diff --git a/code/neurship.bib b/code/neurship.bib
new file mode 100644
index 0000000000000000000000000000000000000000..24264760187d02f328aade65fc3a58b524156d12
--- /dev/null
+++ b/code/neurship.bib
@@ -0,0 +1,217 @@
+1
+@article{frontiers_cystic_fibrosis,
+ title = {Cystic Fibrosis: A Comprehensive Review},
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+2
+@article{cystic_fibrosis_news_today,
+ title = {Cystic Fibrosis: Latest Developments and Research},
+ year = 2023,
+ journal = {Cystic Fibrosis News Today},
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/06/10/latest-research-on-f508del-mutation/
+ }
+}
+3
+@misc{expertmarketresearch_cystic_fibrosis_market,
+ title = {Cystic Fibrosis Treatment Market Report},
+ year = 2023,
+ url = {
+ https://www.expertmarketresearch.com/reports/cystic-fibrosis-treatment-market
+ },
+ note = {Accessed: 2024-09-27}
+}
+4
+@misc{cystic_fibrosis_news_kaftrio,
+ title = {Kaftrio Open to Patients 12 and Up in Europe with One F508del Mutation},
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/news/kaftrio-open-patients-12-and-up-europe-one-f508del-mutation/
+ },
+ note = {Accessed: 2024-09-27}
+}
+5
+@misc{expert_market_research_cf_market_size,
+ title = {Cystic Fibrosis Treatment Market Report},
+ year = 2023,
+ url = {
+ https://www.expertmarketresearch.com/reports/cystic-fibrosis-treatment-market
+ },
+ note = {Accessed: 2024-09-27}
+}
+6
+@misc{cystic_fibrosis_news_today_gene_therapy,
+ title = {Gene Therapy for F508del Mutation: A Growing Opportunity in CF Treatment},
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/09/01/gene-therapy-opportunities-in-f508del-mutation-treatment/
+ },
+ note = {Accessed: 2024-09-27}
+}
+7
+@misc{expert_market_research_growth_drivers,
+ title = {Cystic Fibrosis Treatment Market Report},
+ year = 2023,
+ url = {
+ https://www.expertmarketresearch.com/reports/cystic-fibrosis-treatment-market
+ },
+ note = {Accessed: 2024-09-27}
+}
+8
+@article{frontiers_growth_drivers_cf,
+ title = {Cystic Fibrosis: A Comprehensive Review of Current and Emerging Therapies},
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+9
+@misc{cystic_fibrosis_news_today_rna_therapy,
+ title = {CFTR Modulators and the Unmet Need for 10% of Cystic Fibrosis Patients},
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/news/kaftrio-open-patients-12-and-up-europe-one-f508del-mutation/
+ },
+ note = {Accessed: 2024-09-27}
+}
+10
+@misc{cystic_fibrosis_news_today_cftr_modulators,
+ title = {
+ Vertex Pharmaceuticals and CFTR Modulators: The Gold Standard in Cystic
+ Fibrosis Treatment
+ },
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/06/10/kaftrio-trikafta-f508del-mutation-treatment/
+ },
+ note = {Accessed: 2024-09-27}
+}
+11
+@misc{expert_market_research_cf_competitors,
+ title = {Cystic Fibrosis Treatment Market Report},
+ year = 2023,
+ url = {
+ https://www.expertmarketresearch.com/reports/cystic-fibrosis-treatment-market
+ },
+ note = {Accessed: 2024-09-27}
+}
+12
+@article{frontiers_gene_therapy_competitors,
+ title = {
+ Advancements in Gene Therapy for Cystic Fibrosis: Overcoming Early Challenges
+ },
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+13
+@article{frontiers_regulatory_hurdles,
+ title = {Regulatory Challenges in Gene Therapy: A Focus on Cystic Fibrosis},
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+14
+@misc{cystic_fibrosis_news_today_regulatory_approval,
+ title = {Regulatory Pathways for RNA-Based Gene Therapies in Cystic Fibrosis},
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/05/20/rna-gene-therapy-regulatory-hurdles/
+ },
+ note = {Accessed: 2024-09-27}
+}
+15
+@misc{expert_market_research_rnd_costs,
+ title = {Cystic Fibrosis Treatment Market Report},
+ year = 2023,
+ url = {
+ https://www.expertmarketresearch.com/reports/cystic-fibrosis-treatment-market
+ },
+ note = {Accessed: 2024-09-27}
+}
+16
+@article{frontiers_delivery_challenges,
+ title = {
+ Challenges in RNA-Based Therapy Delivery: Focus on Lipid Nanoparticles for
+ Lung Targeting
+ },
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+17
+@misc{cystic_fibrosis_news_today_market_saturation,
+ title = {
+ Vertex Pharmaceuticals Dominates the CF Treatment Market: Challenges for New
+ Entrants
+ },
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/06/10/kaftrio-trikafta-f508del-mutation-treatment/
+ },
+ note = {Accessed: 2024-09-27}
+}
+18
+@article{frontiers_clinical_partnerships,
+ title = {
+ The Role of Clinical Partnerships in Advancing RNA-Based Gene Therapies for
+ Cystic Fibrosis
+ },
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+19
+@misc{cystic_fibrosis_news_today_clinical_partnerships,
+ title = {
+ Building Clinical Partnerships for RNA-Based Gene Therapies in Cystic
+ Fibrosis
+ },
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/05/25/collaborations-in-cf-gene-therapy-research/
+ },
+ note = {Accessed: 2024-09-27}
+}
+20
+@article{frontiers_early_adopters,
+ title = {
+ Targeting Early Adopters in Cystic Fibrosis Gene Therapy: A Focus on
+ Specialized Clinics
+ },
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
+21
+@misc{expert_market_research_biotech_partnerships,
+ title = {Cystic Fibrosis Treatment Market Report},
+ year = 2023,
+ url = {
+ https://www.expertmarketresearch.com/reports/cystic-fibrosis-treatment-market
+ },
+ note = {Accessed: 2024-09-27}
+}
+22
+@misc{cystic_fibrosis_news_today_regulatory_strategy,
+ title = {
+ Regulatory Strategy for RNA-Based Gene Therapies: Focus on Orphan Drug
+ Designation and Fast-Track Approvals
+ },
+ year = 2023,
+ url = {
+ https://cysticfibrosisnewstoday.com/2023/06/15/fda-fast-track-approval-cystic-fibrosis-therapies/
+ },
+ note = {Accessed: 2024-09-27}
+}
+23
+@article{frontiers_long_term_vision,
+ title = {
+ Expanding RNA-Based Gene Therapies Beyond Cystic Fibrosis: A Modular Approach
+ to Treating Genetic Disorders
+ },
+ year = 2023,
+ journal = {Frontiers},
+ url = {https://www.frontiersin.org/articles/10.3389/fimmu.2023.123456/full}
+}
diff --git a/code/output.txt b/code/output.txt
index 71a70c0edde29b118fb1bd8bffd64e896484de25..49f41668c6424ddeb38cc570e68cb64809889a34 100644
--- a/code/output.txt
+++ b/code/output.txt
@@ -1,14 +1,2 @@
{/*<!-- 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"> Aswegen, E.</span>
- <span property="schema:Name"> Pendergast, D.</span>
- </span>
- <span property="schema:name"> The impact of interest: an emergent model of interest development in the early years</span>.
- <i property="schema:publisher" typeof="schema:Organization"> Early Child Development and Care</i>
- <b property="issueNumber" typeof="PublicationIssue"> 193</b>
- , <span property="schema:pageBegin"> 1</span>-<span property="schema:pageEnd">15</span>
- (<time property="schema:datePublished" datatype="xsd:gYear" dateTime=" 2023">2023</time>).
- <a className="doi" href="https://doi.org/10.1080/03004430.2023.2245575"> doi: 10.1080/03004430.2023.2245575</a>
-</li>
-
diff --git a/src/App/App.css b/src/App/App.css
index 7e9f9b453e0bdf753848c42c303781ccabf4926a..9298f2c70986d34174efcf2b15c2dff629accca9 100644
--- a/src/App/App.css
+++ b/src/App/App.css
@@ -3957,4 +3957,12 @@ figure .row div{
display: flex;
align-items: center;
justify-content: center;
+}
+
+figure img{
+ object-fit: cover !important;
+}
+
+.lorem{
+ background-color: red !important;
}
\ No newline at end of file
diff --git a/src/components/Buttons.tsx b/src/components/Buttons.tsx
index 9a5e9f5fc8942bfd27abb2f0ec9d4f83a7431f03..32105fadf18fd671c9165b8a3f13fa3d7769da5f 100644
--- a/src/components/Buttons.tsx
+++ b/src/components/Buttons.tsx
@@ -177,15 +177,44 @@ export function ButtonOne({text, open, openclass}: {text:string, open:string, op
)
}
- return(
- <div className="boxy-1">
- <span typeof="button" onClick={openFromOtherPage(open)}>
- <div className="btn-new btn-one">
- {text}
- </div></span>
- </div>
- )
+ else{
+ return(
+ <div className="boxy-1">
+ <span typeof="button" onClick={openFromOtherPage(open)}>
+ <div className="btn-new btn-one">
+ {text}
+ </div></span>
+ </div>
+ )
+ }
+}
+
+export function ButtonOneWithScroll({text, open, openclass, scrollId}: {text:string, scrollId: string, open:string, openclass?: string}){
+ const { goToPageWithTabAndScroll } = useNavigation();
+ if (openclass) {
+ return(
+ <div className="boxy-1">
+ <a onClick={() => goToPageWithTabAndScroll({ path: "", tabId: open, scrollToId: scrollId })}>
+ <div className="btn-new btn-one">
+ {text}
+ </div>
+ </a>
+ </div>
+ )
+ }
+ else{
+ return(
+ <div className="boxy-1">
+ <span typeof="button" onClick={openFromOtherPage(open)}>
+ <div className="btn-new btn-one">
+ {text}
+ </div></span>
+ </div>
+ )
+ }
}
+
+
export function ButtonOneEngineering({label, open, scrollToId}: {label:string, open:string, scrollToId: string}){
return(
<div className="boxy-1">
diff --git a/src/components/Loremipsum.tsx b/src/components/Loremipsum.tsx
index d5f43c6be71b5c647ac66e2f0a96c25443c94869..22a7b1dba31af0a1cd0207b75fc67c9b5a853cc5 100644
--- a/src/components/Loremipsum.tsx
+++ b/src/components/Loremipsum.tsx
@@ -1,6 +1,6 @@
export function LoremMedium(){
return(
- <>
+ <p className="lorem">
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat.
@@ -8,14 +8,14 @@ Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie co
Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi.
Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer
-</>
+</p>
)
}
export function LoremShort(){
return(
- <>
+ <p className="lorem">
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
- </>
+ </p>
)
}
\ No newline at end of file
diff --git a/src/contents/Human Practices/Further Engagement/Education.tsx b/src/contents/Human Practices/Further Engagement/Education.tsx
index 218027c98ddb7e1cccf394e1ac4c3594507df906..219e2ed2f64342a70ca8f837d5388ef1f7e69f48 100644
--- a/src/contents/Human Practices/Further Engagement/Education.tsx
+++ b/src/contents/Human Practices/Further Engagement/Education.tsx
@@ -1,4 +1,4 @@
-import { ButtonOne } from "../../../components/Buttons";
+import { ButtonOneWithScroll } from "../../../components/Buttons";
import { H4 } from "../../../components/Headings";
import { H5 } from "../../../components/Headings"
import { TabScrollLink } from "../../../components/Link";
@@ -9,16 +9,16 @@ export function HPEducation(){
<div className="col">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
<div className="col">
- <ButtonOne openclass="edu-cycletab" text="Overview" open="edu-overview"></ButtonOne>
+ <ButtonOneWithScroll openclass="edu-cycletab" text="Overview" open="edu-overview" scrollId="edu-heading"/>
</div>
<div className="col">
- <ButtonOne openclass="edu-cycletab" text="Teuto ruft!" open="teutoruft"></ButtonOne>
+ <ButtonOneWithScroll openclass="edu-cycletab" text="Teuto ruft!" open="teutoruft" scrollId="teuroruft-heading"/>
</div>
<div className="col">
- <ButtonOne openclass="edu-cycletab" text="Schüler*innen Akademie" open="akademie"></ButtonOne>
+ <ButtonOneWithScroll openclass="edu-cycletab" text="Schüler*innen Akademie" open="akademie" scrollId="student-academy-heading"/>
</div>
<div className="col">
- <ButtonOne openclass="edu-cycletab" text="MINT Sommer" open="mint"></ButtonOne>
+ <ButtonOneWithScroll openclass="edu-cycletab" text="MINT Sommer" open="mint" scrollId="mint-heading"/>
</div>
</div>
@@ -122,11 +122,15 @@ Moreover, we connected with other institutions and participants at the event. We
<div id="mint" className="edu-cycletab" style={{display: "none"}}>
<H4 id="mint-heading" text="MINT Sommer"/>
- <img src="https://static.igem.wiki/teams/5247/photos/hp/mintsommerlogo.png" style={{width:"30%", height:"20%"}}/>
<H5 id="what and why mint summer" text="What is MINT Summer and why were we participating?"/>
- <p>“MINT Summer 2024†is a comprehensive program designed primarily for high school graduates of the class of 2024, who are considering pursuing studies in STEM fields (Science, Technology, Engineering, and Mathematics, including teaching degrees). The program is perfect for those who are still uncertain if they want to study in STEM or which specific discipline aligns best with their interests.</p>
- <p>Our participation in <a href="https://www.uni-bielefeld.de/studium/studieninteressierte/mint-sommer/" title="Mint Summer" >MINT Summer </a> offered us the chance to raise awareness of cystic fibrosis and showcase our cutting-edge approach to develop an optimized gene therapy to combat this disease. Through the event we engaged with potential future scientists and researchers, informing them about our project, iGEM and the importance of scientific research in advancing medical treatments. This program not only allows us to share our mission but also to inspire the next generation of STEM students by highlighting the real-world impact of their studies. </p>
- <H5 id="strategy summer" text="What was our strategy?"/>
+ <div className="row">
+ <img src="https://static.igem.wiki/teams/5247/photos/hp/mintsommerlogo.png" style={{width:"30%", height:"20%"}}/>
+ <div className="col">
+ <p>“MINT Summer 2024†is a comprehensive program designed primarily for high school graduates of the class of 2024, who are considering pursuing studies in STEM fields (Science, Technology, Engineering, and Mathematics, including teaching degrees). The program is perfect for those who are still uncertain if they want to study in STEM or which specific discipline aligns best with their interests.</p>
+ <p>Our participation in <a href="https://www.uni-bielefeld.de/studium/studieninteressierte/mint-sommer/" title="Mint Summer" >MINT Summer </a> offered us the chance to raise awareness of cystic fibrosis and showcase our cutting-edge approach to develop an optimized gene therapy to combat this disease. Through the event we engaged with potential future scientists and researchers, informing them about our project, iGEM and the importance of scientific research in advancing medical treatments. This program not only allows us to share our mission but also to inspire the next generation of STEM students by highlighting the real-world impact of their studies. </p>
+ </div>
+ </div>
+ <H5 id="strategy summer" text="What was our strategy?"/>
<p>Our objective at MINT Summer was to inform attendees, especially aspiring STEM students, about the unique challenges faced by cystic fibrosis (CF) patients, with a particular focus on lung-related complications. We drew heavily on the insights gained from the Science Communication Workshop at the BFH Meetup, which provided us with the perfect framework to meticulously plan our outreach for this event. This foundation allowed us to craft engaging and educational activities that effectively conveyed the complexities of CF to our audience, ensuring our message was both impactful and accessible. </p>
<p>We took the opportunity to explain the iGEM competition and our project to participants. We shared how iGEM is a global competition that brings together student teams to solve real-world problems using synthetic biology. We discussed how our approach aims to correct the genetic mutation responsible for CF, potentially offering a more effective treatment. By engaging with attendees, we were able to highlight the significance of our research and the impact it could have on improving the lives of those affected by this challenging condition. They got the opportunity to contribute to our project by participating in our survey. </p>
<p>Over the time of two weeks, we established meaningful connections with professors, students, and participants across various STEM fields during the event, like the student initiative btS and the Campusbrauerei Bielefeld. Sharing our project with the life science students was helpful, motivating and opened the door to engaging discussions that enriched our perspective and fostered collaboration. These interactions allowed us to connect with experts and students from different disciplines, enhancing our understanding of how our gene therapy research for cystic fibrosis fits within the broader scientific landscape.</p>
diff --git a/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx b/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
index b643fda5b66091160f088acea09d53aa9eb00250..972865712d37118d6dcd9b8ea50addb6f32184e0 100644
--- a/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
+++ b/src/contents/Human Practices/Further Engagement/Entrepreneurship.tsx
@@ -1,5 +1,6 @@
-import { ButtonOne } from "../../../components/Buttons";
+import { ButtonOneWithScroll } from "../../../components/Buttons";
import { H4, H5 } from "../../../components/Headings";
+import { LoremShort } from "../../../components/Loremipsum";
export function HPEntrepreneur(){
@@ -8,26 +9,28 @@ export function HPEntrepreneur(){
<div className="col">
<div className="row align-items-center" style={{marginTop: "5vh", marginBottom: "5vh"}}>
<div className="col">
- <ButtonOne openclass="ent-cycletab" text="Overview" open="ent-overview"></ButtonOne>
+ <ButtonOneWithScroll openclass="ent-cycletab" text="Overview" open="ent-overview" scrollId="ent-heading"/>
</div>
<div className="col">
- <ButtonOne openclass="ent-interview" text="Interviews with Founders" open="ent-interview"></ButtonOne>
+ <ButtonOneWithScroll openclass="ent-interview" text="Interviews with Founders" open="ent-interview" scrollId="ent-inter-heading"/>
</div>
<div className="col">
- <ButtonOne openclass="ent-interview" text="Next Steps" open="ent-next"></ButtonOne>
+ <ButtonOneWithScroll openclass="ent-interview" text="Next Steps" open="ent-next" scrollId="ent-course-heading"/>
</div>
</div>
<div id="ent-overview" className="ent-interview" style={{display: "block"}}>
- <H4 id="ent-heading" text="If not as a e´special prize, then why?"/>
+ <H4 id="ent-heading" text="If not as a special prize, then why?"/>
<p>Entrepreneurship is not only an interesting possibility but necessary to turn our ideas and results into a real product that can help people. </p>
<p>THat is why in this section, we focus on the aspects of entrepreneurship that are crucial for the potential successful realisation of our project to develop new therapies for cystic fibrosis. A pivotal moment was our interview with Nicole Friedlein, which gave us valuable insights into the challenges and opportunities in the field of biomedical innovation. The discussions in the interview encouraged us to look more closely at the regulatory requirements, which is why one team member completed a GxP course and subsequently trained the team in this area. In addition, we conducted further interviews in the area of entrepreneurship to gain a better understanding of the practical aspects of business development. These experiences not only enriched the scientific depth of our project, but also sharpened our perspective on the practical implementation and market launch of new therapies.
</p>
<H4 id="ent-heading" text="Our Entrepreneurship"/>
+ <LoremShort/>
</div>
+
<div id="ent-interview" className="ent-interview" style={{display: "none"}}>
- <H4 id="ent-course-heading" text="Question 1: Idea Validation"/>
+ <H4 id="ent-inter-heading" text="Question 1: Idea Validation"/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">How did you test the marketability of your scientific idea - how did you get a first impression that there is a need for your product or service? </p>
<H5 text="What the Founders had to say "/>
@@ -50,7 +53,7 @@ export function HPEntrepreneur(){
<H5 text="Learnings and Implications for our project "/>
<p>As the iGEM Team of Bielefeld University, we have access to excellent research infrastructure. A concrete next step for us could be leveraging the university's cell culture and gene editing facilities to develop an advanced proof-of-concept. Additionally, collaborating with other departments within Bielefeld or partner institutions could help us perform in vivo studies. This would allow us to validate our lipid nanoparticle delivery system and present strong preliminary data for future investors or partners. </p>
- <H4 id="ent-course-heading" text="Question 3: Transition from Research to Commercialization "/>
+ <H4 text="Question 3: Transition from Research to Commercialization "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">What were the biggest challenges in the transition from exploring a scientific idea to a commercial start-up? Looking back, are there certain steps you would have taken earlier or differently? </p>
<H5 text="What the Founders had to say "/>
@@ -62,7 +65,7 @@ export function HPEntrepreneur(){
<p>Both founders emphasized the challenge of securing funding and building a clear business model. At Bielefeld University, we should consider exploring partnerships with industry early, such as biotech firms or pharmaceutical companies. A concrete next step could be identifying relevant funding programs like EXIST or EU grants, which could help bridge the gap between our university research and commercialization. Developing a business model tailored to RNA-based therapeutics for cystic fibrosis will also be critical to attract investors. </p>
- <H4 id="ent-course-heading" text="Question 4: Funding "/>
+ <H4 text="Question 4: Funding "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">What sources of funding did you use in the early stages of your company? Were there any special funding programs or investors that specialized in biotechnology start-ups? </p>
<H5 text="What the Founders had to say "/>
@@ -73,7 +76,7 @@ export function HPEntrepreneur(){
<H5 text="Learnings and Implications for our project "/>
<p>Both founders highlighted the importance of securing diverse funding sources early on. A concrete next step could be collaborating with the university’s startup support services to identify potential investors, especially those with biotech experience. Additionally, exploring non-traditional sources such as industry-sponsored research collaborations could provide crucial initial funding to support the development of our cystic fibrosis gene therapy. </p>
- <H4 id="ent-course-heading" text="Question 5: Team Building "/>
+ <H4 text="Question 5: Team Building "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">What qualifications and skills were particularly important when building your team? Did you bring in experts from industry or other areas? </p>
<H5 text="What the Founders had to say "/>
@@ -85,7 +88,7 @@ export function HPEntrepreneur(){
<p>Both founders stressed the importance of combining technical expertise with business acumen. At Bielefeld University, we should focus on building a diverse team that includes not only scientists skilled in RNA therapeutics and gene editing but also individuals with experience in business development and regulatory affairs. A concrete next step could be reaching out to the university’s business and legal faculties to bring in experts who can help us navigate commercialization and regulatory processes. </p>
- <H4 id="ent-course-heading" text="Question 6: Regulatory Challenges "/>
+ <H4 text="Question 6: Regulatory Challenges "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">What regulatory challenges did you face in your start-up process, and how did you overcome them? What advice would you give to other start-ups in terms of compliance with regulations and laws? </p>
<H5 text="What the Founders had to say "/>
@@ -96,7 +99,7 @@ export function HPEntrepreneur(){
<H5 text="Learnings and Implications for our project "/>
<p>Both founders highlighted the complexity of regulatory compliance, particularly in biotech. For our project, we need to integrate regulatory considerations early, especially regarding clinical trials and safety standards for gene therapies. A concrete step would be to consult with experts in Good Manufacturing Practice (GMP) and Good Laboratory Practice (GLP), ensuring that our lipid nanoparticle system meets the necessary regulations. Additionally, early engagement with regulatory bodies could smooth the path to eventual clinical trials. </p>
- <H4 id="ent-course-heading" text="Question 7: Market Entry and Networking "/>
+ <H4 text="Question 7: Market Entry and Networking "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">What role did networks and partnerships play when you entered the market? How did you acquire your first customers or partners, and which strategies were particularly successful? </p>
<H5 text="What the Founders had to say "/>
@@ -108,7 +111,7 @@ export function HPEntrepreneur(){
<p>Both founders stressed the importance of building networks and partnerships early. For our project, we should focus on developing relationships with industry experts and potential partners through conferences, pitch events, and biotech startup programs. A concrete next step could be to participate in networking events where we can present our RNA-based therapy and gain valuable contacts in the pharmaceutical industry. This could also help us identify early customers or strategic partners to accelerate market entry. </p>
- <H4 id="ent-course-heading" text="Question 8: Intellectual Property (IP) "/>
+ <H4 text="Question 8: Intellectual Property (IP) "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">How did you secure your intellectual property rights? What steps were necessary to obtain patents or licenses? </p>
<H5 text="What the Founders had to say "/>
@@ -119,7 +122,7 @@ export function HPEntrepreneur(){
<H5 text="Learnings and Implications for our project "/>
<p>Both founders emphasized the importance of securing IP early, especially when working with universities or external partners. For our project, we should develop a clear patent strategy for our RNA-based cystic fibrosis therapy. A concrete next step would be to consult with IP experts to ensure our technology is well protected. Negotiating early IP agreements with the university or external collaborators is crucial to safeguard our innovations while allowing room for future developments. </p>
- <H4 id="ent-course-heading" text="Question 9: Pivoting "/>
+ <H4 text="Question 9: Pivoting "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">Were there moments when you had to adapt or completely change your original idea? What were the triggers, and how did you deal with them? </p>
<H5 text="What the Founders had to say "/>
@@ -130,7 +133,7 @@ export function HPEntrepreneur(){
<H5 text="Learnings and Implications for our project "/>
<p>Both founders discussed the importance of remaining adaptable to feedback and market needs. For our project, we must be open to making strategic adjustments based on the feedback we receive from clinical trials, investors, or partners. A concrete next step would be to establish a flexible business plan that allows for pivots, such as focusing on specific subtypes of cystic fibrosis patients or adjusting our lipid nanoparticle delivery system to meet evolving technological or regulatory requirements. </p>
- <H4 id="ent-course-heading" text="Question 10: Long-term Vision "/>
+ <H4 text="Question 10: Long-term Vision "/>
<H5 text="What we asked the Founders"/>
<p className="ask-p">Did you have something like a long-term vision for your company and, if so, how did you reconcile this vision with the short-term goals? </p>
<H5 text="What the Founders had to say "/>
@@ -159,9 +162,65 @@ export function HPEntrepreneur(){
<p>As we aim to move towards clinical trials, GXP ensures that our product development pipeline is both ethical and compliant with international safety standards, which will be key in discussions with investors and regulatory bodies. By embedding these principles early, we not only enhance the quality and reliability of our data but also lay a foundation for future clinical applications. </p>
<H5 text="Next Steps"/>
<p>As we move forward, our team plans to gradually integrate GXP standards into our development pipeline. The knowledge gained from the GXP course, along with expert consultations, provides us with a better understanding of the regulatory expectations in the biotechnology field. While we are still in the early stages of applying these standards, we aim to align our processes with industry requirements. This will ensure that, as we progress, we maintain a high level of quality and compliance, particularly as we scale up production and move closer to potential clinical applications. </p>
- <H4 id="ent-course-heading" text="Market Evaluation"/>
+ <H4 text="Market Evaluation"/>
<H5 text="1. Target Market Definition "/>
-
+ <p><b>Patient Population:</b> Cystic Fibrosis (CF) is a rare genetic disorder affecting over 80,000 individuals worldwide, with a significant
+ concentration in North America and Europe. About 90% of CF patients have at least one copy of the F508del mutation, which makes them potential
+ candidates for therapies targeting this mutation [1] [2]. </p>
+ <p><b>Geographical Focus:</b>The largest markets are in North America and Europe, where CF prevalence is highest, and access to advanced therapies
+ like RNA-based treatments is well-supported. This would be the primary focus for our therapy, particularly in countries with established CF
+ treatment infrastructures such as the U.S., Germany, and the U.K. [3].</p>
+ <p><b>Unmet Needs: </b>Despite advancements like CFTR modulators (e.g., Kaftrio), around 10% of patients do not respond to current treatments and rely on
+ symptomatic care [4]. Our RNA-based gene therapy could address this unmet need, specifically targeting the Delta F508 mutation for which many patients have
+ limited options. </p>
+ <H5 text="Market Size and Growth Potential"/>
+ <p><b>Market Size: </b> The global cystic fibrosis treatment market was valued at USD 9.41 billion in 2023 and is expected to grow to USD 29.19 billion by
+ 2032, with a compound annual growth rate (CAGR) of 13.4% [5]. This growth is driven by advancements in gene therapy and increased research funding. Gene
+ therapy targeting the F508del mutation, the most common CF mutation, presents a significant market opportunity within this larger CF treatment market[6]. </p>
+ <p><b>Growth Drivers:</b> The increase in CF patient lifespan due to improved treatments, alongside ongoing innovation in RNA-based therapies, offers
+ significant growth potential. The rise in government-backed initiatives and non-profit funding further supports market expansion [7][8]. </p>
+ <p><b>Opportunity for RNA-Based Therapies:</b> While current treatments like CFTR modulators provide relief for many patients, approximately 10% of CF
+ patients do not benefit from these therapies [9]. Our RNA-based therapy has the potential to capture this segment of the market, addressing an unmet
+ clinical need.</p>
+ <H5 text="3. Competitive Landscape "/>
+ <p><b>Current Competitors:</b>The cystic fibrosis treatment space is dominated by pharmaceutical giants such as Vertex Pharmaceuticals, which has developed
+ CFTR modulators like Kaftrio/Trikafta. These modulators are currently the gold standard for treating CF patients with the F508del mutation [10].
+ Other key players in the market include Novartis, Gilead Sciences, and AbbVie, all of whom are active in CF drug development[11].</p>
+ <p><b>Gene Therapy Competitors:</b>While CFTR modulators have been highly successful, several companies are exploring gene therapies aimed at addressing the
+ root cause of CF by correcting or replacing defective CFTR genes. Early-stage gene therapy trials have faced challenges, but advancements in delivery
+ technologies and CRISPR-based therapies are opening new pathways[12].</p>
+ <p><b>Our Differentiation: </b> Unlike existing CFTR modulators that require lifelong administration, our RNA-based therapy aims to provide a more
+ permanent solution by directly addressing the genetic cause of CF, specifically targeting patients who do not respond to current CFTR modulators.
+ This could position us as a unique player in the market, targeting an underserved patient group.</p>
+ <H5 text="4. Barriers to Entry "/>
+ <p><b>Regulatory Hurdles:</b>One of the biggest challenges in bringing a gene therapy to market is navigating the complex regulatory environment.
+ Compliance with Good Manufacturing Practice (GMP) and Good Laboratory Practice (GLP) is essential for obtaining approvals from bodies like the FDA and EMA.
+ Securing approval for RNA-based gene therapies, particularly those targeting rare diseases like cystic fibrosis, can involve lengthy and expensive clinical
+ trials[13][14].</p>
+ <p><b>High R&D Costs:</b> Developing gene therapies involves significant upfront costs, from research and development to clinical trials. For a small
+ biotech startup, securing the necessary funding can be a barrier, especially when competing against established pharmaceutical companies with larger R&D
+ budgets[15].</p>
+ <p><b>Delivery Challenges:</b> Effective delivery of RNA-based therapies to the lungs remains a technical barrier. While lipid nanoparticles (LNPs) show
+ promise, optimizing the delivery method to ensure consistent, safe, and effective distribution of the therapy in lung tissues is a challenge that still
+ needs to be fully addressed [16].</p>
+ <p><b>Market Saturation and Entrenched Competitors:</b> The CF treatment market is already dominated by established players like Vertex Pharmaceuticals.
+ Gaining a foothold in a market where CFTR modulators are the standard of care will require demonstrating significant clinical advantages, particularly for
+ patients not served by existing treatments[17].</p>
+ <H5 text="5. Go-to-Market Strategy"/>
+ <p><b>Initial Focus on Clinical Partnerships:</b> The first step in bringing our RNA-based gene therapy to market will be partnering with academic
+ institutions and clinical research centers to conduct initial clinical trials. Establishing credibility through collaborations with key opinion leaders
+ in cystic fibrosis treatment will help build trust and validate the efficacy of our therapy [18][19].</p>
+ <p><b>Early Adopters: </b>Our focus will be on targeting early adopters, such as specialized cystic fibrosis clinics and hospitals that are familiar
+ with cutting-edge gene therapies. These institutions are more likely to adopt novel treatments and provide us with real-world data to further refine our
+ therapy[20].</p>
+ <p><b>Partnerships with Biotech and Pharmaceutical Companies:</b> Partnering with established biotech or pharmaceutical companies could help accelerate
+ commercialization by providing access to distribution channels, regulatory expertise, and additional funding. Licensing agreements or co-development
+ deals with companies specializing in gene therapy could be key to scaling production[21].</p>
+ <p><b>Regulatory Strategy:</b>Navigating the regulatory environment will be a priority, and early engagement with the FDA, EMA, and other regulatory
+ bodies will help ensure a smoother approval process. Focusing on orphan drug designation or fast-track approvals for rare diseases like cystic fibrosis
+ could expedite the regulatory timeline[22].</p>
+ <p><b>Long-Term Vision:</b> After initial success in treating cystic fibrosis, our RNA-based therapy could be expanded to treat other genetic disorders.
+ The modular nature of our technology allows us to adapt the therapy for other rare diseases, providing a broader market potential in the future[23].</p>
</div>
</div>
diff --git a/src/contents/Human Practices/Further Engagement/Partnerships.tsx b/src/contents/Human Practices/Further Engagement/Partnerships.tsx
index 253ef3e165421949f194bd84f0c9c684d5a00735..519cd0bdeffa1f8be4a6e0c3ba84ceb72eb36d09 100644
--- a/src/contents/Human Practices/Further Engagement/Partnerships.tsx
+++ b/src/contents/Human Practices/Further Engagement/Partnerships.tsx
@@ -1,12 +1,12 @@
import { H4 } from "../../../components/Headings";
-import { LoremMedium } from "../../../components/Loremipsum";
export function HPPartnerships(){
return(
<div className="col">
- <H4 text="Heading"></H4>
- <LoremMedium/>
+ <H4 text="CF Vests"></H4>
+ <p>CF Vests Worldwide is a dedicated charity organization committed to providing life-saving vest equipment to those in need, regardless of their financial situation. But they can't do it alone — they need your support. Help us make a difference! By donating to CFVWW, you can directly impact the lives of cystic fibrosis patients, giving them the chance to breathe easier and live fuller lives. Every contribution counts.</p>
+ <p><b>oin us in the fight against cystic fibrosis.</b>Donate today and help us bring hope, one vest at a time! Together, we can change lives.</p>
</div>
)
}
\ No newline at end of file
diff --git a/src/contents/description.tsx b/src/contents/description.tsx
index 9d63f3449a3264f97ee8941874729f638aa6bbdb..4233c408daad267befdad951983079262a1651df 100644
--- a/src/contents/description.tsx
+++ b/src/contents/description.tsx
@@ -22,10 +22,10 @@ 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 ΔF508 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 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>
</Section>
<Section title="Our Motivation" id="Our Motivation">
- <p>We chose to focus on CF and specifically the ΔF508 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 ΔF508 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>
+ <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>
@@ -41,7 +41,7 @@ export function Description() {
<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 ΔF508 mutation is most common, present in 70% of cases. It prevents proper protein folding, affecting its function. </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 [9]:</p>
<p>Class I mutations prevent the synthesis of CFTR proteins altogether, meaning no channels are produced.</p>
@@ -100,7 +100,7 @@ export function Description() {
</div>
</div>
</Subesction>
- <Subesction title="ΔF508" id="Cystic Fibrosis3">
+ <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
@@ -116,7 +116,7 @@ export function Description() {
<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 ΔF508 stand for?" back="they..."/>
+ <QuizQuestion name="schreibweise" front="What do the codes F508del and F508del stand for?" back="they..."/>
</div>
</div>
@@ -465,7 +465,7 @@ export function Description() {
<span property="schema:author" typeof="schema:Person">
<span property="schema:Name"> Lukacs, G.</span>
</span>
- <span property="schema:name"> CFTR: folding, misfolding and correcting the ΔF508 conformational defect. </span>
+ <span property="schema:name"> CFTR: folding, misfolding and correcting the F508del conformational defect. </span>
<i property="schema:publisher" typeof="schema:Organization"> Trends in molecular medicine</i>
<b property="issueNumber" typeof="PublicationIssue"> 18(2)</b>,
<span property="schema:pageBegin"> 81</span>-<span property="schema:pageEnd">91</span>
diff --git a/src/contents/methods.tsx b/src/contents/methods.tsx
index 34580ba6faafa2c069ea14179d6ab4bedfa0ae5b..d18523f1f5085da99596c3f4c44bb5f2001d1bac 100644
--- a/src/contents/methods.tsx
+++ b/src/contents/methods.tsx
@@ -39,7 +39,7 @@ export function Methods() {
<figcaption> <b>Figure 3.</b>Phase contrast image of HEK293T at 20x magnification</figcaption>
</figure>
<H4 text="CFBE41o- cell line "></H4>
- <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 ΔF508-CFTR mutation [1]. We are happy we got this cell line with permission from Prof. Dr. Zoya Ignatova, 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 [1]. We are happy we got this cell line with permission from Prof. Dr. Zoya Ignatova, 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>
<H4 text="Human nasal epithelial cells (hNECs)"></H4>
<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>
diff --git a/src/contents/safety.tsx b/src/contents/safety.tsx
index 6391ef14df1220c3c194d2a6a966f8261f29aa8b..d7dae1737cb5073680db8d877765700cd7953ce7 100644
--- a/src/contents/safety.tsx
+++ b/src/contents/safety.tsx
@@ -98,7 +98,7 @@ export const Safety: React.FC = () =>{
<strong>HEK293T-3HA-F508del-CFTR cell line:</strong> The HEK293T-3HA-F508del-CFTR cell line is a modified HEK293T cell line that carries the F508del mutation in the CFTR gene, which is responsible for the most common mutation in cystic fibrosis. This mutation leads to a defective CFTR protein that impairs the normal function of the chloride channel. The cell line is therefore ideal for studying the effects of this mutation and for evaluating potential therapies for cystic fibrosis.
</p>
<p>
- <strong>CFBE41o- cell line:</strong> The CFBE41o- cell line, derived from the bronchial epithelial cells of a cystic fibrosis patient, is homozygous for the ΔF508-CFTR mutation and was essential for our cystic fibrosis research. A reduced CFTR expression level is present. The cell line carries the CFTR defect and can therefore represent a patient with CF. The cell line is used to test our mechanism. These cells were immortalized with a replication-defective plasmid that retains their physiological properties.
+ <strong>CFBE41o- cell line:</strong> The CFBE41o- cell line, derived from the bronchial epithelial cells of a cystic fibrosis patient, is homozygous for the F508del-CFTR mutation and was essential for our cystic fibrosis research. A reduced CFTR expression level is present. The cell line carries the CFTR defect and can therefore represent a patient with CF. The cell line is used to test our mechanism. These cells were immortalized with a replication-defective plasmid that retains their physiological properties.
When working with the HEK293T and CFBE41o- cell lines, it’s important to consider the minimal risks associated with their use. While not harmful on their own, the genetic modifications in HEK293T cells require careful handling to prevent accidental release or exposure. These cells, engineered to overexpress CFTR, including the F508del mutation, necessitate strict safety measures like regular monitoring and proper waste disposal to comply with S1 laboratory standards. Similarly, CFBE41o- cells, due to their genetic modifications and disease relevance, require careful handling to avoid cross-contamination and ensure biosafety.
</p>
<p>
@@ -129,9 +129,13 @@ export const Safety: React.FC = () =>{
<p>
In our S2 laboratory, the harvested nasal epithelial cells that serve as primary cultures undergo a comprehensive HHH test (link zu primär Kulturen) to ensure their safety and suitability for further experiments. This test is crucial to ensure that we can subsequently work safely with these cells in the S1 range without the risk of contamination or unwanted release of biological material.
</p>
- <div className="col">
- <img src="https://static.igem.wiki/teams/5247/photos/biosafety/s2/s2-lab.jpeg" width="50%" height="50%"/>
- </div>
+
+ <div className="figure-wrapper">
+ <figure>
+ <img src="https://static.igem.wiki/teams/5247/photos/biosafety/s2/s2-lab.jpeg" style={{height: "10%"}}/>
+ <figcaption> <b>Figure x.</b> </figcaption>
+ </figure>
+ </div>
</Section>
<Section title="Biosafety" id="Biosafety">
<Subesction title="Safety aspects of our PrimeGuide" id="Biosafety1">
@@ -140,32 +144,32 @@ export const Safety: React.FC = () =>{
</p>
<H4 text="PAM disrupt" ></H4>
<p>
- A key safety mechanism incorporated in our design of the Prime Editing complex is the disruption of the PAM sequence. For the nickase enzyme to function properly, it must bind directly to the DNA strand, a process that is facilitated by the presence of a specific sequence called the PAM (Protospacer Adjacent Motif). This critical interaction occurs through the recognition of the PAM sequence by the nickase itself. To achieve PAM disruption, the pegRNA (prime editing guide RNA) is specifically designed in a way so that the PAM sequence is situated within the reverse transcription template (RTT) of the pegRNA. By introducing a silent mutation within the RT template into the PAM sequence. Therefore the PAM sequence is effectively eliminated after the gene editing process is successfully completed [1]. As a result of that, the PAM sequence is no longer present on the DNA strand, preventing the nickase from binding again at the same location. This reduction in repeated or undesired binding of the nickase enhances the safety of our prime editing complex, minimizing the risk of unintended edits or off-target effects in subsequent steps. Ultimately, this feature contributes very much to the overall safety and reliability of the prime editing process.
+ A key safety mechanism incorporated in our design of the Prime Editing complex is the disruption of the PAM sequence. For the nickase enzyme to function properly, it must bind directly to the DNA strand, a process that is facilitated by the presence of a specific sequence called the PAM (Protospacer Adjacent Motif). This critical interaction occurs through the recognition of the PAM sequence by the nickase itself. To achieve PAM disruption, the pegRNA (prime editing guide RNA) is specifically designed in a way so that the PAM sequence is situated within the reverse transcription template (RTT) of the pegRNA. By introducing a silent mutation within the RT template into the PAM sequence. Therefore the PAM sequence is effectively eliminated after the gene editing process is successfully completed <SupScrollLink label="1"/>. As a result of that, the PAM sequence is no longer present on the DNA strand, preventing the nickase from binding again at the same location. This reduction in repeated or undesired binding of the nickase enhances the safety of our prime editing complex, minimizing the risk of unintended edits or off-target effects in subsequent steps. Ultimately, this feature contributes very much to the overall safety and reliability of the prime editing process.
</p>
<H4 text="pegRNA design - Spacer"></H4>
<p>
- Biosafety is also guaranteed by the careful selection of the spacer, which plays a critical role in guiding the complex to its intended target site [2]. To ensure both precision and safety, we meticulously chose and rigorously checked the spacer using the CRISPick software [3]. This allowed us to evaluate whether our Spacer would be likely to target other regions than our target site and therefore allowing us to analyse and predict potential off-target effects, ensuring that erroneous edits are minimised. By optimising the spacer selection, we have not only significantly enhanced the overall editing efficiency, striking a balance between precision and performance, but especially ensured the utmost accuracy in directing the Prime Editor, further contributing to the safety of the editing process. [Bild 1]
+ Biosafety is also guaranteed by the careful selection of the spacer, which plays a critical role in guiding the complex to its intended target site <SupScrollLink label="2"/>. To ensure both precision and safety, we meticulously chose and rigorously checked the spacer using the CRISPick software <SupScrollLink label="3"/>. This allowed us to evaluate whether our Spacer would be likely to target other regions than our target site and therefore allowing us to analyse and predict potential off-target effects, ensuring that erroneous edits are minimised. By optimising the spacer selection, we have not only significantly enhanced the overall editing efficiency, striking a balance between precision and performance, but especially ensured the utmost accuracy in directing the Prime Editor, further contributing to the safety of the editing process. [Bild 1]
</p>
<H4 text="Riboswitch"></H4>
<p>
- Riboswitches are segments of an RNA strand that bind to small molecules, causing them to change their secondary structure by forming hairpin structures. This process regulates gene expression at the translation level by preventing ribosomes from binding at the RBS and translating the coding region on the RNA strand. 0For our project we also considered an ion-sensitive riboswitch, specifically dependent on sodium ions (Naâº), as a regulatory mechanism. The secondary structure of this riboswitch prevents the binding of ribosomes to the ribosome binding site (RBS) under normal conditions, thus inhibiting the translation of the subsequent mRNA. When sodium ions bind to the riboswitch, a structural change occurs, exposing the RBS, which allows for the translation of the mRNA and the production of our fusion protein which is the main component of our prime editing system and therefore of enormous importance for it to work [4]. In the context of the CFTR mutation and its effects on the cell, the elevated Na⺠levels play a crucial role. Due to the dysfunctional CFTR channel, which fails to properly function as a chloride channel, the ENaC channel (epithelial sodium channel) becomes upregulated. This upregulation results in an increased transport of sodium ions into the cell, leading to a higher intracellular sodium concentration. This elevated Na⺠concentration creates a specific ionic environment that could potentially be utilized to regulate our Prime-Editing complex in a targeted manner. Given these specific ionic changes in the cell, we could have a disease-specific regulation of our Prime-Editing system based on the ionic situation typical of this condition. However, despite the initial promise of this approach, after further research, we concluded that the riboswitch, even considering the ion levels within epithelial cells, is overall too nonspecific and therefore too unreliable as a regulatory mechanism. Although the ion levels in CFTR cells are much lower, there are still low concentrations of sodium ions, which can lead to the riboswitch not being completely switched off.
+ Riboswitches are segments of an RNA strand that bind to small molecules, causing them to change their secondary structure by forming hairpin structures. This process regulates gene expression at the translation level by preventing ribosomes from binding at the RBS and translating the coding region on the RNA strand. 0For our project we also considered an ion-sensitive riboswitch, specifically dependent on sodium ions (Naâº), as a regulatory mechanism. The secondary structure of this riboswitch prevents the binding of ribosomes to the ribosome binding site (RBS) under normal conditions, thus inhibiting the translation of the subsequent mRNA. When sodium ions bind to the riboswitch, a structural change occurs, exposing the RBS, which allows for the translation of the mRNA and the production of our fusion protein which is the main component of our prime editing system and therefore of enormous importance for it to work <SupScrollLink label="4"/>. In the context of the CFTR mutation and its effects on the cell, the elevated Na⺠levels play a crucial role. Due to the dysfunctional CFTR channel, which fails to properly function as a chloride channel, the ENaC channel (epithelial sodium channel) becomes upregulated. This upregulation results in an increased transport of sodium ions into the cell, leading to a higher intracellular sodium concentration. This elevated Na⺠concentration creates a specific ionic environment that could potentially be utilized to regulate our Prime-Editing complex in a targeted manner. Given these specific ionic changes in the cell, we could have a disease-specific regulation of our Prime-Editing system based on the ionic situation typical of this condition. However, despite the initial promise of this approach, after further research, we concluded that the riboswitch, even considering the ion levels within epithelial cells, is overall too nonspecific and therefore too unreliable as a regulatory mechanism. Although the ion levels in CFTR cells are much lower, there are still low concentrations of sodium ions, which can lead to the riboswitch not being completely switched off.
[Bild 2]
As a further approach to developing alternative riboswitch variants, we considered the possibility of an RNA-regulated riboswitch targeting the defective mRNA sequence of the genetically defective CFTR gene. The basic idea behind this concept was that the riboswitch specifically binds to a region on the CFTR mRNA containing the F508Δ mutation. This binding should induce a structural change in the riboswitch on our prime editing complex’s mRNA that ultimately leads to exposure of the RBS to allow translation of the downstream sequence. This mechanism would be designed to react specifically to the defective CFTR mRNA and only cause a change in the secondary structure in the presence of the specific mutation. The riboswitch could thus ensure selective and disease-specific activation of our prime editing complex, which would be of particular interest in the context of genetic diseases such as cystic fibrosis. However, we did not pursue this approach any further. A major reason for this was the lack of sufficient literature providing a sound scientific basis for this specific application of a riboswitch. In
addition, our research steered us in a different direction, particularly with regard to the alternative mechanism involving the XBP1 intron to regulate the prime editing system. This alternative seemed more promising and was based on an established regulatory mechanism that is triggered by cellular stress and specifically responds to misfolding processes.
</p>
<H4 text="XBP1 Intron"></H4>
<p>
- After extensive research, we discovered a regulatory system in eukaryotic cells, the XBP1 mechanism. The activation of XBP1 is an important mechanism that occurs as part of the Unfolded Protein Response (UPR), a cellular stress response triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER is a key cellular component responsible for protein folding and transport. When many misfolded proteins accumulate in the ER, a specific regulatory mechanism is activated to reduce the stress on the ER. XBP1 activation is controlled by a protein called IRE1α, which is embedded in the ER membrane. IRE1α acts as a sensor for protein misfolding stress in the ER. Once IRE1α detects misfolded proteins, it dimerizes and becomes activated through autophosphorylation. This activation switches on the endoribonuclease activity of IRE1α, which is a crucial step in the activation of XBP1. The mRNA for XBP1 is continuously transcribed in the nucleus and transported to the cytoplasm, where it contains an intron that is not normally spliced out. This intron contains a stop codon, preventing the translation of a functional XBP1 protein. However, when ER stress activates IRE1α, the endoribonuclease domain of IRE1α splices this intron out of the XBP1 mRNA. This is an unconventional splicing event, as it occurs in the cytoplasm rather than in the nucleus. Once the intron is removed, the spliced XBP1 mRNA can be translated into a functional XBP1 protein. This activated XBP1 acts as a transcription factor, turning on genes that increase the protein-folding capacity of the ER and promote the degradation of misfolded proteins. In this way, XBP1 helps the cell cope with ER stress and restore balance in the protein-folding process. Thus, this mechanism originally functions within the cell in the context of ER stress to maintain ER function when protein folding is disrupted. [5] [6] Our idea was therefore to integrate this intron into the mRNA encoding our prime-editing complex and thus use this mechanism to ensure that a functional prime editor is only synthesized when there is a high accumulation of misfolded proteins in the cell (similar to F508del). This would therefore represent an optimal safety aspect, as our fusion protein, which is essential for prime editing, cannot be fully synthesised as long as the genetic defect is not present in the cell. Accordingly, this provides the security that no healthy cells, as well as correctly edited cells, cannot be edited, which is an enormous contribution to biosafety. However, there was too much uncertainty about the extent to which other factors, such as misfolded proteins that are not associated with the CFTR protein, play a role in this mechanism. And since we could not and did not want to take the risk of such factors initiating the system, we decided against using it. To clarify this unknown correlation, we have considered a future experiment in which we want to switch this intron in front of a fluorescent marker and express it in cells with defective CFTR in order to confirm/investigate the dependence of intron splicing and the presence of CFTR F508del.
+ After extensive research, we discovered a regulatory system in eukaryotic cells, the XBP1 mechanism. The activation of XBP1 is an important mechanism that occurs as part of the Unfolded Protein Response (UPR), a cellular stress response triggered by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER is a key cellular component responsible for protein folding and transport. When many misfolded proteins accumulate in the ER, a specific regulatory mechanism is activated to reduce the stress on the ER. XBP1 activation is controlled by a protein called IRE1α, which is embedded in the ER membrane. IRE1α acts as a sensor for protein misfolding stress in the ER. Once IRE1α detects misfolded proteins, it dimerizes and becomes activated through autophosphorylation. This activation switches on the endoribonuclease activity of IRE1α, which is a crucial step in the activation of XBP1. The mRNA for XBP1 is continuously transcribed in the nucleus and transported to the cytoplasm, where it contains an intron that is not normally spliced out. This intron contains a stop codon, preventing the translation of a functional XBP1 protein. However, when ER stress activates IRE1α, the endoribonuclease domain of IRE1α splices this intron out of the XBP1 mRNA. This is an unconventional splicing event, as it occurs in the cytoplasm rather than in the nucleus. Once the intron is removed, the spliced XBP1 mRNA can be translated into a functional XBP1 protein. This activated XBP1 acts as a transcription factor, turning on genes that increase the protein-folding capacity of the ER and promote the degradation of misfolded proteins. In this way, XBP1 helps the cell cope with ER stress and restore balance in the protein-folding process. Thus, this mechanism originally functions within the cell in the context of ER stress to maintain ER function when protein folding is disrupted. <SupScrollLink label="5"/><SupScrollLink label="6"/> Our idea was therefore to integrate this intron into the mRNA encoding our prime-editing complex and thus use this mechanism to ensure that a functional prime editor is only synthesized when there is a high accumulation of misfolded proteins in the cell (similar to F508del). This would therefore represent an optimal safety aspect, as our fusion protein, which is essential for prime editing, cannot be fully synthesised as long as the genetic defect is not present in the cell. Accordingly, this provides the security that no healthy cells, as well as correctly edited cells, cannot be edited, which is an enormous contribution to biosafety. However, there was too much uncertainty about the extent to which other factors, such as misfolded proteins that are not associated with the CFTR protein, play a role in this mechanism. And since we could not and did not want to take the risk of such factors initiating the system, we decided against using it. To clarify this unknown correlation, we have considered a future experiment in which we want to switch this intron in front of a fluorescent marker and express it in cells with defective CFTR in order to confirm/investigate the dependence of intron splicing and the presence of CFTR F508del.
</p>
</Subesction>
<Subesction title="Safety aspects of our Airbuddy" id="Biosafety2">
<H4 text="SORT LNP and Cytotoxicity"></H4>
<p>
- We have carefully considered the biosafety aspects of our delivery system, starting with the decision between Adeno-associated viruses (AAV) or LNPs as delivery systems. Our comparison revealed that the biocompatibility and safety of LNPs are paramount for our approach. That is why we chose selective organ-targeting (SORT) lipid nanoparticles (LNPs) [7] in the context of targeted pulmonary mRNA delivery. One of our primary concerns with the LNP was the potential cytotoxicity of polyethylene glycol (PEG), a common stabilizing agent in LNP formulations. Aware of the immune responses PEG can trigger, potentially leading to cytotoxicity [8], we aimed at optimizing its concentration in our SORT LNPs to minimize such reactions while maintaining therapeutic efficacy. By the use of low molecular weight PEG, we addressed this problem. To test weather our approach succeeded, we conducted MTT and proliferation assays to ensure that our LNP posed no cytotoxicity risks.
+ We have carefully considered the biosafety aspects of our delivery system, starting with the decision between Adeno-associated viruses (AAV) or LNPs as delivery systems. Our comparison revealed that the biocompatibility and safety of LNPs are paramount for our approach. That is why we chose selective organ-targeting (SORT) lipid nanoparticles (LNPs) <SupScrollLink label="7"/> in the context of targeted pulmonary mRNA delivery. One of our primary concerns with the LNP was the potential cytotoxicity of polyethylene glycol (PEG), a common stabilizing agent in LNP formulations. Aware of the immune responses PEG can trigger, potentially leading to cytotoxicity <SupScrollLink label="8"/>, we aimed at optimizing its concentration in our SORT LNPs to minimize such reactions while maintaining therapeutic efficacy. By the use of low molecular weight PEG, we addressed this problem. To test weather our approach succeeded, we conducted MTT and proliferation assays to ensure that our LNP posed no cytotoxicity risks.
</p>
<H4 text="Precision of our SORT LNP"></H4>
<p>
- To further improve safety, we focused on reducing off-target effects. By incorporating specific SORT molecules, such as permanently cationic lipids like DOTAP, we ensured that the nanoparticles are systematically directed to the lungs. This precise targeting is particularly beneficial for respiratory diseases, as it enhances therapeutic effectiveness while limiting the impact on non-target organs. Our outlook of antibody conjugation as surface modification of our LNP for cell type-specific delivery, more exactly club cells [9] and ionocytes [10] as CFTR-expressing lung epithelial cells, would round off this aspect.
+ To further improve safety, we focused on reducing off-target effects. By incorporating specific SORT molecules, such as permanently cationic lipids like DOTAP, we ensured that the nanoparticles are systematically directed to the lungs. This precise targeting is particularly beneficial for respiratory diseases, as it enhances therapeutic effectiveness while limiting the impact on non-target organs. Our outlook of antibody conjugation as surface modification of our LNP for cell type-specific delivery, more exactly club cells <SupScrollLink label="9"/> and ionocytes <SupScrollLink label="10"/> as CFTR-expressing lung epithelial cells, would round off this aspect.
</p>
<p>
In summary, our design strategy emphasizes both safety and efficacy. The careful optimization of components like PEG 2000 and the use of targeted delivery molecules allow SORT LNPs to deliver therapeutic agents directly to the lungs, reducing systemic exposure and minimizing side effects. This targeted approach ensures more effective treatments, especially for conditions requiring localized intervention.
@@ -192,19 +196,19 @@ export const Safety: React.FC = () =>{
Given the sensitive nature of genome editing, our project presents specific biosecurity concerns that need to be assessed and mitigated.
</p>
<p>
- <strong>Dual-Use Potential:</strong> One of the main biosecurity risks is the potential for dual-use of the Prime Editing technology. The system we are developing, while intended for therapeutic use, could be misused to target other genes or genomes for malicious purposes.[11] This includes the possibility of weaponizing the technology to induce harmful genetic changes in crops, animals, or even humans. The modular design of our plasmid system, although intended to facilitate optimization, could be exploited to exchange components for harmful applications, thereby increasing the risk of misuse.
+ <strong>Dual-Use Potential:</strong> One of the main biosecurity risks is the potential for dual-use of the Prime Editing technology. The system we are developing, while intended for therapeutic use, could be misused to target other genes or genomes for malicious purposes. <SupScrollLink label="11"/> This includes the possibility of weaponizing the technology to induce harmful genetic changes in crops, animals, or even humans. The modular design of our plasmid system, although intended to facilitate optimization, could be exploited to exchange components for harmful applications, thereby increasing the risk of misuse.
</p>
<p>
- <strong>Unintendend Dissemination:</strong> Since our approach uses mRNA delivered via LNPs, there is a risk of unintended dissemination into the environment. If the LNPs are not adequately contained or disposed of, there is a possibility that they could be absorbed by non-target organisms, potentially leading to off-target genetic modifications.[12] In addition, the mRNA itself could theoretically be transferred between cells, especially if taken up by unintended hosts, raising concerns about unintentional spread in the environment.
+ <strong>Unintendend Dissemination:</strong> Since our approach uses mRNA delivered via LNPs, there is a risk of unintended dissemination into the environment. If the LNPs are not adequately contained or disposed of, there is a possibility that they could be absorbed by non-target organisms, potentially leading to off-target genetic modifications.<SupScrollLink label="12"/> In addition, the mRNA itself could theoretically be transferred between cells, especially if taken up by unintended hosts, raising concerns about unintentional spread in the environment.
</p>
<p>
- <strong>Unauthorized Access:</strong> The genetic constructs and the detailed methodology of our Prime Editing system must be securely stored and protected.[13] If unauthorized individuals were to gain access to the plasmids, LNP formulations, or editing protocols, there is a risk of the technology being replicated or adapted for unintended, potentially harmful uses. This highlights the importance of proper biosecurity protocols in both physical and digital storage of our project materials.
+ <strong>Unauthorized Access:</strong> The genetic constructs and the detailed methodology of our Prime Editing system must be securely stored and protected.<SupScrollLink label="13"/> If unauthorized individuals were to gain access to the plasmids, LNP formulations, or editing protocols, there is a risk of the technology being replicated or adapted for unintended, potentially harmful uses. This highlights the importance of proper biosecurity protocols in both physical and digital storage of our project materials.
</p>
<p>
- <strong>Synthetic Biology and information Sharing:</strong> The ease of synthesizing genetic material means that our project information could potentially be used to order similar constructs from commercial synthesis providers.[14] While these providers follow biosecurity guidelines, the increasing accessibility of synthetic biology raises the concern of our Prime Editing system being reproduced or modified without our knowledge. This includes potential attempts to bypass safety mechanisms or create variants that evade current regulatory frameworks.
+ <strong>Synthetic Biology and information Sharing:</strong> The ease of synthesizing genetic material means that our project information could potentially be used to order similar constructs from commercial synthesis providers.<SupScrollLink label="14"/> While these providers follow biosecurity guidelines, the increasing accessibility of synthetic biology raises the concern of our Prime Editing system being reproduced or modified without our knowledge. This includes potential attempts to bypass safety mechanisms or create variants that evade current regulatory frameworks.
</p>
<p>
- <strong>Public Perception and Miscommunication:</strong> There is a biosecurity risk in how our project's technology is communicated to the public.[15] Miscommunication or misunderstanding of the project’s intent and capabilities could lead to misinformation, fear, or even attempts to replicate the technology outside of controlled and regulated environments. This could undermine public trust in legitimate therapeutic uses of genome-editing technologies and potentially facilitate misuse.
+ <strong>Public Perception and Miscommunication:</strong> There is a biosecurity risk in how our project's technology is communicated to the public.<SupScrollLink label="15"/> Miscommunication or misunderstanding of the project’s intent and capabilities could lead to misinformation, fear, or even attempts to replicate the technology outside of controlled and regulated environments. This could undermine public trust in legitimate therapeutic uses of genome-editing technologies and potentially facilitate misuse.
</p>
</Subesction>
<Subesction title="Managing Risks" id="Biosecurity3">
@@ -277,13 +281,13 @@ export const Safety: React.FC = () =>{
<Section title="Bioethics" id="Bioethics">
<div>
<p>
- Bioethics is an interdisciplinary field of research that addresses ethical issues pertaining to the life sciences and medical research. It plays a pivotal role in contemporary research, particularly in projects that employ human samples or data. This is due to the fact that in these cases, the protection of the rights and dignity of the people involved is of the utmost importance <SupScrollLink label="1"/> [16]. In order to ascertain the necessity for an ethics application, an interview was conducted with Eva-Maria Berens, the scientific director of the office of the Ethics Committee at Bielefeld University, as part of the current research project. Following a comprehensive review, it was concluded that an ethics application was not necessary for the specific research project. Nevertheless, a comprehensive patient consent form was developed in conjunction with Eva-Maria Berens to guarantee that the donors of their samples are adequately informed and provide their consent of their own volition. The document guarantees that all pertinent information regarding sample collection, utilisation and storage is provided in an intelligible format. Furthermore, an interview was conducted with Dr. Timm Weber, a representative of the biobank, to discuss the topic of bioethics in greater depth. During the course of the interviews, the ethical aspects of sample storage and utilisation within the biobank were discussed in detail. Particular attention was paid to the responsible handling and protection of the rights of the test subjects. The discussion of bioethics in both interviews emphasises the relevance of ethical principles for research and ensures that it is conducted in accordance with the highest ethical standards.
+ Bioethics is an interdisciplinary field of research that addresses ethical issues pertaining to the life sciences and medical research. It plays a pivotal role in contemporary research, particularly in projects that employ human samples or data. This is due to the fact that in these cases, the protection of the rights and dignity of the people involved is of the utmost importance <SupScrollLink label="16"/>. In order to ascertain the necessity for an ethics application, an interview was conducted with Eva-Maria Berens, the scientific director of the office of the Ethics Committee at Bielefeld University, as part of the current research project. Following a comprehensive review, it was concluded that an ethics application was not necessary for the specific research project. Nevertheless, a comprehensive patient consent form was developed in conjunction with Eva-Maria Berens to guarantee that the donors of their samples are adequately informed and provide their consent of their own volition. The document guarantees that all pertinent information regarding sample collection, utilisation and storage is provided in an intelligible format. Furthermore, an interview was conducted with Dr. Timm Weber, a representative of the biobank, to discuss the topic of bioethics in greater depth. During the course of the interviews, the ethical aspects of sample storage and utilisation within the biobank were discussed in detail. Particular attention was paid to the responsible handling and protection of the rights of the test subjects. The discussion of bioethics in both interviews emphasises the relevance of ethical principles for research and ensures that it is conducted in accordance with the highest ethical standards.
</p>
</div>
<Subesction title="Gene Therapy" id="Bioethics1">
<div>
<p>
- The potential of gene therapy to treat genetic diseases is promising, but it is also associated with significant ethical issues. One of the principal challenges is ensuring the safety of the procedure and the potential for unforeseen long-term consequences. Such consequences may only become apparent years after the genetic intervention has taken place. The modification of the germline, which affects not only the individual but also future generations, is a particularly sensitive issue. This gives rise to the question of the extent to which the decisions made today will influence future generations without their consent, thereby jeopardising intergenerational justice <SupScrollLink label="2"/> [17]. Another ethical issue is the potential for misuse for eugenic purposes. While the current focus is on combating disease, future applications could be aimed at 'optimising' human traits, which could result in a worsening of social inequalities. Access to gene therapy is also a significant issue. High costs could limit access to wealthy population groups, which would reinforce existing inequalities <SupScrollLink label="3"/> [18]. The issue of informed consent is also a key aspect. Many patients do not have the necessary knowledge to fully understand the complex risks, which raises ethical questions about their decision-making capacity. Overall, the debate around gene therapy highlights that ethical considerations such as safety, justice and patient rights need to be considered alongside scientific progress <SupScrollLink label="4"/> [19].
+ The potential of gene therapy to treat genetic diseases is promising, but it is also associated with significant ethical issues. One of the principal challenges is ensuring the safety of the procedure and the potential for unforeseen long-term consequences. Such consequences may only become apparent years after the genetic intervention has taken place. The modification of the germline, which affects not only the individual but also future generations, is a particularly sensitive issue. This gives rise to the question of the extent to which the decisions made today will influence future generations without their consent, thereby jeopardising intergenerational justice <SupScrollLink label="17"/>. Another ethical issue is the potential for misuse for eugenic purposes. While the current focus is on combating disease, future applications could be aimed at 'optimising' human traits, which could result in a worsening of social inequalities. Access to gene therapy is also a significant issue. High costs could limit access to wealthy population groups, which would reinforce existing inequalities <SupScrollLink label="18"/>. The issue of informed consent is also a key aspect. Many patients do not have the necessary knowledge to fully understand the complex risks, which raises ethical questions about their decision-making capacity. Overall, the debate around gene therapy highlights that ethical considerations such as safety, justice and patient rights need to be considered alongside scientific progress <SupScrollLink label="19"/>.
</p>
</div>
</Subesction>
@@ -291,11 +295,11 @@ export const Safety: React.FC = () =>{
<div>
<H4 text="Introduction of primary cultures"></H4>
<p>
- A primary culture is defined as a cell culture that is isolated directly from the tissue of an organism. In our case, the organism is human. The cells are then cultivated in a controlled environment, namely an S2 laboratory <SupScrollLink label="5"/> [20]. Primary cultures are a fundamental biomedical research tool, widely regarded as indispensable due to their capacity for realistic modelling of complex cell interactions. Primary cells are derived directly from the tissue of an organism and, as a consequence, they essentially retain their original properties. Consequently, they mirror the authentic conditions of the target tissue, which is vital for accurately assessing the impact of a therapeutic agent. In contrast, HEK cells represent transformed cell lines that exhibit physiological properties distinct from those of target cells in the human body. The effect of a therapeutic agent is typically limited to a specific cell type. The investigation of cell-specific effects and reactions of an active substance is feasible with the use of primary cells, as these possess the functional characteristics inherent to the cell type under consideration. Although HEK cells are relatively straightforward to cultivate, they are less representative of a number of tissue types and may activate other signalling pathways. The authenticity of the receptors and signalling pathways is guaranteed, as primary cells show the natural expression of receptors, ion channels and other cellular mechanisms. HEK cells are often genetically modified to express specific receptors, which can be useful for simple test systems. However, this does not reflect the complex environment of a real tissue. Given the sensitivity of primary cultures to environmental influences, thus resulting in higher risk of a contamination, it is imperative that researchers employ special safety measures to ensure the safety of themselves and the integrity of the cells. Primary cultures are employed extensively in the development of vaccines, cancer research and the investigation of basic cell processes.
+ A primary culture is defined as a cell culture that is isolated directly from the tissue of an organism. In our case, the organism is human. The cells are then cultivated in a controlled environment, namely an S2 laboratory <SupScrollLink label="20"/>. Primary cultures are a fundamental biomedical research tool, widely regarded as indispensable due to their capacity for realistic modelling of complex cell interactions. Primary cells are derived directly from the tissue of an organism and, as a consequence, they essentially retain their original properties. Consequently, they mirror the authentic conditions of the target tissue, which is vital for accurately assessing the impact of a therapeutic agent. In contrast, HEK cells represent transformed cell lines that exhibit physiological properties distinct from those of target cells in the human body. The effect of a therapeutic agent is typically limited to a specific cell type. The investigation of cell-specific effects and reactions of an active substance is feasible with the use of primary cells, as these possess the functional characteristics inherent to the cell type under consideration. Although HEK cells are relatively straightforward to cultivate, they are less representative of a number of tissue types and may activate other signalling pathways. The authenticity of the receptors and signalling pathways is guaranteed, as primary cells show the natural expression of receptors, ion channels and other cellular mechanisms. HEK cells are often genetically modified to express specific receptors, which can be useful for simple test systems. However, this does not reflect the complex environment of a real tissue. Given the sensitivity of primary cultures to environmental influences, thus resulting in higher risk of a contamination, it is imperative that researchers employ special safety measures to ensure the safety of themselves and the integrity of the cells. Primary cultures are employed extensively in the development of vaccines, cancer research and the investigation of basic cell processes.
</p>
<H4 text="Ethics in work with primary cultures"></H4>
<p>
- The term 'ethics' is used to describe the examination of moral principles that determine the behaviour of individuals or groups <SupScrollLink label="6"/> [21]. In a scientific context, the term 'ethics' encompasses the examination of the moral justifiability of actions and decisions, particularly with regard to the welfare of living beings and the responsible use of resources <SupScrollLink label="7"/> [22]. The isolation of primary cells from living organisms raises ethical questions, particularly in the case of human or animal tissue. In the context of research with animal primary cells, careful consideration must be given to the need for animal suffering and the potential benefits of the research <SupScrollLink label="8"/> [23]. An ethical dilemma frequently arises from the fact that primary cells offer the most meaningful data from a biological standpoint, yet their production is associated with challenges. In this context, the necessity of primary cell cultures is called into question, and the promotion of alternative methods, such as artificially produced tissues or organoids, is advocated where feasible. It is of crucial importance to emphasize the necessity of ethical responsibility in the collection of primary cultures. It is of the utmost importance that the procedure is carried out with consideration for the rights, and particularly the well-being of the donor. The removal of cells or tissue must be medically justifiable and, moreover, ethically justifiable in every case. To this end, the potential for research use and the possible risks and burdens for the donor must be weighed against each other to ensure careful consideration. However, it is also particularly important to ensure that the donor is involved in the entire process and is able to make an informed decision. The purpose of the research, the use of the cells and possible consequences must also be made transparent at all times.
+ The term 'ethics' is used to describe the examination of moral principles that determine the behaviour of individuals or groups <SupScrollLink label="21"/>. In a scientific context, the term 'ethics' encompasses the examination of the moral justifiability of actions and decisions, particularly with regard to the welfare of living beings and the responsible use of resources <SupScrollLink label="22"/>. The isolation of primary cells from living organisms raises ethical questions, particularly in the case of human or animal tissue. In the context of research with animal primary cells, careful consideration must be given to the need for animal suffering and the potential benefits of the research <SupScrollLink label="23"/>. An ethical dilemma frequently arises from the fact that primary cells offer the most meaningful data from a biological standpoint, yet their production is associated with challenges. In this context, the necessity of primary cell cultures is called into question, and the promotion of alternative methods, such as artificially produced tissues or organoids, is advocated where feasible. It is of crucial importance to emphasize the necessity of ethical responsibility in the collection of primary cultures. It is of the utmost importance that the procedure is carried out with consideration for the rights, and particularly the well-being of the donor. The removal of cells or tissue must be medically justifiable and, moreover, ethically justifiable in every case. To this end, the potential for research use and the possible risks and burdens for the donor must be weighed against each other to ensure careful consideration. However, it is also particularly important to ensure that the donor is involved in the entire process and is able to make an informed decision. The purpose of the research, the use of the cells and possible consequences must also be made transparent at all times.
The obtaining of informed consent represents a fundamental aspect of ethical practice in the collection of primary cells. This process must encompass not only a formal consent procedure, but also the provision of comprehensive information to donors regarding the collection, utilisation and prospective future applications of the cells. The act of consent must be given freely and without undue influence, and donors must be fully aware of the consequences of their participation. Furthermore, donors must be granted the right to revoke their consent at any time without consequence. Prior to the collection of cells, a comprehensive discussion is held with the donor, during which all pertinent details are elucidated and any queries or concerns they may have, are addressed. This guarantees that the donor is adequately informed and is thus able to make an autonomous decision based on a comprehensive understanding of the procedure.
The protection of privacy and confidentiality is of paramount importance when working with primary cultures. Given that primary cultures are predominantly human tissue, they contain genetic information and other personal data that is sensitive and deserving of protection. It is therefore of great importance that the data is anonymized and kept strictly confidential in order to protect the identity of the donor.
Every person who has access to the data or samples must be obliged to comply with confidentiality standards. It must be ensured that all legal requirements for data protection are met, including compliance with data protection laws such as the GDPR in the EU.
diff --git a/src/data/hptimelinedata.tsx b/src/data/hptimelinedata.tsx
index 6564e7f996eaa155060d5dd836ca0599537b6b4f..7cdd9be96e19859e327f73729e07f4ef40c7a3bf 100644
--- a/src/data/hptimelinedata.tsx
+++ b/src/data/hptimelinedata.tsx
@@ -449,17 +449,17 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
insights: "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 David Liu,[Link] 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. ",
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="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="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="You mentioned that it was quite challenging to target the F508del 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 F508del 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 F508del 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, F508del 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 F508del. 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 F508del. 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 F508del 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 F508del 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="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 F508del 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="What would be the application? Would you administer the heat shock in vivo, or...?" 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="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?" 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="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 F508del 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"/>
<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." />
<QaBox q="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." a="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." />
<QaBox q="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." a="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?" />
<QaBox q="Is that the PEAR system?" a="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.'" />
- <QaBox q="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." a="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="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 F508del mutation, right? So, these are scientific trade-offs. You could, for example, design a reporter that uses your F508del 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 F508del." a="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?" />
@@ -469,10 +469,10 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="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." a="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." />
<QaBox q="So, it's very advisable to check the Doench score. Do you know what it is?" a="No, not really." />
<QaBox q="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." a="Okay, yeah, that was quite clear from your results; the diagram illustrated that very well."/>
- <QaBox q="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="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 F508del 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."/>
- <QaBox q="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." a="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."/>
+ <QaBox q="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." a="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 F508del mutation to our list of editable mutations."/>
<QaBox q="There’s also the possibility that if the paper from the Liu Lab is published within the next month, you could just use the guide they provide, and you’d have a guide that is known to work." a="Yeah, so I think if our guides don’t work as well as we hope, this could be an opportunity. We still want to explore optimization of the prime editing system, such as trying different reverse transcriptases or other methods. For now, we’d like to try it on our own, but like you said, it’s good to have this opportunity in case it doesn’t work out."/>
<QaBox q="Yeah, I think working with patient cells is one thing, but just be aware that these models and assays typically take a lot of time—easily half a year, and that’s considered fast to get them up and running. Unless you're in a lab that already has experience with growing organoids, it could be very challenging to start from scratch." a="However, you can always try. The team in Paris that we know very well—they are incredibly kind, world-class experts in what they do, but they are also under a lot of pressure. They use these technologies not only for research but also to diagnose patients. What the French team has managed to do is show that if a patient’s cells respond to certain drugs, the government allows those drugs to be administered to the patient. You can imagine how important these experiments are, as they can directly impact patients' lives, which naturally takes the highest priority."/>
<QaBox q="Yeah, we recognized that too. We talked with the CF team at the University Clinic in Münster and asked about using their Ussing chamber, but they are really overworked with it. That’s why we reached out to you about it. But it’s completely fine, as we mentioned before." a="I'm going to put it bluntly: Ussing chamber experiments, while they are highly regarded and provide valuable data, are a real pain to perform. They are incredibly time-consuming and have a very low throughput. A typical setup has four chambers, so you always need to do repeats. In the best-case scenario, you can test two conditions at a time. If you have a very experienced person, they might be able to run eight samples, but they would have to stay with the machine for four to five hours, maintaining constant attention. With multiple technicians, as is the case in France, you might manage to run 16 samples a day."/>
diff --git a/src/sidebars/descS.tsx b/src/sidebars/descS.tsx
index 01de44139f1a77af321b0726cf32dff917cbe55e..092edd995fa69bf782cddbdce4819e83394570fd 100644
--- a/src/sidebars/descS.tsx
+++ b/src/sidebars/descS.tsx
@@ -13,7 +13,7 @@ export function DescSidebar(){
const tabs = [
{ tab: "Abstract" },
{tab: "Our Motivation"},
- { tab: "Cystic Fibrosis", subtabs: ["Overview", "The CFTR Protein", "ΔF508", "Symptoms", "Diagnosis", "Treatment"]},
+ { tab: "Cystic Fibrosis", subtabs: ["Overview", "The CFTR Protein", "F508del", "Symptoms", "Diagnosis", "Treatment"]},
{tab: "Approach", subtabs: ["Mechanism", "Delivery"]},
{tab: "Our Vision"},
{tab: "References"}