective knowledge from multiple teams, including ours. We were excited to contribute and help future teams navigate these challenges more easily. The handbook would serve as a valuable tool. </p>],
- insights: [<p>Throughout the collaboration, we gained significant insights, both scientific and collaborative. Initially, our meetings with the Linköping team and other participating teams - Patras, Radboud-University and TERMOSZ-Selye-HUN - were invaluable. These sessions allowed us to exchange ideas and learn how each team planned to use lipid-based delivery systems in their own projects. This mutual sharing of knowledge opened our eyes to new methodologies and potential applications of LNPs and liposomes. We also gained a deeper appreciation for the interdisciplinary nature of these systems. From the challenges of formulating stable particles to optimizing their efficiency in targeting cells, we realized the complexity of the field and how collaboration could help overcome many of these obstacles. By discussing our respective approaches, we were able to pool our expertise, which not only improved our understanding but also ensured that the handbook would be comprehensive and valuable for various iGEM teams, regardless of their specific project focus.
- In summary: </p>,
- <ul>
- <li>Learned different approaches to using LNPs and liposomes in iGEM projects.</li>
- <li>Discovered new methods for optimizing LNPs.</li>
- <li>Recognized challenges in particle stability and targeted delivery.</li>
- <li>Gained appreciation for the interdisciplinary complexity of these systems.</li>
- <li>Focused on documenting work to benefit future iGEM teams.</li>
- </ul>
- ],
- implementation: [<p>The collaboration expanded our understanding of what's possible, inspiring us to consider new ideas for how we might integrate advanced techniques into our nanoparticle systems in future projects. The collaborative process also encouraged us to document our work more thoroughly, ensuring that future iGEM teams could benefit from both our successes and the challenges we encountered along the way. Beyond the technical improvements, the experience taught us the value of teamwork across borders and disciplines. Each team brought a unique perspective, and by working together, we were able to develop a resource that was far greater than the sum of its parts</p>],
- type: "meta",
- summary: "This collaboration with Linköping and the other iGEM teams was an incredibly enriching experience. Together, we developed a “Delivery-Based Handbookâ€[link Handbook] that will serve as a valuable resource for future teams working with LNPs and liposomes. The knowledge we gained not only enhanced our project but also strengthened our sense of community within iGEM. We are excited to present the handbook at the Grand Jamboree, where we will finally meet our collaborators in person and celebrate the culmination of our collective efforts. This partnership has shown us the immense power of collaboration, and we are proud to have been part of such a meaningful initiative.",
- months: "February",
- pictureurl_aim: "https://static.igem.wiki/teams/5247/photos/hp/linkoping-handbook-lnp.webp",
- pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/hp/linkoping-cooperation-photo.png",
- },
- {
- vorname: "GxP course",
- nachnname:"",
- pictureurl: pics['gxpcourse'],
- tag: "Other",
- heading: "Successful participation of a team member in a 5 day GxP course",
- interviewtabid: "gxpcourse",
- cardtext: "",
- quote: "The GXP course was extremely useful as it provided us with important knowledge that supports our entire team in complying with quality standards. This knowledge will help us to organise our processes efficiently and in accordance with regulations in the future.",
- quoteVorname:"Kaya",
- quoteNachname:"Lange",
- aimofcontact: "",
- insights: "",
- implementation: "",
- text: [<p>I, Kaya, Team Member of iGEM Bielefeld 2024, recently participated in an intensive one-week GXP (Good Practice) training course, which was pivotal experience for both me and our project. The course covered essential regulatory frameworks, including</p>,
- <ul>
- <li>Good Laboratory Practice (GLP)</li>
- <li>Good Clinical Practice (GCP)</li>
- <li>Good Manufacturing Practice (GMP)</li>
- </ul>,
- <p>
- which are all designed to ensure quality, safety, and compliance across every phase of scientific research and development.
- As the head of Integrated Human Practices, I found this training particularly valuable. It provided me with a deeper understanding of the rigorous standards that need to be maintained in research, especially concerning ethics, data integrity, and patient safety. I learned how to properly document research processes, ensure the reproducibility of results, and assess and mitigate risks, all while keeping the ethical considerations of our project at the forefront.
- I have acquired the ability to create standard operating procedures (SOPs) that guarantee the transparent and traceable documentation of each stage of the research process. This not only facilitates internal organisation but is also crucial for subsequent approvals and audits by regulatory authorities.
- It is of paramount importance to ensure the reproducibility of our experiments by maintaining accurate protocols and meticulously documenting all variables. This is of particular importance should the intention be to pursue clinical research at a later stage, as the reproducibility of experiments is a crucial factor in the validity of the results.
- I acquired knowledge of techniques for risk assessment, including Failure Mode and Effects Analysis (FMEA). This process enables the identification of potential risks in a project at an early stage, thus facilitating the development of strategies to minimise them. This approach allows us to identify and address potential sources of error before they lead to significant issues.
- This knowledge is crucial as we think about the future of our project, particularly if we aim to move our gene therapy approach for cystic fibrosis closer to clinical trials and real-world applications. My participation in the GXP training has equipped me with the necessary tools to potentially guide our team through the complex regulatory landscape, ensuring our work remains aligned with industry standards and ready for the next steps in development.
- One of the key speakers during the GXP course was Dr. Marcus Berger [LINK INtreview Beerger], whose expertise was invaluable to me and the entire team. After the course, I had the opportunity to ask Dr. Berger some questions, further deepening my understanding of the practical applications of GXP in research. The connection with Dr. Berger has been highly beneficial, as his insights helped shape key aspects of our project’s development and compliance with industry standards. His guidance will continue to be a valuable resource for our team moving forward.
- Through this training, I feel better positioned to contribute to the team’s efforts, ensuring our project adheres to global safety and ethical guidelines. This experience has strengthened our approach and set a solid foundation for future progress, ensuring that our research, public engagement, and potential clinical applications continue to meet the highest regulatory standards. </p>],
- type: "meta",
- summary: "Kaya, a member of the iGEM Bielefeld 2024 team, completed an intensive one-week GXP (Good Practice) training, which covered Good Laboratory Practice (GLP), Good Clinical Practice (GCP), and Good Manufacturing Practice (GMP). The training provided valuable insights into maintaining high standards of quality, safety, and ethics throughout the research process. Kaya learned crucial skills, such as documenting research processes for reproducibility, creating standard operating procedures (SOPs), and conducting risk assessments using techniques like Failure Mode and Effects Analysis (FMEA). This knowledge is essential for advancing their cystic fibrosis gene therapy project toward clinical trials and ensuring compliance with regulatory standards. Dr. Marcus Berger, a key speaker in the course, provided additional guidance, offering valuable insights that will continue to benefit the team.",
+ quote: "X",
+ aimofcontact: "The aim of the interview was to gain deeper insights into the topic of protein engineering, especially with regard to Fanzor (SpuFz) and to get feedback on our existing approaches for possible nickases, as well as for the planned nickase assay. ",
+ insights: "The interview provided the iGEM team with valuable advice regarding their Prime Editing project and especially on their planned nickase assay. Dr Saito gave detailed feedback on technical challenges, especially with protein expression in E. coli, and suggested switching to yeast for better results. He also encouraged the team to plan carefully, given the project's complexity, and offered guidance on future experiments.  ",
+ implementation: "We have adapted our planned nickase assay according to Dr Saito's advice and changed it accordingly to expression of the RNP complex using yeast.",
+ summary: "The interview aimed to gain insights into protein engineering, particularly regarding Fanzor (SpuFz), and to get feedback on potential nickases and a planned nickase assay. Dr. Saito provided valuable advice, suggesting the use of yeast for protein expression over E. coli due to technical challenges and encouraged careful planning. Based on his feedback, the iGEM team has adapted their nickase assay to express the RNP complex in yeast, aligning with Dr. Saito's recommendations.",
months: "august",
- pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/for-wiki-texts/gxp/gxp-course-kaya.webp",
- pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/for-wiki-texts/gxp/gxp-course-group.webp",
+ interview:<>
+ <QaBox q="Are you familiar with iGEM, by the way?" a="Of course, I know it."/>
+ <QaBox q="Did you participate yourself at some point?" a="Unfortunately, I didn't. I belong to an earlier generation. iGEM actually started relatively recently."/>
+ <QaBox q="We thought for the structure of the interview, we would start by giving you a brief overview of our project so far to familiarize you with it. Then, we’ll move on to the questions. Is that okay?" a="Yes, of course, please go ahead."/>
+ <QaBox q="We've been working on this project for more than half a year now. It began because one of our team members has a friend with cystic fibrosis. That got us interested in the topic. We started by investigating how gene editing technologies like CRISPR-Cas9 could be applied to cystic fibrosis. Then, we explored prime editing and considered if it could be used for this disease or adapted for other applications. Initially, we wondered if we could make prime editing more compact, especially since delivery is challenging due to its large complex size. We looked into various delivery methods, including AAVs (Adeno-Associated Viruses). Our first approach was to explore alternative nickases and possibly engineer new ones. That's how we came across your research – Fanzor. We also considered other candidates like CasX. Are you familiar with CasX?" a="Yes, I am."/>
+ <QaBox q="We're also experimenting with changes to the editing complex itself. In addition, we aim to deliver the editing complex using nanoparticles. We chose to focus on the lungs, hoping that targeting this area would reduce the need for AAV viruses, making the delivery less immunogenic and not as limited by size." a="So, in this iGEM project, you're working on both reducing the size of the prime editor and developing nanoparticles for delivery?"/>
+ <QaBox q="Yes, that’s the plan. Before we start with the main questions, how much time do you have? Is half an hour okay?" a="No problem, half an hour is fine."/>
+ <QaBox q="Great! Then, let’s start with the first question. Our approach to modifying the endonuclease FANZOR started with understanding its mechanism. Could we go over this mechanism with you to ensure we understood it correctly?" a="Of course, please go ahead."/>
+ <QaBox q="As we understand it, the FANZOR protein has different domains, including the RuvC and the NUC domain. The RuvC domain cuts the DNA after binding. Is that correct?" a="Actually, we don't call this domain the NUC domain anymore. In the past, about eight years ago, researchers thought it was a nuclease domain, called the 'NUC' domain. However, now we know that this domain itself does not have catalytic activity. We call it the TNB domain, derived from the protein's ancestor, TNPB."/>
+ <QaBox q="I see. Thank you for clarifying. We also noticed that Cas9 has two catalytic domains, which allow for mutation of one or two of them to create a nickase, making single-strand cuts. Is this similar with Cas12 elements?" a="It's a bit more complicated with Cas12. This project, in particular, is very advanced and involves understanding the nuances of these domains. The paper on Cas12 prime editing discusses how mutations can affect functionality. In FANZOR, you might be able to attempt similar mutations, though I haven't personally tried them."/>
+ <QaBox q="That aligns with our thinking. We recently looked at a paper describing the engineering of Cas12a into a nickase. Our approach involves investigating similar patterns in FANZOR. For example, we identified two key amino acids — glutamine and arginine — that appear to interact with the DNA." a="I agree that targeting specific domains is a potential approach. However, altering an enzyme to gain a new function is challenging. The Cas12a paper provides a path forward by showing how certain domains can be mutated to create nickases. Actually, this project is really important and at the forefront of science. Researchers worldwide are working on developing smaller CRISPR-Cas-like prime editors."/>
+ <QaBox q="That’s awesome to hear, thank you for this feedback. We plan to test this concept. One of our ideas is to mutate specific amino acids in the TNB domain of FANZOR to see if it changes its functionality. We have ordered these different versions of FANZOR." a="That’s a reasonable approach. However, be cautious. If the mutation destabilizes the protein, it might not be expressed correctly. But it's worth trying, as the outcome can vary depending on the mutation and the protein."/>
+ <QaBox q="Yes, we are aware of that risk. We’re also planning to use in vitro assays to test our candidates. We designed guide RNAs and will use gel electrophoresis to analyze the results, looking for nicking or double-strand cuts." a="Interesting. Are you planning to purify each candidate protein?"/>
+ <QaBox q="Yes, but we cannot use yeast, so we will try producing the proteins in E. coli and then purify them. We’ll combine them with in vitro-transcribed omega RNA. Do you think that would work?" a="It might not work with E. coli for FANZOR. In our experience, E. coli cannot produce the holoenzyme of FANZOR without its associated RNA. We initially tried E. coli but then switched to yeast, which allowed us to obtain functional protein-RNA complexes."/>
+ <QaBox q="That’s valuable insight. Is the reason E. coli fails because it cannot properly form the protein-RNA complex?" a="Possibly. The exact reason isn’t clear, but we found that only in yeast, where the protein and RNA are co-expressed from their native loci, could we obtain a functional complex. We also tried replicating this setup in E. coli, but it didn't work."/>
+ <QaBox q="This is really helpful information. We will consider switching to yeast. Do you have any advice on how to quickly transition our approach from E. coli to yeast?" a="You can certainly use yeast. It’s not too difficult. You could order the plasmids from Addgene and start culturing yeast. Does your iGEM team have the ability to work with yeast?"/>
+ <QaBox q="Yes, we can. We've just never worked with yeast before because it seemed easier to use E. coli. But it’s good to know that it’s manageable." a="Yes, it is. We used a yeast strain called BCY123, which contains the galactose induction system. For protein induction in yeast, this system is necessary. If you use another yeast strain, make sure it has the capability for galactose induction."/>
+ <QaBox q="Got it, we will consider using Gibson assembly. Once we clone our mutation candidates into your plasmid, we’ll express the RNA-protein complex in yeast and purify it from there. Is that correct?"
+ a="Yes, that's the right approach. The plasmid we used has an MBP-tag for purification, which works better than a His-tag. It avoids the high background that His-tags often introduce." />
+ <QaBox q="Thank you for this advice. We also have a question about the yeast strain you used, BCY123. Is it crucial to use this specific strain, or could we use an alternative as long as it supports galactose induction?"
+ a="In theory, you can use any yeast strain that allows galactose induction. However, I recommend following the working protocol with BCY123 since it has already been proven to work. It’s the safest way to ensure consistency in your experiments." />
+ <QaBox q="Perfect, thank you very much for all your detailed answers! That would be it for the interview, it was a pleasure getting to know you!"
+ a="Thank you! I’ll be in Tokyo at RIKEN, one of the top science institutes in Japan. If you know any German students interested in coming to Japan, please let them know. We have various opportunities for internships or short stays." />
+ <QaBox q="That’s wonderful to hear. Thank you so much for your time and valuable insights."
+ a="My pleasure. I wish you the best of luck with your project. Feel free to reach out anytime. Goodbye!" />
+ </>,
+ pictureurl_interview:"https://static.igem.wiki/teams/5247/integrated-human-practices/saito.webp",
},
{
title: "Dr. rer. nat. ",
@@ -1504,7 +1664,8 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
job: "GXP ",
affiliation: "Expert",
pictureurl: pics['gxpexpert'],
- tag: "Academia",
+ tag: "Industry",
+ language: "de",
heading: "Deep Dive into Good Practise, GxP ",
interviewtabid: "gxpexpert",
cardtext: "",
@@ -1534,6 +1695,70 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="What initial steps do we need to take to analyse the market and prepare for market access?" a="A stakeholder analysis of the market participants and a comparison of the new therapy with the standard therapy are required. Physician networks should be identified and the pricing strategy defined, taking into account the GBA and the health insurance funds."/>
<QaBox q="How do we develop a strategy for the protection of intellectual property and patents?" a="The strategy should include patent applications in the following order: First for the active ingredient and the formulation (product patent), then the manufacturing route as process patent and followed by indication as use patent. Finally, a utility model may also be useful."/>
</>
- }
+ },
+ {
+ vorname: "Physical ",
+ nachnname: "and Biophysical Chemistry ",
+ job: "Working group ",
+ affiliation: "University Bielefeld ",
+ language: "en",
+ pictureurl: pics['physik'],
+ tag: "Academia",
+ heading: "Performance of Experiments for LNP characterization ",
+ interviewtabid: "biophysik",
+ cardtext: "",
+ quote: "x",
+ aimofcontact: [<p>For our project, we collaborated closely with the Physical Chemistry workgroup to properly categorize our lipid nanoparticles (LNPs). We reached out to them to leverage their expertise and ensure that our characterization was thorough and precise. Marco, Uwe, and Yvonne were instrumental in this effort, not only advising us on appropriate characterization methods but also actively assisting us during the experimental process and data analysis. </p>],
+ insights: [<p>We employed several analytical techniques, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential analysis. TEM and SEM allowed us to visualize the structural morphology of the LNPs, providing detailed images to understand their size and shape on the nanometer scale. DLS was used to measure the size distribution of the particles in solution, while the zeta potential analysis gave us insight into the surface charge, which is crucial for understanding stability in suspension. </p>],
+ implementation: [<p>Thanks to the guidance and hands-on support of the Physical Chemistry team, we successfully completed these tests, gaining detailed insights into our LNPs that will be crucial for our project's further development. Their expertise not only streamlined the process but also ensured the reliability and accuracy of our results. Here a sneak peak of the results – take a look at the image of our SORT LNP taken via TEM. </p>],
+ summary: "We collaborated with the Physical Chemistry workgroup to accurately characterize our lipid nanoparticles (LNPs). Their expertise, particularly from Marco, Uwe, and Yvonne, was invaluable in selecting and applying various analytical techniques, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential analysis. This collaboration not only enhanced our understanding of the LNPs' size, shape, and stability but also ensured the reliability of our results. With their guidance, we successfully completed our tests, providing crucial insights for the project's advancement.",
+ months: "September",
+ pictureurl_interview: "https://static.igem.wiki/teams/5247/delivery/cryo-em.webp",
+ pictureurl_implementation: " https://static.igem.wiki/teams/5247/delivery/tem-sort-lnp1.webp",
+ },
+ {
+ title: "M.Sc.",
+ vorname: "Hakan",
+ nachnname: "Soytürk",
+ job: "PhD Student Faculty of Biology / Working Group",
+ affiliation: "University Bielefeld ",
+ language: "en",
+ pictureurl: pics['hakan'],
+ tag: "Academia",
+ heading: "Helping and conducting experiments with yeast cells",
+ interviewtabid: "hakan",
+ cardtext: "",
+ quote: "Just hand me over the strain and the vector, I will try to take care of the rest.",
+ aimofcontact: [<p>After our interview with Makoto Saito[link] we learned, that he was not able to express the SpuFz1 protein in E. coli and recommended we used yeast to produce it. We were provided with a yeast expression strain and a suitable vector to clone the coding sequence into, but we lacked the necessary know-how and the facilities to transform yeast, select for positive transformants and cultivate the yeast. </p>],
+ insights: [<p>Hakan generously agreed to carry out the transformation and prepare potential positive transformants for cultivation for us, leaving only the purification of the proteins from the supernatant for us to do. </p>],
+ implementation: [<p>Hakan performed the transformation of a pPIC9K-n3SpuFz1 construct we created into Yeast. Unfortunately, the first attempt of transformation did not yield any positive clones. However, we value his spontaneous and extensive support as a great contribution to our project. </p>],
+ summary: "We collaborated with the Physical Chemistry workgroup to accurately characterize our lipid nanoparticles (LNPs). Their expertise, particularly from Marco, Uwe, and Yvonne, was invaluable in selecting and applying various analytical techniques, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential analysis. This collaboration not only enhanced our understanding of the LNPs' size, shape, and stability but also ensured the reliability of our results. With their guidance, we successfully completed our tests, providing crucial insights for the project's advancement.",
+ months: "September",
+ },
+ {
+ title: "M.Sc.",
+ vorname: "Benjamin",
+ nachnname: "Moorlach",
+ job:"PhD student Working Group Patel 'Fermentation and Formulation of Biologicals and Chemicals'",
+ affiliation:"at FH Bielefeld",
+ pictureurl: pics['moorlach'],
+ tag: "Academia",
+ heading: "Gathering information about Chitosan coating for RNA protection",
+ interviewtabid: "moorlach",
+ cardtext: "",
+ language: "de",
+ quote: "x",
+ aimofcontact: [<p>The aim of the contact with Benjamin Willem Moorlach, M.Sc., from the Department of Engineering and Mathematics, was to gain a deeper understanding of how Chitosan could be applied in lipid-based nanoparticles (LNPs) and to explore its potential role in our project. We had several questions focusing on the properties of Chitosan, its advantages and disadvantages, and how it could be integrated into LNPs. Benjamin Moorlach provided extensive insights into Chitosan’s interactions with RNA, its behavior, and how we might leverage it for our formulations. </p>],
+ insights: [<p>From our discussion, we gained valuable insights into the unique properties of Chitosan, a cationic polymer with significant potential to stabilize RNA. Notably, Chitosan offers strong protection against RNases, making it highly beneficial for formulations like lipid-based nanoparticles (LNPs). Another key feature is its heat stability, withstanding temperatures up to 121°C, which makes it suitable for processing methods such as spray drying. However, at higher concentrations (0.5% or more), Chitosan can become toxic, suffocating cells and displaying antimicrobial properties. While it differs from PEG and cannot serve as a direct alternative, Chitosan can be a valuable complement, especially in stabilizing RNA within LNPs.
+ A critical point Benjamin emphasized is that Chitosan must be in an acidic environment, typically with a pH range of 4 to 6, to remain positively charged. This positive charge is essential for its effective interaction with RNA and successful integration into the LNP system.
+ One of the most important attributes of Chitosan is its ability to form complexes with RNA, offering a high degree of protection, which is crucial for the stability of LNP formulations. This characteristic makes Chitosan particularly advantageous in enhancing RNA stability during processes like spray drying. However, incorporating Chitosan directly into the lipid shell of LNPs poses challenges due to its hydrophilic nature and incompatible charge ratios, which prevent its use as an external coating on LNPs. Instead, it is more suitable for forming stable RNA-Chitosan complexes that can be encapsulated within the LNP structure, ensuring improved stability and protection.</p>],
+ implementation: [<p>We have integrated the information by primarily using Chitosan as an RNA stabilizer, rather than embedding it directly into the LNP lipid shell. Benjamin suggested forming Chitosan-RNA complexes first and then encapsulating them within LNPs to ensure the RNA remains stable and functional. For this, Chitosan with a low molecular weight (around 5 kDa) is ideal, as it helps produce smaller particles that can be efficiently encapsulated.
+ Additionally, Benjamin recommended starting with small-scale tests (about 100 µL) before moving to larger formulations. The ratio of RNA to Chitosan is key to creating negatively charged particles, and a 2:1 ratio should be maintained. We will verify successful encapsulation using microscopic analysis and gel electrophoresis.
+ This knowledge has directly shaped our approach to using Chitosan. Our focus is now on forming stable RNA-Chitosan complexes, which can be encapsulated in LNPs. We’ve also learned the importance of optimizing concentrations to prevent aggregation or toxicity while ensuring the particles stay within the desired nanometer range. Microscopy and electrophoresis will now be key methods in our protocol to confirm complete RNA encapsulation within the LNPs. </p>],
+ summary: "In summary, the insights from Benjamin’s expertise were crucial in shaping our understanding of how to integrate Chitosan into our LNP formulations. Chitosan’s protective abilities for RNA, along with its heat stability, make it a valuable component in our project. However, its hydrophilic and cationic nature presents challenges for direct integration into LNP lipid shells, so we are focusing on its use as an encapsulation for the RNA. Benjamin’s advice on concentrations, molecular weight, and complex formation gave us a clear path forward, which will be validated through experimental testing. ",
+ months: "september"
+ },
+
+
]
diff --git a/src/data/steckbriefe.ts b/src/data/steckbriefe.ts
index 58c14e439bc8a675c874a2a593ed5b9c3bc76764..de52bb657d282db6ebe739f09acccc5e411bc114 100644
--- a/src/data/steckbriefe.ts
+++ b/src/data/steckbriefe.ts
@@ -586,7 +586,7 @@ export const teammembers: Array<SteckbriefInterface> = [
"The biggest challenge definitely is to handle the neverending amount of tasks, most of which are completely new challenges you have to learn by yourself",
],
favlabmusic: "Technoo",
- funfacts: ["I am to uncreative to submit a fun fact"],
+ funfacts: ["I have been, no joke, dreaming of primers lately"],
islands: [
"a saw",
"a metal pot",
diff --git a/src/pages.ts b/src/pages.ts
index 0fc08e120f5c9b3b553f536e9378fcd6cba943fe..d5ed3061471a96c3dd044621bf12a490e1ab40fd 100644
--- a/src/pages.ts
+++ b/src/pages.ts
@@ -490,7 +490,27 @@ export const NavPages: (Page | PageRef | Folder)[] = [
name: "BFH European MeetUp",
title: "MeetUp",
path: "contribution/?scrollTo=BFH European MeetUp"
- }
+ },
+ {
+ name: "Safety Guideline",
+ title: "Safety Guideline",
+ path: "contribution/?scrollTo=primary-culture-safety-guideline"
+ },
+ {
+ name: "Collaborations",
+ title: "Collaborations",
+ path: "contribution/?scrollTo=collaborations"
+ },
+ {
+ name: "Patient Matters",
+ title: "Patient Matters",
+ path: "contribution/?scrollTo=patient-matters"
+ },
+ {
+ name: "Wiki",
+ title: "Wiki",
+ path: "contribution/?scrollTo=wiki"
+ },
]
},
];
diff --git a/src/sidebars/conS.tsx b/src/sidebars/conS.tsx
index 428699632e6c778459503d6a703e2ab6deb9e83a..10d61ddbf4bf1b161554497da5d58a0c3f6db887 100644
--- a/src/sidebars/conS.tsx
+++ b/src/sidebars/conS.tsx
@@ -12,7 +12,8 @@ export function ConSidebar(){
const tabs = [
{ tab: "BFH European MeetUp", subtabs: ["Aftervideo", "Timeline", "About", "Posters", "Guideline", "Gallery", "Downloads"]},
- {tab: "Safety Guidelines"},
+ {tab: "Safety Guideline"},
{tab: "Collaborations"},
+ {tab: "Patient Matters"},
{tab: "Wiki"}
];
diff --git a/src/sidebars/engS.tsx b/src/sidebars/engS.tsx
index 57e8cd58c04733a048c450737bf2fcc4afd306b8..0f27b51c74e08ae1c1d4b09a371e1438eb102e21 100644
--- a/src/sidebars/engS.tsx
+++ b/src/sidebars/engS.tsx
@@ -6,8 +6,8 @@ import { useNavigation } from "../utils";
export function EngSide(){
- let nums = [ "cyc1", "cyc2", "cyc3", "cyc4", "pe1", "pe2", "pe3", "peg1", "peg2", "peg3", "peg4", "peg5", "peg6", "peg7",
- "nik1", "nik2", "nik3", "nik4", "nik5", "nik6","del1", "del2", "del3", "del4", "del5"]
+ let nums = ["rep1", "rep2", "rep3", "rep4", "rep5", "rep6", "trf1", "trf2", "trf3", "trf4", "pe1", "pe2", "pe3", "peg1", "peg2", "peg3", "peg4", "peg5",
+ "nic1", "nic2", "nic3", "nic4", "nic5", "nic6","del1", "del2", "del3", "del4", "del5"]
useEffect(() => {
window.addEventListener('scroll', handleScroll);
return () => window.removeEventListener('scroll', handleScroll);
@@ -34,10 +34,10 @@ export function EngSide(){
<div className="sticky-top">
<nav className="sidebar" id="eng-sidebar">
<div>
- <a onClick={openThem({it: "our-cycle", scrollToId: "ourcycle"})}>
+ <a onClick={openThem({it: "our-cycle", scrollToId: "our-cycle-header"})}>
<div className="detail-sideitem">
<div id="parent-our-cycle" className="sideitem active-sideitem">
- <summary>Our Cycle</summary>
+ <summary>Our Cycles</summary>
<span id="our-cycle" className="sidesubtab" style={{display: "block"}}>
</span>
@@ -46,23 +46,54 @@ export function EngSide(){
</a>
</div>
<div>
- <a onClick={openThem({it: "proof-of-concept", scrollToId: "Proof of Concept"})}>
+ <a onClick={openThem({it: "reporter", scrollToId: "reporter-header"})}>
<div className="detail-sideitem">
- <div id="parent-proof-of-concept" className="sideitem">
- <summary>Proof Of Concept</summary>
- <span id="proof-of-concept" className="sidesubtab" style={{display: "none"}}>
+ <div id="parent-reporter" className="sideitem">
+ <summary>Reporter System</summary>
+ <span id="reporter" className="sidesubtab" style={{display: "none"}}>
<ul>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-proof-of-concept", scrollToId: "cyc1head"})}>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-reporter", scrollToId: "rep1head"})}>
<span id="subtitle0" className='sideitem'>Iteration 1</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-proof-of-concept", scrollToId: "cyc2head"})}>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-reporter", scrollToId: "rep2head"})}>
<span id="subtitle1" className='sideitem'>Iteration 2</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-proof-of-concept", scrollToId: "cyc3head"})}>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-reporter", scrollToId: "rep3head"})}>
<span id="subtitle2" className='sideitem'>Iteration 3</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-proof-of-concept", scrollToId: "cyc4head"})}>
- <span id="subtitle3" className='sideitem'>Iteration 4 </span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-reporter", scrollToId: "rep4head"})}>
+ <span id="subtitle3" className='sideitem'>Iteration 4</span>
+ </a></li>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-reporter", scrollToId: "rep5head"})}>
+ <span id="subtitle4" className='sideitem'>Iteration 5</span>
+ </a></li>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-reporter", scrollToId: "rep6head"})}>
+ <span id="subtitle5" className='sideitem'>Outlook</span>
+ </a></li>
+ </ul>
+ </span>
+ </div>
+ </div>
+ </a>
+ </div>
+ <div>
+ <a onClick={openThem({it: "transfection", scrollToId: "transfection-header"})}>
+ <div className="detail-sideitem">
+ <div id="parent-transfection" className="sideitem">
+ <summary>Transfection</summary>
+ <span id="transfection" className="sidesubtab" style={{display: "none"}}>
+ <ul>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-transfection", scrollToId: "trf1head"})}>
+ <span id="subtitle6" className='sideitem'>Iteration 1</span>
+ </a></li>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-transfection", scrollToId: "trf2head"})}>
+ <span id="subtitle7" className='sideitem'>Iteration 2</span>
+ </a></li>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-transfection", scrollToId: "trf3head"})}>
+ <span id="subtitle8" className='sideitem'>Iteration 3</span>
+ </a></li>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-transfection", scrollToId: "trf4head"})}>
+ <span id="subtitle9" className='sideitem'>Iteration 4</span>
</a></li>
</ul>
</span>
@@ -71,20 +102,20 @@ export function EngSide(){
</a>
</div>
<div>
- <a onClick={openThem({it: "pe-systems", scrollToId: "PE Systems"})}>
+ <a onClick={openThem({it: "pe-systems", scrollToId: "pe-systems-header"})}>
<div className="detail-sideitem">
<div id="parent-pe-systems" className="sideitem">
<summary>PE Systems</summary>
<span id="pe-systems" className="sidesubtab" style={{display: "none"}}>
<ul>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pe-systems", scrollToId: "pe1head"})}>
- <span id="subtitle4" className='sideitem'>Iteration 1</span>
+ <span id="subtitle10" className='sideitem'>Iteration 1</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pe-systems", scrollToId: "pe2head"})}>
- <span id="subtitle5" className='sideitem'>Iteration 2</span>
+ <span id="subtitle11" className='sideitem'>Iteration 2</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pe-systems", scrollToId: "pe3head"})}>
- <span id="subtitle6" className='sideitem'>Iteration 3</span>
+ <span id="subtitle12" className='sideitem'>Outlook</span>
</a></li>
</ul>
</span>
@@ -93,32 +124,26 @@ export function EngSide(){
</a>
</div>
<div>
- <a onClick={openThem({it: "pegrna", scrollToId: "pegRNA"})}>
+ <a onClick={openThem({it: "pegrna", scrollToId: "pegrna-header"})}>
<div className="detail-sideitem">
<div id="parent-pegrna" className="sideitem">
<summary>pegRNA</summary>
<span id="pegrna" className="sidesubtab" style={{display: "none"}}>
<ul>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg1head"})}>
- <span id="subtitle7" className='sideitem'>Iteration 1</span>
+ <span id="subtitle13" className='sideitem'>Iteration 1</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg2head"})}>
- <span id="subtitle8" className='sideitem'>Iteration 2</span>
+ <span id="subtitle14" className='sideitem'>Iteration 2</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg3head"})}>
- <span id="subtitle9" className='sideitem'>Iteration 3</span>
+ <span id="subtitle15" className='sideitem'>Iteration 3</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg4head"})}>
- <span id="subtitle10" className='sideitem'>Iteration 4</span>
+ <span id="subtitle16" className='sideitem'>Iteration 4</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg5head"})}>
- <span id="subtitle11" className='sideitem'>Iteration 5</span>
- </a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg6head"})}>
- <span id="subtitle12" className='sideitem'>Iteration 6</span>
- </a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-pegrna", scrollToId: "peg7head"})}>
- <span id="subtitle13" className='sideitem'>Iteration 7</span>
+ <span id="subtitle17" className='sideitem'>Iteration 5</span>
</a></li>
</ul>
</span>
@@ -127,29 +152,29 @@ export function EngSide(){
</a>
</div>
<div>
- <a onClick={openThem({it: "nikase", scrollToId: "Nikase"})}>
+ <a onClick={openThem({it: "nickase", scrollToId: "nickase-header"})}>
<div className="detail-sideitem">
- <div id="parent-nikase" className="sideitem">
- <summary>Nikase</summary>
- <span id="nikase" className="sidesubtab" style={{display: "none"}}>
+ <div id="parent-nickase" className="sideitem">
+ <summary>Nickase</summary>
+ <span id="nickase" className="sidesubtab" style={{display: "none"}}>
<ul>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nikase", scrollToId: "nik1head"})}>
- <span id="subtitle14" className='sideitem'>Iteration 1</span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nickase", scrollToId: "nic1head"})}>
+ <span id="subtitle18" className='sideitem'>Iteration 1</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nikase", scrollToId: "nik2head"})}>
- <span id="subtitle15" className='sideitem'>Iteration 2</span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nickase", scrollToId: "nic2head"})}>
+ <span id="subtitle19" className='sideitem'>Iteration 2</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nikase", scrollToId: "nik3head"})}>
- <span id="subtitle16" className='sideitem'>Iteration 3</span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nickase", scrollToId: "nic3head"})}>
+ <span id="subtitle20" className='sideitem'>Iteration 3</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nikase", scrollToId: "nik4head"})}>
- <span id="subtitle17" className='sideitem'>Iteration 4</span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nickase", scrollToId: "nic4head"})}>
+ <span id="subtitle21" className='sideitem'>Iteration 4</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nikase", scrollToId: "nik5head"})}>
- <span id="subtitle18" className='sideitem'>Iteration 5</span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nickase", scrollToId: "nic5head"})}>
+ <span id="subtitle22" className='sideitem'>Iteration 5</span>
</a></li>
- <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nikase", scrollToId: "nik6head"})}>
- <span id="subtitle19" className='sideitem'>Iteration 6</span>
+ <li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-nickase", scrollToId: "nic6head"})}>
+ <span id="subtitle23" className='sideitem'>Iteration 6</span>
</a></li>
</ul>
</span>
@@ -158,26 +183,26 @@ export function EngSide(){
</a>
</div>
<div>
- <a onClick={openThem({it: "delivery", scrollToId: "Delivery"})}>
+ <a onClick={openThem({it: "delivery", scrollToId: "delivery-header"})}>
<div className="detail-sideitem">
<div id="parent-delivery" className="sideitem">
<summary>Delivery</summary>
<span id="delivery" className="sidesubtab" style={{display: "none"}}>
<ul>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-delivery", scrollToId: "del1head"})}>
- <span id="subtitle21" className='sideitem'>Iteration 1</span>
+ <span id="subtitle24" className='sideitem'>Iteration 1</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-delivery", scrollToId: "del2head"})}>
- <span id="subtitle22" className='sideitem'>Iteration 2</span>
+ <span id="subtitle25" className='sideitem'>Iteration 2</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-delivery", scrollToId: "del3head"})}>
- <span id="subtitle23" className='sideitem'>Iteration 3</span>
+ <span id="subtitle26" className='sideitem'>Iteration 3</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-delivery", scrollToId: "del4head"})}>
- <span id="subtitle24" className='sideitem'>Iteration 4</span>
+ <span id="subtitle27" className='sideitem'>Iteration 4</span>
</a></li>
<li><a onClick={() => goToPageWithTabAndScroll({path: "", tabId: "tab-delivery", scrollToId: "del5head"})}>
- <span id="subtitle25" className='sideitem'>Outlook</span>
+ <span id="subtitle28" className='sideitem'>Outlook</span>
</a></li>
</ul>
</span>
diff --git a/src/sources/eng-delivery-sources.tsx b/src/sources/eng-delivery-sources.tsx
index e0ae8003b030bfd6e732e64e892042a4631668db..3f7104b66e4c632b0ba17c4ec43643672ade3525 100644
--- a/src/sources/eng-delivery-sources.tsx
+++ b/src/sources/eng-delivery-sources.tsx
@@ -1,6 +1,6 @@
import BibtexParser from "../components/makeSources";
-export default function Engdeliverysources(){
+export default function EngDelsources(){
return (
<div>
<BibtexParser bibtexSources={bibtexSources} />
diff --git a/src/sources/eng-nickases-sources.tsx b/src/sources/eng-nickases-sources.tsx
index f46c39b0f248ea98bad81d74d53f6f6110550e2e..f3e8228ef1e3e86ba70ef3388af625462a003b03 100644
--- a/src/sources/eng-nickases-sources.tsx
+++ b/src/sources/eng-nickases-sources.tsx
@@ -1,6 +1,6 @@
import BibtexParser from "../components/makeSources";
-export default function Engnickasesources(){
+export default function EngNicksources(){
return (
<div>
<BibtexParser bibtexSources={bibtexSources} />
diff --git a/src/sources/eng-peg-sources.tsx b/src/sources/eng-peg-sources.tsx
index 59fed905dbbc5b3baf8bcc1544cc81120255fb39..d429a543a8e1229ad513ea418f7fd5e341b81eba 100644
--- a/src/sources/eng-peg-sources.tsx
+++ b/src/sources/eng-peg-sources.tsx
@@ -1,6 +1,6 @@
import BibtexParser from "../components/makeSources";
-export default function Engpegsources(){
+export default function EngPegsources(){
return (
<div>
<BibtexParser bibtexSources={bibtexSources} />
diff --git a/src/sources/eng-reporter-sources.tsx b/src/sources/eng-reporter-sources.tsx
new file mode 100644
index 0000000000000000000000000000000000000000..71a37a95eda4a20a607e3b60025d043113ce18aa
--- /dev/null
+++ b/src/sources/eng-reporter-sources.tsx
@@ -0,0 +1,43 @@
+import BibtexParser from "../components/makeSources";
+
+export default function EngRepsources(){
+ return (
+ <div>
+ <BibtexParser bibtexSources={bibtexSources} />
+ </div>
+ );
+}
+
+
+const bibtexSources = [
+`
+@article{Heim_Prasher_Tsien_1994,
+ title = {Wavelength mutations and posttranslational autoxidation of green fluorescent protein.},
+ author = {Heim, R and Prasher, D C and Tsien, R Y},
+ year = 1994,
+ month = dec,
+ journal = {Proceedings of the National Academy of Sciences},
+ publisher = {Proceedings of the National Academy of Sciences},
+ volume = 91,
+ number = 26,
+ pages = {12501–12504},
+ doi = {10.1073/pnas.91.26.12501},
+ abstractnote = {The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a p-hydroxybenzylidene-imidazolidinone chromophore, which is generated by cyclization and oxidation of the protein’s own Ser-Tyr-Gly sequence at positions 65-67. Cloning of the cDNA and heterologous expression of fluorescent protein in a wide variety of organisms indicate that this unique posttranslational modification must be either spontaneous or dependent only on ubiquitous enzymes and reactants. We report that formation of the final fluorophore requires molecular oxygen and proceeds with a time constant (approximately 4 hr at 22 degrees C and atmospheric pO2) independent of dilution, implying that the oxidation does not require enzymes or cofactors. GFP was mutagenized and screened for variants with altered spectra. The most striking mutant fluoresced blue and contained histidine in place of Tyr-66. The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.}
+}
+`,`
+ @article{Simon,
+ title = {PEAR, a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells},
+ author = {Simon, Dorottya Anna and Tálas, András and Kulcsár, Péter István and Biczók, Zsuzsanna and Krausz, Sarah Laura and Várady, György and Welker, Ervin},
+ year = 2022,
+ month = feb,
+ journal = {eLife},
+ publisher = {eLife Sciences Publications, Ltd},
+ volume = 11,
+ pages = {e69504},
+ doi = {10.7554/eLife.69504},
+ issn = {2050-084X},
+ abstractnote = {Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%–30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.},
+ editor = {Lapinaite, Audrone and Stainier, Didier YR and Hamilton, Jennifer R}
+ }
+`
+]
\ No newline at end of file
diff --git a/src/sources/eng-poc-sources.tsx b/src/sources/eng-trf-sources.tsx
similarity index 99%
rename from src/sources/eng-poc-sources.tsx
rename to src/sources/eng-trf-sources.tsx
index c78172cfc1acc6a86bb56068cf642bf73d726ae9..866091d438d9ce97a34834934c39f5507568638d 100644
--- a/src/sources/eng-poc-sources.tsx
+++ b/src/sources/eng-trf-sources.tsx
@@ -1,6 +1,6 @@
import BibtexParser from "../components/makeSources";
-export default function EngPoCsources(){
+export default function EngTrfsources(){
return (
<div>
<BibtexParser bibtexSources={bibtexSources} />
diff --git a/src/sources/joshua-inv-sources.tsx b/src/sources/joshua-inv-sources.tsx
index c99fd02a58a4777d02ffbb66db41e5e28b9ddead..dd4665e5125446fdebb96d9758c77db7ceba911c 100644
--- a/src/sources/joshua-inv-sources.tsx
+++ b/src/sources/joshua-inv-sources.tsx
@@ -32,9 +32,9 @@ doi = {https://doi.org/10.1016/j.xcrm.2024.101544},
url = {https://www.sciencedirect.com/science/article/pii/S2666379124002349},
-author = {Mattijs Bulcaen and Phéline Kortleven and Ronald B. Liu and Giulia Maule and Elise Dreano and Mairead Kelly and Marjolein M. Ensinck and Sam Thierie and Maxime Smits and Matteo Ciciani and Aurelie Hatton and Benoit Chevalier and Anabela S. Ramalho and Xavier {Casadevall i Solvas} and Zeger Debyser and François Vermeulen and Rik Gijsbers and Isabelle Sermet-Gaudelus and Anna Cereseto and Marianne S. Carlon},}
+author = {Mattijs Bulcaen and Phéline Kortleven and Ronald B. Liu and Giulia Maule and Elise Dreano and Mairead Kelly and Marjolein M. Ensinck and Sam Thierie and Maxime Smits and Matteo Ciciani and Aurelie Hatton and Benoit Chevalier and Anabela S. Ramalho and Xavier {Casadevall i Solvas} and Zeger Debyser and François Vermeulen and Rik Gijsbers and Isabelle Sermet-Gaudelus and Anna Cereseto and Marianne S. Carlon},
-`,`
+}`,`
@article{zewi,
author = {Teeratakulpisarn, Jamaree and Kosuwon, Pensri and Srinakarin, Jiraporn and Panthongviriyakul, Charnchai and Sutra, Sumitr},
diff --git a/src/sources/liu-inv-sources.tsx b/src/sources/liu-inv-sources.tsx
new file mode 100644
index 0000000000000000000000000000000000000000..07b47b1207a33294301a46e04c438ac0aa639060
--- /dev/null
+++ b/src/sources/liu-inv-sources.tsx
@@ -0,0 +1,76 @@
+import BibtexParser from "../components/makeSources";
+
+export default function LiuInterviewSources(){
+ return (
+ <div>
+ <BibtexParser bibtexSources={bibtexSources} />
+ </div>
+ );
+}
+
+
+const bibtexSources = [
+`
+@article{eins,
+title = {Search-and-replace genome editing without double-strand breaks or donor DNA},
+volume = {576},
+rights = {2019 The Author(s), under exclusive licence to Springer Nature Limited},
+ISSN = {1476-4687},
+DOI = {10.1038/s41586-019-1711-4},
+abstractNote = {Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few byproducts, the primary genetic causes of sickle cell disease and Tay–Sachs disease; to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.},
+number = {7785},
+journal = {Nature},
+publisher = {Nature Publishing Group},
+author = {Anzalone, Andrew V. and Randolph, Peyton B. and Davis, Jessie R. and Sousa, Alexander A. and Koblan, Luke W. and Levy, Jonathan M. and Chen, Peter J. and Wilson, Christopher and Newby, Gregory A. and Raguram, Aditya and Liu, David R.},
+year = {2019},
+month = {dec},
+pages = {149–157},
+language = {en}
+}`,`
+
+@article{zwei,
+title = {Phage-assisted evolution and protein engineering yield compact, efficient prime editors},
+volume = {186},
+ISSN = {0092-8674, 1097-4172},
+DOI = {10.1016/j.cell.2023.07.039},
+number = {18},
+journal = {Cell},
+publisher = {Elsevier},
+author = {Doman, Jordan L. and Pandey, Smriti and Neugebauer, Monica E. and An, Meirui and Davis, Jessie R. and Randolph, Peyton B. and McElroy, Amber and Gao, Xin D. and Raguram, Aditya and Richter, Michelle F. and Everette, Kelcee A. and Banskota, Samagya and Tian, Kathryn and Tao, Y. Allen and Tolar, Jakub and Osborn, Mark J. and Liu, David R.},
+year = {2023},
+month = {aug},
+pages = {3983-4002.e26},
+language = {English}
+}`,`
+
+@article{drei,
+title = {Phage-assisted continuous and non-continuous evolution},
+volume = {15},
+rights = {2020 The Author(s), under exclusive licence to Springer Nature Limited},
+ISSN = {1750-2799},
+DOI = {10.1038/s41596-020-00410-3},
+abstractNote = {Directed evolution, which applies the principles of Darwinian evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using continuous evolution, which enables the steps of directed evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted continuous evolution (PACE) enables continuous directed evolution in bacteria by mapping the steps of Darwinian evolution onto the bacteriophage life cycle and allows directed evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-continuous evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a continuous flow apparatus and the performance and analysis of evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.},
+number = {12},
+journal = {Nature Protocols},
+publisher = {Nature Publishing Group},
+author = {Miller, Shannon M. and Wang, Tina and Liu, David R.},
+year = {2020},
+month = {dec},
+pages = {4101–4127},
+language = {en}
+}`,`
+@article{vier,
+title = {Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells},
+rights = {2024 The Author(s)},
+ISSN = {2157-846X},
+DOI = {10.1038/s41551-024-01233-3},
+abstractNote = {Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE—engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal ‘dead’ single-guide RNAs—we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.},
+journal = {Nature Biomedical Engineering},
+publisher = {Nature Publishing Group},
+author = {Sousa, Alexander A. and Hemez, Colin and Lei, Lei and Traore, Soumba and Kulhankova, Katarina and Newby, Gregory A. and Doman, Jordan L. and Oye, Keyede and Pandey, Smriti and Karp, Philip H. and McCray, Paul B. and Liu, David R.},
+year = {2024},
+month = {jul},
+pages = {1–15},
+language = {en}
+}`
+]
\ No newline at end of file
diff --git a/src/sources/methods-sources.tsx b/src/sources/methods-sources.tsx
index 3a291e0c01a6023af99fcc52a8b0c86bf3b95887..ecfe2eb453dbace246642ed959b8fb03d2d433b7 100644
--- a/src/sources/methods-sources.tsx
+++ b/src/sources/methods-sources.tsx
@@ -237,6 +237,26 @@ new8.
}
`,
+`@article{ehrhardt_towards_2006,
+ title = {Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line {CFBE41o}-},
+ shorttitle = {Towards an in vitro model of cystic fibrosis small airway epithelium},
+ author = {Ehrhardt, Carsten and Collnot, Eva-Maria and Baldes, Christiane and Becker, Ulrich and Laue, Michael and Kim, Kwang-Jin and Lehr, Claus-Michael},
+ year = 2006,
+ month = mar,
+ journal = {Cell and Tissue Research},
+ volume = 323,
+ number = 3,
+ pages = {405--415},
+ doi = {10.1007/s00441-005-0062-7},
+ issn = {0302-766X, 1432-0878},
+ url = {http://link.springer.com/10.1007/s00441-005-0062-7},
+ urldate = {2024-09-09},
+ copyright = {http://www.springer.com/tdm},
+ language = {en},
+ file = {Ehrhardt et al. - 2006 - Towards an in vitro model of cystic fibrosis small.pdf:C\:\\Users\\Isabell\\Zotero\\storage\\RXLMCE3V\\Ehrhardt et al. - 2006 - Towards an in vitro model of cystic fibrosis small.pdf:application/pdf}
+}
+`
+,
`
@book{Mennella_2024,
title = {Cilia: methods and protocols},