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import { ScrollLinkWithChild } from "../components/ScrollLink";
import JoshuaInterviewSources from "../sources/joshua-inv-sources";
import MattijsInterviewSources from "../sources/mattij-inv-sources";
import RnhaleSources from "../sources/rnhale-sources";
import WischmeyerSources from "../sources/wimscheyer-sources";
function HPLinktoOtherHPTab({tab, text}:{tab: string, text: string}){
const {goToPagesAndOpenTab} = useNavigation();
return(
<a onClick={() => goToPagesAndOpenTab(tab, "")}> {text} </a>
)
}
function HPgoToPageAndScroll({id, text, path}:{id: string, text: string, path: string}){
const {goToPageAndScroll} = useNavigation();
return(
<a onClick={() => goToPageAndScroll(id, path)}> {text} </a>
)
}
/*function HPgoToPageAndOpenTab({id, text, path}:{id: string, text: string, path: string}){
const {goToPagesAndOpenTab} = useNavigation();
return(
<a onClick={() => goToPagesAndOpenTab(id, path)}> {text} </a>
)
}
function HPgoToPageAndOpenTabAndScroll({tab, text, path, scrollId}:{tab: string, text: string, path: string, scrollId: string}){
const {goToPageWithTabAndScroll} = useNavigation();
return(
<a onClick={() => goToPageWithTabAndScroll({tabId: tab, path: path, scrollToId: scrollId})}> {text} </a>
)
vorname: string;
nachnname: string;
pictureurl: string;
tag: StakeholderTag;
heading: string;
quoteVorname?: string; /* Wenn die quote nicht von der Person ist über die der Text ist */
aimofcontact?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
insights?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
clarification?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
implementation?: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
pictureurl_interview?: string; /* Picture that goes into the paragraph "Insights" */
pictureurl_aim?: string; /* Picture that goes into the paragraph "Aim of contact" */
pictureurl_implementation?: string; /* Picture that goes into the paragraph "Implementation" */
more_pictures?: Array<string> ;
references?: React.ReactNode; /* Muss HTML Code sein - Liliana erstellt den aus Bib dateien */
text?: string | Array<string> | Array<React.ReactNode> |React.ReactNode ; /* Extra Text */
summary: string | Array<string> | Array<React.ReactNode>; /* Sobald Zitierungen drin sind oder Links muss es HTML Code sein, ansonsten gehen strings */
months: string,
type StakeholderTag = 'Industry' | 'Academia' | 'Patient' | 'Medical Professional' | 'Milestone' | 'Education'| 'Outreach'| 'Other';
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olariu: "https://static.igem.wiki/teams/5247/photos/hp/olariu-cristian.jpg",
westhoff: "https://static.igem.wiki/teams/5247/photos/hp/hp-katrin-portrait.jpg",
mattijs: "https://static.igem.wiki/teams/5247/photos/hp/mattijs.jpg",
julia: "https://static.igem.wiki/teams/5247/photos/hp/julia.jpg",
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kuehnel: "https://static.igem.wiki/teams/5247/photos/hp/hp-philippk-hnel.jpeg ",
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nicole: "https://static.igem.wiki/teams/5247/photos/hp/hp-friedlein-nicole.jpg",
joshua: "https://static.igem.wiki/teams/5247/photos/hp/joshua.jpg",
hammer: "https://static.igem.wiki/teams/5247/photos/hp/hp-hammer.webp",
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kühnel: "https://static.igem.wiki/teams/5247/photos/hp/hp-philippk-hnel.jpeg ",
moorlach: "https://static.igem.wiki/teams/5247/delivery/moorlach.webp ",
corden: "https://static.igem.wiki/teams/5247/delivery/corden-hp.webp",
marco: " https://static.igem.wiki/teams/5247/photos/hp/marco-raukic.webp ",
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kaihammer:"https://static.igem.wiki/teams/5247/photos/hp/interwiev-kai.jpeg",
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gxpcourse:"https://static.igem.wiki/teams/5247/photos/hp/kayagxpexpert.webp",
linköping:"https://static.igem.wiki/teams/5247/photos/hp/liu2024-rund.webp",
biobank:"https://static.igem.wiki/teams/5247/photos/hp/biobank.webp",
bethel: "https://static.igem.wiki/teams/5247/photos/hp/logo-evangelisches-klinikum-bethel.webp",
saito:"https://static.igem.wiki/teams/5247/photos/hp/hp-makoto-saito.jpg",
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sriram: "https://static.igem.wiki/teams/5247/photos/sriram.svg",
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tag: "",
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cardtext: "",
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/* WICHTIG!
Fehlende Infos einfach leer lassen und keine Dummy-Texte einfügen!
*/
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
heading: "Development of a multidisciplinary team structure",
interviewtabid: "recruiting",
quoteNachname:"Zimmermann, Intructor",
quoteVorname:"Sinan",
quote: "My first challenge was selecting the iGEM team. I focused on choosing candidates with strong technical skills and the ability to work well in an interdisciplinary setting, aiming to create a diverse team with complementary strengths.",
Building a successful interdisciplinary team, like the one involved in this iGEM project, requires careful coordination and a shared vision for addressing complex scientific challenges. This team is an excellent example of how bringing together individuals from various academic backgrounds fosters innovation and facilitates problem-solving in the fields of synthetic biology and biotechnology.</p>,
<p><strong>The team members come from diverse fields of study, including Molecular Biotechnology, Bioinformatics, Molecular Cell Biology, Genome-Based System Biology, Interdisciplinary Biomedicine, and even Media Production.</strong></p>, <p>This variety of expertise allows them to approach problems from multiple perspectives, ensuring that the project is well-rounded and capable of addressing both
experimental and computational challenges. For instance, while the biotechnologists and molecular biologists focus on designing and conducting lab experiments, the bioinformaticians contribute crucial insights for data analysis and genome sequencing. The inclusion of a media production expert adds another layer to the team’s capabilities, ensuring that science communication and
project documentation are handled professionally, which is particularly important for presenting their work at conferences and competitions.
The multidisciplinary structure of this team evolved naturally as they identified the project’s needs and recruited members who could fill specific roles. Over time, this multidisciplinary structure has continued to develop as the team’s project evolved. Roles became more defined as team members took on specific responsibilities, whether it was refining prime editing systems or crafting
communication strategies for iGEM. The range of experience across the team, from younger students like Vincent Carl Stöckl in the early stages of their academic careers to more advanced students in later semesters, allows for a blend of fresh perspectives and seasoned expertise. This balance helps ensure both the immediate success of the project and the continuity of its progress in the future.
In summary, this interdisciplinary iGEM team demonstrates how a diverse group of individuals with specialized skills can come together to tackle a complex biological project. By combining their expertise in molecular biology, bioinformatics, cell biology, and media production, the team is able to execute their project holistically, ensuring that each aspect, from experimental design to public outreach, is handled with precision and creativity.</p>],
months: "February"
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
heading: "Getting Acquainted with Cystic Fibrosis",
interviewtabid: "firstpresi",
quote: "Firstly, we discussed various project ideas, including the use of magnetic microswimmers for targeted medical applications, gene editing approaches for cystic fibrosis, treatments for muscular dystrophy and combating cyanobacteria with algae.",
summary: [<p>During our initial discussions about project ideas, the team explored several innovative concepts before honing in on <strong>cystic fibrosis</strong>. Each project presented unique scientific challenges and potential impacts.</p>,
<p>One idea involved the development of <strong>magnetic microswimmers</strong> designed for targeted cancer therapy, particularly for ovarian cancer. The proposal aimed to overcome the limitations of traditional treatments, such as radiotherapy and cisplatin, which often damage healthy cells and result in numerous side effects. The microswimmers would be biodegradable algae-based vehicles, magnetically guided to deliver drugs directly to cancerous cells while minimizing damage to surrounding tissue. This approach offered a novel, non-invasive targeting mechanism, leveraging the potential of magnetic fields to direct the swimmers precisely to the affected areas.</p>,
<p>Another compelling project was focused on <strong>Duchenne muscular dystrophy (DMD)</strong>. This genetic disorder leads to muscle degeneration as muscle tissue is gradually replaced by fibrotic tissue. The team proposed several treatment approaches, including exon skipping and gene therapy using viral vectors. Key strategies included enhancing Cas protein efficiency through engineering, using liposomal vectors for gene delivery, and combining CRISPR technology with integrase fusion proteins. The team also discussed previous iGEM projects that had explored similar methodologies, emphasizing the need for specificity and efficacy in delivering therapeutic genes.</p>,
<p>We also considered a project aimed at preventing <strong>cyanobacteria blooms</strong> using peptides or proteins derived from algae. Cyanobacteria can produce harmful toxins that degrade water quality and threaten ecosystems. The proposed approach focused on utilizing natural algae to develop mechanisms that inhibit the growth of harmful cyanobacteria without disrupting the ecosystem. This project emphasized conservation and biodiversity while addressing a significant environmental issue linked to the climate crisis.</p>,
<p>Throughout our discussions, we recognized that while each project had merit, the <strong>cystic fibrosis</strong> initiative offered the most promise for a targeted approach to a well-defined problem. CF, affecting a substantial number of individuals, provided a clear opportunity for meaningful impact through gene therapy. The insights gained from our examination of other projects informed our approach to CF, allowing us to integrate knowledge from diverse fields and methodologies, ultimately shaping a comprehensive and innovative project direction.</p>
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
heading: "Brainstorming and selection of ideas and concepts at team weekend in Hamburg",
quote: "Thanks to our in-depth research phase, we were able to gather many insightful ideas. The workshop was highly productive and moved us significantly forward, allowing us to gain a clear vision of our project by the end of the day.",
quoteVorname:"Kathleen",
quoteNachname: "Susat, Teammember",
summary: [<p>From April 5th to 7th, we embarked on an enriching excursion dedicated to project work and team building. Our primary goal was to engage in meaningful brainstorming sessions focused on our iGEM project and to explore human practices thoroughly. We kicked off our weekend with a series of discussions that emphasized the importance of considering the impact of our work on the world.
Throughout our time in Hamburg, we dedicated several hours to reading relevant literature and research papers, both individually and in groups. This collaborative effort allowed us to deepen our understanding of existing projects and innovations in our field. We critically analyzed previous iGEM projects and explored who had worked on similar ideas, which helped us identify gaps in the research and potential avenues for our own project.
Our discussions naturally flowed into brainstorming sessions where we reflected on the impacts we wanted to achieve. We explored essential questions: What effects do we aim to have on society? What activities can we plan to gather evidence about how our work will resonate with the community? How can we effectively engage with stakeholders and share our findings?
In addition to structured brainstorming, we exchanged ideas informally during breaks and meals, fostering a creative atmosphere. By the end of the weekend, we had not only strengthened our team bonds but also clarified our vision for the project ahead. The insights gained from our discussions and readings laid a solid foundation for our future work, allowing us to approach our project with renewed focus and purpose.</p>],
job: "Patient and Student",
affiliation: "Bielefeld University",
pictureurl: pics['max'],
tag: "Patient",
heading: "Gathering valuable insights from the patient’s perspective",
interviewtabid: "maxfirst",
quoteNachname:"Beckmann, Patient",
quoteVorname: "Max",
quote: "A friend of mine with cystic fibrosis recently got a fungal infection that he can't get rid of. His situation really struck me; it showed how quickly a seemingly minor issue can worsen a person's life, especially for someone like us. It’s a strong reminder of how fragile our health is and how fast things can change without warning.",
aimofcontact: [<p>When CF came up as a possible topic, we reached out to a teammate's friend Max in the hopes of getting insights into the needs of CF patients and current treatments to verify the need for further treatment options.
Since he was much more enthusiastic and open for discussion than we dared to hope, we extended our exchanges into the realms of the reality of life for CF patients, possible progressions, organizations and doctors in our area and his personal perspectives and values.
The interest in meeting him grew in the whole team and we invited him to one of our meetings. </p>],
insights: [<><p>His honest and open answers to us, mostly nothing more than strangers to him, were touching and let the seriousness of CF set in.
Learning about the challenges he faced felt heavy, besides him being in relatively good health and having a good life quality for a CF patient.
<p>Additional to the interpersonal effects of our discussion, Max gave us the reasons to continue with gene therapy approach while focusing on the lung:
Modulators do not erase all symptoms.
There is a keen interest for new treatments in the CF community.
The the decreasing lung functionality it the most limiting.
The immense impact of treatments on the life quality. </p>
<p>We got new insights that motivated us to develop a new treatment, to treat the severe symptomes
The need for a calorie rich diet and digestive problems.
The frequency of checkups needed.
How vastly different the progressions can be.
The increased need for hygiene to prevent infections.
The high price of medicines and induvial therapeutics. </p>
<p>Afterwards, we reflected on the discussion and asked our team members what stuck with them:
“How much attention has to be paid to everything in everyday life, I hadn't even thought about problems at the hairdresser.”
“Simply that he was there and reported everything in such detail. From minute 1, I had permanent goosebumps because I was so moved by this story. I think it's great how he stands his ground in life, does what he wants to do and what defines him as a person. It didn't seem as if his life was determined by CF. I somehow expected it to be different, even if that sounds a bit silly.”
“The amount of medication and how expensive it is.”
"The statement that left the biggest impression for me was when Max was telling about a friend of his and fellow CF patient who caught a fungi infection which he now cannot get rid of anymore, showing how fast a seemingly little infection can change the life of a CF patient for the worse without any kind of warning.”
“The variance in the extent of the limitations of the disease in different patients, including how the disease differs in its severity, even in patients of the same age.”
“How positively and calmly Max deals with his illness but has also pointed out that he is lucky, and that other people are much worse off - how much you have to pay attention to little things that you wouldn't have expected as a healthy person.” </p>
</>],
implementation: [<> <p>This most important aspect of this meeting was less an insight, but the fact Max helped us to put a face to an abstract idea. Many of our ideas to treat CF were interesting and adventurous but meeting him put a lot into perspective. </p>
<p>Max played a significant role in shaping our project from the outset, particularly in guiding our focus on Integrated Human Practices [Link Best HP] and Safety & Security [Link Best Biosafety] as special prizes. Through our discussions, we recognized the importance of these aspects in developing the best possible cure. This collaborative effort led us to pivot our target from the pancreas to the lung and move away from a diagnostic approach. His contributions not only provided valuable insights but also fostered a strong personal investment in our project.</p></>],
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interview: <><QaBox q="How and when were you first diagnosed? " a="When I was about one year old. My mother did not do any screenings or prenatal testing. I was in pain but as an infant you cannot say that, so I screamed a lot. Many doctors shrug that off in small children but after some time a sweat test was done at the children's clinic." />
<QaBox q="What do you think about diagnosing via sweat tests?" a="I am a clear opponent of diagnosing via sweat tests, especially if it is used to rule out CF and people have atypical CF, because of which they do not get diagnosed because of that." />
<QaBox q="What symptoms do you have?" a="Before taking modulators, I was underweight and did not feel hunger. I also had no sense of taste. Now, I have a healthy weight and still have respiratory symptoms such as very sticky mucus and digestive issues." />
<QaBox q="You are taking individual meds, correct? They are individual in respect to the mutation, not the person, right?" a="Yes, and yes, I am. " />
<QaBox q="What other medications are you taking? " a="Nasal spray, pancreatic enzymes, saline solution for inhalation and pantoprazole, used to reduce stomach acid production. " />
<QaBox q="Do you know how exactly they work?" a="Yes, I wrote a report on that during school. In the children's clinic they explained it like this: The CFTR channel is like a door and people with CF don’t have that many doors and some of the doors are broken. The medication makes more doors that function." />
<QaBox q="What changed when you started taking the modulators? " a="Everything. Most of symptoms are minor now and I have a better lung function and quality of life. I even grew taller." />
<QaBox q="Did you formerly take other medication?" a="I don’t remember anything like that, but I also always had good medical care. " />
<QaBox q="Do you experience any side effects from your medications?" a="At first yes, a lot. Stomach cramps and difficulty breathing for example." />
<QaBox q="Is diabetes a concern of yours?" a="Yes, it is common. I have to go to a diabetes checkup once a year. That happens together with all the other checkups like sonographies." />
<QaBox q="Do you know fellow patients that took part in clinical study for gene therapy or at least thought about doing so?" a="I know no one that took part in one but definitely people who would like to do so." />
<QaBox q="Do you know other patients that would want to use gene therapy?" a="Yes, most definitely." />
<QaBox q="Since your sweat is different, do you have trouble with your temperature regulation?" a="No and I do not know any patients with an issue like that. But it still is uncomfortable in the summer, because the sweat is thick, and it can smell stronger, too." />
<QaBox q="How many hours a day are devoted to your illness?" a="Good question, but wrong patient. I am blessed with good health while other people my age may have to be on a ventilator. I currently only have to inhale for 20 minutes every day, take my medication and be conscious about hygiene. I would say 30 minutes a day. " />
<QaBox q="That means you do not have many limitations due to CF, is that right?" a="Yes. There are many things I am concerned about but often there is not a different way." />
<QaBox q="What are some of the limitations you do have?" a="Of course, I am still concerned about my health and using public bathrooms for example. And I still do not go swimming in lakes and things like that. But all in all, I feel like I can live a very normal life. " />
<QaBox q="One concern is hygiene. Our university for example does not have toilet seats in most bathrooms. Do you think there should be?" a="That does not concern healthy people, who are the majority. But specifically for CF-people? No, there are too few at the university. It would be more hygienic overall, though. A “CF-toilet” would be nice as a form of a disabled bathroom." />
<QaBox q="How was your childhood as a sick child and how did your parents act with you? " a="My mother is active in the Muko e.V. and has been for some time. My parents always lead by example about what to do and not to do and dealt with it in a good way. My mother was always very committed and involved in giving me good care. I always knew about my illness but felt it was not that bad, because I received good care and education about my illness." />
<QaBox q="What is a typical age for a diagnosis in your experience?" a="Somewhere between the pregnancy and one year. It is obvious if the children do not gain weight and there are genetic screenings one can do prenatally or after birth. " />
<QaBox q="If a diagnosis is possible during pregnancy, do you know of any treatments during pregnancy?" a="No, I think the youngest age for modulators is 3 years. But people can do genetic testing and counselling before pregnancy." />
<QaBox q="What does a high-fat diet entail?" a="For me, it was a lot of oil and butter and high-calory drinks. " />
<QaBox q="What would happen if you stopped taking your medications?" a="The first thing to happen would be heavy and dry coughing, because the mucus would not be removed properly anymore. Thus, bacteria would not be properly removed from the lungs anymore either and an infection would become more likely. And I would not be able to really process food anymore, so no nutrients, feeling weak and stomach problems. " />
<QaBox q="Physical therapy is a part of your treatment – what exactly do you do there? " a="Breathing exercises and training my lung volume to keep it on the same level. " />
<QaBox q="Do you have further wished for your therapy?" a="Not really. I am very lucky and am free of heavy symptoms on most days. " />
<QaBox q="Is that the norm or do you know people who do want new therapies?" a="No, there is a need for new therapies. " />
<QaBox q="Are these people with different mutations or worse health? " a="I don’t know, the progression is so individual, and infections can create big changes. " />
<QaBox q="A therapy for which organ would benefit most people that have worse health than you do?" a="Probably the lung. The pancreas is important too, but stomach problems are usually less pressing than difficulty in breathing." />
<QaBox q="You mentioned that doing sport is difficult with CF, why?" a="Hygiene. In the lockers and the showers but also with the equipment." />
<QaBox q="Do you feel restricted in your free time activities?" a="No, I always had good alternatives. For example, going swimming at an open-air swimming pool instead of a lake. " />
<QaBox q="Would you have more freedom when you are better protected from Pseudomonas spcc. and other potential infections? "a="Definitely. That is a big increase in the quality of life and that is a win. It also changes the picture people have of the illness. Of course being protected by prevention is good already but effective therapies for infections increase the sense of freedom even more. " />
<QaBox q="You said you are afraid every time you must go for a swab, why is that? " a="I am afraid of getting an infection. That still could be a death sentence. " />
<QaBox q="Are rooms with air conditioning a problem due to the possible germs in the air conditioners? " a="No, there is usually enough movement. But humidifiers are bad because of the pond water. " />
<QaBox q="You mentioned going to the hairdresser is problematic. Could you elaborate? " a="There are many possible sources of ponding water and with that, infections. That and the hygiene aspect in general. I am visited by my hairdresser, and he only uses a specific spray bottle to wet my hair that I keep and dry thoroughly between uses." />
<QaBox q="Are you the first person in your family that has CF? " a="Yes. But I suspect my father has a light or atypical form because he has suspicious mucus." />
<QaBox q="With life expectancies looking better, do many patients want to have biological children?" a="Not all but some. I think some would be interested in a therapy that can be done on the fertilized egg to have a healthy child. " />
<QaBox q="Do you know the film “Five feet apart”? If so, what do think about it, is it accurate? " a="Yes. It does not paint a wrong picture; their progression is possible." />
<QaBox q="Do you think there has to be more effort concerning diagnostics?" a="Early diagnosis is covered by the screenings." />
<QaBox q="Since you almost had to sue for your medication, do you know if there are any lawyers specializing in cases like this? " a="No, I don’t. " />
<QaBox q="Are most of the other patients you know in good health like you?" a="No. Another boy my age got a fungal infection and does not have long time left to live. " />
summary: "We reached out to Max, a friend of a teammate, to gain insights into the needs and experiences of CF patients. Our discussions revealed the challenges faced by CF patients, even those in relatively good health, and emphasized the ongoing need for new treatment options. Max's candid sharing of his experiences highlighted the limitations of current modulators, the importance of lung function, and the impact of treatments on quality of life. This meeting transformed our project perspective, urging us to prioritize safety and real-world benefits in our design. Ultimately, Max's influence led us to focus on lung-targeted gene therapy instead of a diagnostic approach, reinforcing our commitment to Integrated Human Practices.",
months: "April",
/*interview:<iframe title="Bielefeld-CeBiTec: Interview with Max Beckmann (2024) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/16501867-a687-4205-949a-51ead876e109" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>,*/
{
vorname: "Visiting ",
nachnname: "the Hannover Messe",
pictureurl: pics['hannovermesse'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Industry",
heading: "Two teammembers visited the fair to talk with potential stakeholder and sponsors",
interviewtabid: "hannovermesse",
cardtext: "",
quote: "Visiting Hannover Messe was inspiring! We connected with industry leaders and discovered exciting innovations in synthetic biology that could shape our future projects.",
quoteNachname: "Stöckl, Teammember",
quoteVorname: "Vincent",
summary: "Our team members Liliana Sanfilippo and Vincent Stöckl had a productive visit to the Hannover Messe, focusing on synthetic biology and research advancements. They explored innovative technologies and networked with potential sponsors, gaining valuable insights to help enhance our project and drive future developments. The event was a great opportunity to connect with industry leaders and learn about cutting-edge solutions in the field.",
months: "April",
pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/hp/hannover.webp",
},
{
title: "Prof. Dr.",
vorname: "Kristian",
nachnname: "Müller",
job: "Research Group Cellular and Molecular Biotechnology",
affiliation: "Technical Faculty of Bielefeld University",
pictureurl: pics['kristian'],
tag: "Academia",
heading: "Discussion about the delivery method- AVV vs. LNPs and prime editing and gene therapy innovations",
interviewtabid: "kristian",
cardtext: "",
language: "de",
quoteNachname: " Müller, Reasearch Expert for Gene Therapy ",
quoteVorname: "Prof. Dr. Kristiann",
quote: "AAVs have been widely used in gene therapy, but their scalability and immunogenicity pose challenges, particularly when re-dosing is required. In contrast, LNPs offer a promising alternative, with a higher packaging capacity and lower immunogenicity. While AAVs excel in targeted delivery, LNPs could become a more scalable and cost-effective solution for delivering gene-editing tools in the future.",
aimofcontact: [<p>The aim of our interview with Prof. Dr. Kristian Müller was to gain expert insights into the potential of prime editing technology and its application in gene therapy, particularly for treating CF. We sought to understand how innovations in prime editing, combined with optimized delivery systems, could enhance therapeutic outcomes. Prof. Müller’s expertise in molecular biology and gene editing provided valuable perspectives on the opportunities and challenges in developing precise, efficient, and safe treatment strategies for genetic disorders.</p>],
insights: [<p>During the interview, Prof. Dr. Müller highlighted the growing importance of prime editing as a powerful tool for gene therapy. Unlike traditional CRISPR-Cas systems, which often result in double-strand DNA breaks, prime editing allows for precise single-strand cuts, minimizing off-target effects and enabling more specific genetic corrections. This technology opens up new possibilities for treating diseases with known mutations, such as cystic fibrosis.
Prof. Dr. Kristian Müller emphasized the critical role of delivery systems in the success of gene therapies, particularly in the context of CF treatment. Two primary delivery mechanisms were discussed: AAVs (Adeno-associated viruses) and LNPs (Lipid nanoparticles), each with distinct advantages and limitations.
AAVs are a well-established vehicle in gene therapy, having been used successfully in various approved treatments. They are highly efficient at delivering genetic material to target cells, especially in well-characterized diseases like CF. One of their key strengths is their ability to precisely target specific tissues, making them particularly valuable for lung delivery in cystic fibrosis. However, AAVs come with notable challenges, primarily their limited packaging capacity (approximately 4.5 kilobases), which constrains the size of the genetic payload they can carry. Additionally, AAVs can elicit immune responses, particularly when multiple doses are required, posing a barrier to their long-term use.
On the other hand, LNPs offer a scalable and re-dosable alternative. LNPs have the advantage of a larger packaging capacity, allowing them to carry more complex genetic instructions or larger gene-editing tools, such as prime editors. They are also easier and cheaper to produce on a large scale, making them an attractive option for widespread clinical applications. A significant benefit of LNPs is their lower immunogenicity, which reduces the risk of adverse immune reactions upon repeated dosing. However, LNPs currently face challenges in specific targeting compared to AAVs. AAVs have a higher precision in targeting specific tissues, while LNPs still need optimization for targeted delivery to areas like the lungs.</p>],
implementation: [<p>Prof. Müller’s insights directly inform the implementation of our iGEM project, where we aim to design novel prime editors that are small enough to be delivered efficiently, while also exploring LNPs[LINK Cycle Delivery] as a scalable and re-dosable alternative to AAVs. By tailoring our approach to address the specific challenges of CF, such as mucus penetration and lung cell targeting, we can enhance the precision and efficacy of gene therapy. These innovations have the potential to set new standards in the field and contribute to broader research on genetic disease treatment.</p>],
summary: "In our interview with Prof. Dr. Kristian Müller, we explored the revolutionary potential of prime editing as a next-generation gene editing technology. Prof. Müller highlighted the advantages of prime editing over traditional CRISPR-Cas systems, particularly its ability to make precise genetic modifications without double-strand breaks, thus reducing off-target effects. He emphasized the importance of optimizing delivery systems, such as AAV and LNPs, and discussed the ethical considerations and biosafety measures crucial for advancing gene therapy. The interview underscored the significance of cystic fibrosis as a model disease, given its prevalence and the potential for impactful treatments through targeted genetic corrections.",
interview:<><iframe title="Bielefeld-CeBiTec: Interview Müller AAV vs LNP (2024) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/0613b6b8-7755-4373-9d86-9910fe30781f" frameBorder="0" allowFullScreen={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe><p>This interview was recorded on video at a later date.</p></>,
{
vorname: "Looking for expertise",
nachnname: "",
pictureurl: pics['logo'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
heading: "Identifying key experts in cystic fibrosis and prime editing",
interviewtabid: "experts",
cardtext: "",
quote: "Focusing on gene therapy for cystic fibrosis has been a meaningful journey for our team. Collaborating with experts has enriched our understanding and helped us refine our approach, especially in exploring prime editing. We're eager to turn our plans into reality and make a real impact",
quoteNachname:"Kanthak, Teammember",
quoteVorname: "Kai",
summary: [<p>After our team came together and thoroughly explored various project ideas, we decided to focus on gene therapy for cystic fibrosis, largely due to a personal connection with a close friend affected by the condition. Up until that point, we had not yet developed a concrete concept, so we sought to engage with experts in order to broaden our understanding of the latest advancements in gene therapy.
In addition to grasping the importance of a functional gene therapy, we delved into different strategies regarding the underlying mechanisms and the best delivery methods for the therapy. While the general topic of our project was clear, we now faced the challenge of working out the details. At this stage, we decided to consult further experts in the field of cystic fibrosis to deepen our knowledge and refine our approach.
Through connections with the University Hospital Münster and our local hospital, we aimed to gain a comprehensive overview of the clinical applications of gene therapy and the current research in cystic fibrosis. These consultations with specialists allowed us to acquire valuable insights into different therapeutic options and laid the groundwork for our own exploration of potential strategies, particularly in the area of prime editing as a promising treatment avenue.</p>,
<ul>
<li>
<strong>Team Formation & Research:</strong> Chose gene therapy for cystic fibrosis and explored mechanisms and delivery strategies.
</li>
<li>
<strong>Expert Engagement:</strong> Consulted with specialists to refine approach, focusing on prime editing.
</li>
<li>
<strong>Medical Collaboration:</strong> Gained clinical insights through partnerships with hospitals.
</li>
<li>
<strong>Project Development:</strong> Developed detailed plans for gene therapy, incorporating expert feedback.
</li>
<li>
<strong>Testing & Future Application:</strong> Plan to test strategies and prepare for potential clinical trials.
</li>
</ul>
],
{
vorname: "Visiting ",
nachnname: "the Labsupply in Münster",
pictureurl: pics['labsupply'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Industry",
heading: "Two team members connected with prospective stakeholders and sponsors.",
interviewtabid: "labsupply",
cardtext: "",
quote: "LabSupply in Münster was a fantastic opportunity to connect with passionate professionals and explore cutting-edge research tools. We're excited about the potential collaborations ahead!",
quoteVorname: "Liliana",
summary: "In April our team visited LabSupply in Münster, where we had the chance to connect with many welcoming and knowledgeable professionals in the lab equipment field. It was an excellent opportunity to explore the latest advancements in laboratory supplies and tools that can enhance our research.",
months: "April",
pictureurl_aim: "https://static.igem.wiki/teams/5247/photos/hp/labsupply.webp",
},
job: "Professor ",
affiliation: "Univesity Münster",
heading: "Feedback Session with Experts for Cystic Fibrosis Treatment",
quote: "This is also an innovative step that you have developed yourselves. That's the part that's really new and significant. A clever and exciting approach with great potential.",
aimofcontact: [<p>The aim of the interview was to gain expert insights on optimizing the delivery of CFTR-mRNA via lung-targeted lipid nanoparticles (LNPs) for cystic fibrosis (CF) treatment.
Specifically, the goal was to explore potential cell targets, challenges in delivery mechanisms, and technical tools for assessing the effectiveness of mRNA therapies like the Ussing chamber system. </p>],
insights: [<p>The experts highlighted the potential of targeting ionocytes, given their key role in CFTR expression, but emphasized the difficulty in accessing them due to their basal position in the respiratory epithelium.
While Prof. Weber found ionocytes to be an intriguing target, Dr. Große-Onnebrink pointed out that there is still limited understanding of their exact role in CF pathology. Both stressed the challenge of penetrating the
mucus barrier in vivo, particularly when using air-liquid interface cultures, and underscored the importance of optimizing particle size to ensure effective delivery to the deeper regions of the lungs.
Prof. Weber also emphasized the need to test whether the system can still transfect cells in the presence of mucus. </p>,
<p>It was suggested to use the Ussing chamber to assess the effectiveness of the delivery system and therapeutic mRNA, though they noted certain challenges with this technique. We also discussed alternatives like organoids,
which offer only indirect measurements of CFTR function, and patch clamping, which, though more precise, is a more complex and expensive method. Additionally, Prof. Weber recommended exploring chitosan-based delivery
systems due to their success in his previous work, suggesting they could be a safer alternative to PEG-lipid systems, which had shown issues with cytotoxicity. </p>],
implementation: [<p>These insights helped refine our iGEM project in several key ways:
<li>Cell Targeting: We decided to continue exploring ionocytes as a target but acknowledged the technical hurdles involved. We also expanded our focus to include multiple cell types to test different delivery systems. </li>
<li>Delivery Systems: We began investigating chitosan-based nanoparticles as a safer alternative to PEG-lipid systems. The suggestion to optimize particle size and delivery for inhalation was also integrated into our design. </li>
<li>Experimental Tools: Based on the discussion, we plan to use an Ussing chamber to measure overall CFTR function in different cell types but will also explore organoid-based approaches for preliminary testing. Additionally, we consulted the medical faculty on the possibility of using patch clamping for more detailed measurements of successful transfection and restored CFTR function. </li>
summary: "The interview provided key insights into targeting ionocytes for CFTR-mRNA delivery, overcoming mucus barriers, and testing delivery effectiveness using Ussing chambers, organoids, and patch clamping, while also suggesting chitosan-based nanoparticles as a safer alternative to PEG-lipid systems.",
{
title: "Dr.",
vorname: "Cristian-Gabriel",
nachnname: "Olariu",
job: "pediatrician",
affiliation: "OWL University Hospital",
pictureurl: pics['olariu'],
tag: "Medical Professional",
heading: "Discussion with a pediatrician and his former patient about treatment challenges and perspectives",
interviewtabid: "olariu",
cardtext: "",
language: "de",
quoteNachname: "Olariu, Clinical Physician and CF Expert",
quoteVorname: "Dr. Cristian-Gabriel",
quote: "For most families, it’s a shock. Cystic fibrosis still has a strong association with being a life-threatening disease, despite the fact that we now have good treatments, and many patients can live healthy lives. The diagnosis puts a huge psychological strain on the family, especially when dealing with very young children.",
aimofcontact: "To gain a deeper insight into the path to diagnosis, we invited pediatrician Dr. Cristian-Gabriel Olariu from the University Department of Pediatrics and Adolescent Medicine to share his experiences with CF patients with us. We interviewed him because of his expertise in the effects of diagnosis on the patient and the family members, but also on daily life. Additionally, we want to close the gap and create a bridge between academic research and clinical applications. Therefore, Dr. Olariu gave us insights about the clinical perspectives on CF patients.",
insights: [<p>We invited Max, our CF patient contact, to join Dr. Olariu in discussing the intersection of academic research, clinical application, and patient needs. Through our connection with <a href="https://www.cfvww.org" >CF Vests Worldwide</a>, an organization dedicated to providing life-saving therapy vests to cystic fibrosis patients globally, we gained insights into the challenges faced by CF patients, particularly in regions like Thailand, where access to advanced treatments and medical devices is limited. The conversation highlighted the critical role of early diagnosis and intervention, as well as the quality-of-life challenges many patients endure due to conventional treatments that may not be effective for everyone. Innovative approaches, such as our SORT LNP (lipid nanoparticle) delivery system, present promising alternatives for CF therapy. This system, which allows for RNA encapsulation and administration via dry spray inhalation, could revolutionize treatment by targeting lung cells more effectively, particularly in resource-limited settings. Dr. Olariu underscored the need for psychological support and coordinated care for CF patients, emphasizing that novel therapies like LNP-based gene treatments have the potential to improve treatment efficacy and accessibility, ultimately reducing the lifelong burden of care for patients and their families. </p>,
<p>We have considered the extent to which an early diagnosis is always an advantage, as some parents perceive an early diagnosis as an additional burden and would prefer to experience the first years of their child's life without constant medical intervention. Especially when there are cases in which patients only show a clear clinical picture at an advanced age. The psychological burden also lies with the children, who often experience medical trauma because they are involved in such intensive medical care from birth. Additionally, the treatment of cystic fibrosis is very expensive, and the costs are covered by health insurance companies to varying degrees. In some countries, such as the USA, Ukraine or Developing countries, many families cannot afford the necessary treatments. But Dr. Olariu also drew our attention to another problem in the treatment of cystic fibrosis. Infections, especially with bacteria such as Pseudomonas spcc., are difficult to treat and often lead to long hospital stays. Max, our patients’ representative, who knows Dr. Olariu through his treatment, shared his experience with Pseudomonas spcc infections, illustrating the reality of an invisible danger that determines a patient's everyday life. Strict hygiene measures are required to prevent infections, such as wearing face masks in hospital and careful handling of potential sources of infection. The clinics where cystic fibrosis patients are treated work closely with a multidisciplinary team of doctors, psychologists, physiotherapists and nutritionists to ensure that patients receive holistic care. </p>,
<p>Pros of Early Diagnosis and Treatment</p>,
<ol>
<li>Timely Intervention: Prevents severe organ damage and improves long-term outcomes.</li>
<li>Holistic Care: Involves a multidisciplinary team for comprehensive patient support.</li>
<li>Access to Innovations: Allows patients to benefit from advancements like nanoparticle drug delivery.</li>
<li>Family Support: Provides education and resources for effective management from the start.</li>
<li>Psychological Burden: May cause stress for parents and children due to constant medical interventions.</li>
<li>Cost Implications: Treatments can be expensive, with varying insurance coverage, leaving many families unable to afford care.</li>
<li>Infection Risks: Patients still face risks from infections like Pseudomonas spp., leading to potential hospitalizations.</li>
<li>Over-medicalization: Continuous focus on treatment can overwhelm families, affecting the quality of early childhood experiences.</li>
implementation: [<p>In summary, our project greatly benefited from the conversation with Dr. Olariu. His insights into the complexities of cystic fibrosis treatment, particularly the significance of early diagnosis, were invaluable and we considered early treatment methods. Max’s personal experiences added a human perspective, illustrating the medical and psychological challenges he faces, including infections with <i>Pseudomonas spp.</i> Dr. Olariu highlightend the importance of a multidisciplinary approach, involving not just medical professionals but also psychologists, physiotherapists, and nutritionists for holistic care. This discussion helped us appreciate the balance between timely interventions and the emotional burden on patients and their families, guiding us to develop a more empathetic understanding of living with cystic fibrosis.</p>],
interview: <>
<QaBox q="Could you please tell us about the journey that parents go through with their CF-sick children from the first visit to diagnosis and treatment?" a="Since 2016, cystic fibrosis (CF) diagnosis has been part of newborn screening. This means that we receive many children right after birth whose screening results were abnormal. These children are then sent to us for further clarification. Not every child with an abnormal screening result is sick, so we perform a sweat test, and about one-third of the children are diagnosed with the disease. The advantage of early diagnosis is that we can intervene and start treatment early to prevent organ damage. However, there are also rare mutations where the course of the disease is difficult to predict." />
<QaBox q="What are the pros and cons of newborn screening for cystic fibrosis?" a="From a medical point of view, it’s beneficial that we can catch many of these cases early, allowing us to act swiftly. There are even medications for small babies, and early intervention can protect organs, preventing conditions that would require transplants later on. On the downside, because of the wide variety of genetic mutations, some cases we identify may not show significant symptoms until adulthood. This creates a dilemma, as we can’t predict how their condition will progress, but we still start treatments early, which can be stressful for families." />
<QaBox q="Can you give us an example of how this stress impacts families?" a="Yes, I’ve been caring for a patient from birth who is now five years old and doing very well. However, from the beginning, she had to undergo physiotherapy, regular check-ups, and blood tests, even though she hasn’t shown any major symptoms. Her mother once told me she wasn't sure if she would make the same decision again, as the early intervention caused a lot of stress. She wondered if she might have enjoyed the first year of her child’s life more if things had been more relaxed. Now, at age five, nothing significant has changed in her condition, and they’ve decided against starting modulator therapy for the time being." />
<QaBox q="How do families typically react when a CF diagnosis is confirmed?" a="For most families, it’s a shock. Cystic fibrosis still has a strong association with being a life-threatening disease, despite the fact that we now have good treatments and many patients can live healthy lives. The diagnosis puts a huge psychological strain on the family, especially when dealing with very young children. The most important factor in managing this, aside from medical treatments, is the support from the medical team. It’s critical to have a team that works well together, not just a single doctor calling all the shots. Families often need much more psychological and nutritional support early on than medical intervention, and this is where having a multidisciplinary team becomes essential." />
<QaBox q="What is the process for diagnosing and treating older patients who haven’t been through newborn screening?" a="Older patients who come to us with complaints may not have undergone newborn screening, so they are diagnosed based on their symptoms. These complaints can range from mild to severe and are often non-specific, like chronic cough or failure to thrive. When the cause of these symptoms isn’t immediately clear, we do a sweat test. Once diagnosed, we can start treatment, which often involves working with a psychologist to help the family process the news." />
<QaBox q="How do you support families during the initial shock of diagnosis?" a="When the diagnosis is particularly difficult for families to process, we sometimes have the patients stay in the hospital for up to a week. This gives us time to meet with them daily, answer questions, and provide guidance. During the first consultation, families often fall into a state of shock, and no matter how carefully the doctor explains things, it’s hard for them to absorb all the information. Meeting with them again over the following days helps, and we have specialists in hygiene, physiotherapy, and social counseling on the team to offer holistic support." />
<QaBox q="What happens if a child gets infected with Pseudomonas or another bacterial culture in the lungs?" a="Pseudomonas is one of the most feared infections for CF patients. It’s a common environmental bacterium that is difficult for CF patients to clear from their lungs. Once we detect it, we treat the patient with specific antibiotics, often through intravenous delivery over two weeks in the hospital. After the initial treatment, patients may continue with inhaled antibiotics for several months to prevent further infection. It’s a very intensive process, taking a lot of time and energy, and even though we may get rid of the infection a few times, eventually the germ can become resistant and stay in the body." />
<QaBox q="Are there any preventative measures to avoid Pseudomonas infection?" a="Yes, there are hygiene measures. For example, CF patients always wear masks in the hospital to avoid infection from other patients. But it’s difficult to avoid Pseudomonas entirely since it’s found in stagnant water and other places in the environment. We advise patients to be cautious with water sources like sinks or ponds. However, we need to balance strict hygiene with quality of life, especially for children, as being overly strict can lead to obsessive-compulsive behaviors without necessarily reducing the risk of infection." />
<QaBox q="Do some families resist the medical advice on preventing infections?" a="On an emotional level, I feel that families who take calculated risks to improve their quality of life tend to cope better. Overprotection can lead to greater psychological stress. However, I don't have enough experience to say for sure whether those who don’t protect themselves as strictly get infected earlier or suffer worse outcomes. It’s also worth noting that new therapies are now available that help reduce infection risks, allowing for a bit more freedom, especially for children." />
<QaBox q="How often do patients need to be tested for infections like Pseudomonas?" a="The official guideline is every two months, but realistically we aim for every 3-4 months. Regular testing is important because Pseudomonas can be present without symptoms. If too much time passes before detection, it becomes harder to remove the infection." />
<QaBox q="How do you manage chronically infected patients?" a="Patients who are chronically infected with Pseudomonas don't stay in the hospital indefinitely. They usually remain at home, inhaling antibiotics daily and taking physiotherapy to help clear mucus from their lungs. Intravenous antibiotic therapy is reserved for more severe cases or during clinical deterioration." />
<QaBox q="Are chronically infected patients allowed to visit your practice?" a="Yes, chronically infected patients are allowed to visit the practice. We try to schedule them at different times to avoid contact between infected and non-infected patients, and we often use separate rooms to minimize risk." />
<QaBox q="How often do children and adults need to have lung function tests?" a="You can’t conduct a good lung function test until the child is around five years old. After that, it becomes part of the routine check-up because it’s non-invasive and provides a good indicator of lung health. We see children every three months, and I believe the protocol is the same for adults." />
<QaBox q="What do you think about support groups or health retreats for CF patients?" a="Support groups are extremely important. Although we are a good medical team, advice from peers often resonates more with patients. We’ve organized two parents' evenings recently, where parents can exchange experiences and support each other. Unfortunately, we can’t invite the children themselves due to the risk of infection, but in rehabilitation settings, they can meet in germ-specific groups and benefit from shared experiences." />
<QaBox q="Is there a risk of antibiotic resistance with repeated treatments?" a="Yes, resistance is a concern, especially with repeated antibiotic treatments. However, there’s often a discrepancy between what we see in lab tests and the clinical outcomes. Even if a germ shows resistance on paper, many patients still respond well to treatment. We base our decisions more on clinical outcomes than lab results, changing antibiotics only if the patient’s condition doesn’t improve." />
<QaBox q="Are there any side effects to the medications?" a="Yes, all medications have potential side effects, though many of them are minor, like rashes or stomachaches. One serious side effect of some antibiotics is hearing damage, which can lead to lifelong hearing loss. This is why we closely monitor patients in the hospital when starting treatments. The newer therapies, like modulators, can cause liver stress, so we regularly check liver enzymes in the blood. However, severe side effects are rare, and the drugs are generally well tolerated." />
</>,
pictureurl_aim: "https://static.igem.wiki/teams/5247/photos/hp/interview-olariu.svg",
pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/olario-abbildung1.svg",
summary: "We interviewed Dr. Cristian-Gabriel Olariu to gain insights into the diagnosis and treatment of cystic fibrosis (CF). He highlighted the importance of early diagnosis through newborn screening, which allows for timely intervention but can also be perceived as a burden by families, especially when symptoms may not manifest until later. Dr. Olariu emphasized the emotional and financial challenges families face, particularly regarding costly treatments and insurance variability. Patient contact Max shared his experiences with infections like Pseudomonas spp., which complicate care and necessitate a multidisciplinary approach involving medical professionals, psychologists, and nutritionists. Overall, the discussion underscored the need to balance medical interventions with the emotional well-being of patients and families, guiding us toward a more compassionate understanding of living with CF.",
months: "may"
},
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
heading: "Further brainstorming on approaches and specifing the project",
interviewtabid: "exploring",
quote: "One of the most valuable aspects of our project is the feedback we've received. It truly motivates us to make a difference in the community.",
quoteNachname:"Sanfilipo, Teammember",
quoteVorname: "Liliana",
summary:[<p>After receiving valuable feedback from both clinical and academic experts, we decided to focus on optimizing Prime Editing strategies for cystic fibrosis treatment. Both experts not only encouraged us in our approach but also provided insightful feedback, which we will integrate into our future project design. Through these discussions, we learned that current treatment strategies are urgently needed in real life but are limited in precision and efficiency.
Additionally, we gained key insights into lung-specific delivery methods, which inspired us to pursue lung-specific correction of the CFTR gene, a critical aspect of cystic fibrosis therapy.
At this stage, we are eager to expand our perspectives by seeking input from industry and business professionals, while also striving to increase our local impact. To evaluate this impact, we plan to develop a survey aimed at understanding the interest in gene therapy and the community’s knowledge of cystic fibrosis within our local area. This will help us gauge awareness and ensure our project addresses both scientific and societal needs effectively.</p>>
<ul>
<li>
<strong>Expert Feedback Integration:</strong> Refined the project focus on optimizing Prime Editing strategies and lung-specific gene delivery based on clinical and academic insights.
</li>
<li>
<strong>Focus on Lung-Specific Correction:</strong> Shifted toward lung-specific CFTR gene correction for cystic fibrosis treatment.
</li>
<li>
<strong>Community Engagement Plan:</strong> Initiated plans for a local survey to assess awareness of cystic fibrosis and interest in gene therapy, aiming to increase local impact.
</li>
</ul>
],
{
vorname: "Katrin",
nachnname: "Westhoff",
job: "Physiotherapist",
affiliation: "Independent",
pictureurl: pics['westhoff'],
tag: "Medical Professional",
heading: "Interview with a specialized physiotherapist regarding breathing therapy for cystic fibrosis patients",
interviewtabid: "westhoffinv",
cardtext: "",
language: "de",
quoteNachname:"Westhoff, Physiotherapist",
quoteVorname: "Katrin",
quote: "The more we know, the more opportunities we have.",
aimofcontact: "The objective of the contact was to gain in-depth insights into the treatment and care of children with CF. The therapist's expertise was intended to help develop a better understanding of the challenges and necessary measures in the treatment of this chronic disease. In addition, the aim was to ascertain how the therapy is implemented in everyday life and which specific approaches and methods are particularly effective.",
insights: "The interview yielded valuable insights into the regular implementation of the therapy, the use of aids and the adaptation of exercises to the individual needs of the patients. It was notable that the therapy has improved over the last years, considerably thanks to better medication and adapted exercises, with a concomitant increase in life expectancy for children affected by CF. Of particular interest was the emphasis on the importance of sport and exercise, which should not only be therapeutically effective, but also increase quality of life. ",
implementation: "The following statement by Katrin Westhoff had a particular impact on our project: ‘The more we know, the more options we have’. We learnt from the interview that the current medication is already helping many patients very well, but that there is still great potential for improvement. Successful gene therapy would significantly improve the quality of life of CF patients. We implemented the findings from this interview in our participation in MukoMove - we also actively took part in cystic fibrosis awareness month and learnt even more about the importance of physiotherapy.",
pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/katrin-westhoff-zoom.webp",
summary: [<p>The objective of our discussion with a therapist was to gain a comprehensive understanding of the treatment and care of children with cystic fibrosis. The interview provided invaluable insights into the therapy's implementation, highlighting the significant advancements in medication and tailored exercises that have led to improved patient outcomes and increased life expectancy. A key takeaway was the emphasis on the role of sports and exercise, not just for therapeutic efficacy but also for enhancing overall quality of life. It let to our participation in the CF awarness month and the outreach project mukomove [Link mukomove]</p>],
<QaBox q="From what age do the patients come to you? How long do they stay? How many patients do you treat?" a="The patients come to us at a very early age. A definite diagnosis is made after 6 weeks at the latest. Once diagnosed, the whole family is genetically tested, and children are sent for physiotherapy, often starting in the hospital. Currently, we have 8 children with CF in our practice, which is relatively small compared to other diseases. We have slightly more CF patients because we specialize in it."/>
<QaBox q="What kind of exercises do you do?" a="We do a lot of breathing therapy and have attended special training courses for CF that introduced new techniques. The current gold standard is autogenous drainage according to Chevallier, which effectively removes mucus. We follow a general routine: 1. wet inhalation to bind mucus, 2. drainage to expel mucus, and 3. antibiotics to work optimally on clean lungs. We also use special belts for compressing 'magic points' to enhance lung ventilation."/>
<QaBox q="When does drainage start?" a="We start drainage in newborns to prevent mucus from settling."/>
<QaBox q="Are there special exercises that can also be done at home?" a="Yes, parents are instructed on exercises that can also be performed at home."/>
<QaBox q="How often does the therapy take place?" a="Therapy usually occurs once a week or every two weeks. Thanks to improved medication, children are now better off. The therapy has evolved significantly, making it easier to cough up mucus and improving life expectancy. Exercise should be enjoyable and a part of daily life from the age of 8 or 9."/>
<QaBox q="What would happen if no physiotherapy was performed?" a="It’s difficult to predict, but without therapy, children often become more mucousy, leading to worsened ventilation. Specific therapy is crucial, especially during infections."/>
<QaBox q="How do you measure success (in terms of lung function test, exercise, etc.)?" a="Success is measured subjectively by listening to breathing and observing skin color. A well-ventilated lung shows a 'full barrel' appearance, while wheezing indicates poor ventilation. In clinics, lung function tests, CO2 measurements, and 'finger clip' tests are used, though results can be influenced by the child."/>
<QaBox q="Do the exercises bring relief or are they preventative for further complaints?" a="The exercises serve both to relieve acute infections and to prevent further issues. Fewer lung infections reduce the likelihood of mucus adhesions."/>
<QaBox q="Are there any tools to perform therapy?" a="Yes, devices like the 'flutter' or 'cornet' help with exhalation. They create vibrations that loosen mucus in the lungs and should be used by all children with lung diseases."/>
<QaBox q="What complaints do patients bring with them?" a="Patients typically have lung problems, dry lung mucosa, and pancreatic issues leading to poor metabolism, requiring enzyme therapy before meals. Some children experience growth disorders and less commonly, excessive perspiration."/>
<QaBox q="Are pancreatic complaints also treated by physiotherapists?" a="Pancreatic complaints are rarely treated with physiotherapy, except in cases of inflammation, where patients may be admitted to the hospital. Techniques like massage or kinesiology tape can help with constipation."/>
<QaBox q="Are there any special hygiene guidelines for you when working with CF patients?" a="Hygiene is crucial when treating CF patients. We separate children with and without infections (e.g., Pseudomonas) and enforce strict disinfection protocols. Only children with similar infection statuses are treated on the same day."/>
<QaBox q="Are the specific exercises customized? And if so, how do you know which therapy is the right one for which patient?" a="Exercises are tailored to each patient's situation, focusing on mucus removal and lung ventilation. Each therapist may have their own preferred exercises and techniques."/>
<QaBox q="Do patients always go to the same physiotherapist?" a="Yes, if therapy is effective, patients tend to remain with the same physiotherapist."/>
<QaBox q="How many physiotherapists offer muco-therapy?" a="The exact number is unknown, but several child therapists in the region provide CF therapy."/>
<QaBox q="How are the relatives educated?" a="Education often begins in the maternity ward with a sweat test. In Gütersloh, all children are referred to Bethel for immediate CF care. Parents often experience trauma as children can be severely ill despite appearing healthy."/>
<QaBox q="What are the limitations of individual medicine?" a="Drug effectiveness can vary, and some are only approved from a certain age. Improved medications can significantly enhance quality of life and life expectancy."/>
</>
},
job: " Scientific Sales Representative for Cell Culture Products",
affiliation: "Stemcell",
heading: "Longstanding Support from Stemcell Expert in Cell Culture Media and Primary Cell Cultivation",
quote: "x",
aimofcontact: "Our primary goal in reaching out to her was to gain insights into optimizing cell culture media, explore potential sponsorship opportunities for our project, and seek expert advice on handling primary cells in the lab.",
insights: "Throughout our multiple discussions, she offered detailed advice on selecting and optimizing cell culture media for our specific needs, while also connecting us with opportunities for sponsorship. Later in the project, she provided technical guidance on the cultivation and handling of primary cells, ensuring that we followed best practices for cell culture.",
implementation: "Based on her feedback, we refined our approach to cell culture media, enhancing our experimental workflows.Her technical support, as well as the support of Julia Watson helped us to establish our experiments in cellculture. Additionally, her advice was critical during the transition to primary cell culture, helping us secure the necessary resources and expertise for our experiments.",
summary: "We have had the privilege of collaborating with a dedicated expert from Stemcell Technologies, who has consistently supported iGEM Bielefeld. She provided valuable guidance on cell culture media, sponsorship opportunities, and later, practical advice for cultivating primary cells. Her contributions have been instrumental in advancing our project.",
months: "several times",
pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/daniela.webp",
},
{
vorname: "Documenting progress",
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
heading: "Tracking progress in expert search and idea development",
interviewtabid: "progress",
quote: "I believe our focus on a human-centric approach is crucial. It ensures that our research not only advances science but also prioritizes the needs and safety of the patients we aim to help.",
quoteNachname:"Lange, Teammember",
quoteVorname: "Kaya",
summary: [<p>
Early in our research, we committed to a human-centric approach, recognizing the importance of working according to Good Laboratory Practice (GLP) standards. At this point, we also identified key Special Prizes to aim for, including the Biosafety and Security Award, Best New Basic Part, and Best Integrated Human Practices. To further advance our project, we partnered with Stemcell Technologies, whose technical support provided valuable insights. This collaboration allowed us to explore the cultivation of primary human nasal epithelial cells, which are crucial for testing our synthetic biology components and Prime Editing technologies. In line with this, we expanded our biosafety standards to ensure compliance with higher safety levels and enhanced our understanding of preclinical trial-like experiments. Moreover, we are committed to raising awareness within our local community while also seeking to broaden our impact internationally. This dual focus helps us ensure both the safety and societal relevance of our project as we move forward.
</p>,
<ul>
<li>
<strong>Commitment to Human-Centric Approach:</strong> Early in the project, the decision to follow a human-centric approach ensured that societal impact, ethical considerations, and the needs of patients would be central to all scientific developments.
</li>
<li>
<strong>Selection of Special Prizes (Biosafety and Security, Best Integrated Human Practices):</strong> Prioritizing biosafety, security, and human practices from the start highlighted the project’s focus on safety, ethical responsibility, and community engagement, aligning with broader human-centric goals.
</li>
<li>
<strong>Collaboration with Stemcell Technologies:</strong> Partnering with industry leaders provided technical expertise that allowed the team to deepen their understanding of human cell cultivation and gene editing, ensuring that the project’s technological developments were informed by real-world applications.
</li>
<li>
<strong>Expansion of Biosafety Standards:</strong> Extending biosafety protocols to mimic preclinical trial conditions reinforced the commitment to safe, ethical research practices and laid the groundwork for clinical relevance, demonstrating responsibility toward future patients.
</li>
<li>
<strong>Local and International Community Engagement:</strong> Efforts to raise awareness at both local and global levels ensured that the project was not only scientifically sound but also socially responsible, with a focus on educating and involving the public in the conversation around cystic fibrosis and gene therapy.
</li>
</ul>
],
vorname: "MukoMove",
nachnname: "",
pictureurl: pics['mukomove'],
tag: "Outreach",
heading: "Moving together for health and hope, our participation in CF awarness month",
interviewtabid: "mukomovehp",
type: "meta",
cardtext: "",
quoteNachname: "Susat, Teammember",
quoteVorname: "Kathleen",
quote: "It was amazing to see how movement can bring people together. I had a great time.",
summary: [<p>MUKOmove[LINK unten mukomove] is a sports initiative by Mukoviszidose e.V., the German Cystic Fibrosis Association, to raise awareness and funds for cystic fibrosis. Team iGEM Bielefeld participated from May 8th to May 12th, promoting the event at their university and city, encouraging others to join and collect sport hours. They organized a team event at their university, involving sports games to promote community engagement. Their efforts helped surpass their goal of 240 hours, with the team achieving 358 sport hours, while the entire event gathered over 36,000 sport hours. The initiative successfully raised awareness about cystic fibrosis and promoted physical activity as a means of community building.</p>],
job: "Intern",
affiliation: " Harvard/ Boston Childrens Hospital",
pictureurl: "https://static.igem.wiki/teams/5247/photos/hp/hp-jpgerhards-potrats.webp",
tag: "Academia",
heading: "Discussion on optimizing our pegRNA Design to improve precision in prime editing",
interviewtabid: "JPpegRNA",
cardtext: "",
language: "de",
quoteNachname: "Lenger, Teammember",
quoteVorname: "Malte",
quote: "The interview proved to be invaluable in gaining an initial understanding of the principles of pegRNA design and optimisation, particularly in the context of silent edits.",
aimofcontact: "The aim of the contact was to engage in a discussion about prime editing and pegRNAs, as the Jan-Phillip Gerhards had used these technologies in his internship at the Boston Childrens Hospital. We sought to exchange ideas, gather insights, and explore potential improvements or strategies for our project, leveraging his experience with prime editing tools. His practical knowledge in this field was very valuable for refining our approach and ensuring we were aligned with the latest advancements and methodologies in prime editing. ",
insights: "During our discussion we gained valuable insights that had a significant impact on our project. One of the most important findings was the effectiveness of silent edits, which will enable us to make our PrimeGuide safer. Silent edits change the sequence of bases in the DNA in such a way that the resulting protein remains unchanged, because the genetic code is redundant. This means that different codons can code for the same amino acid. By making silent edits in addition to correcting the CFTR gene, we can prevent the pegRNA from rebinding. We have also learned that the length of the primer binding site (PBS) plays a crucial role in determining optimal results and that it is recommended to keep the PBS temperature close to 37°C. Specifically, PBS lengths of 17nt (38.3°C) and 16nt (36.4°C) were found to be ideal options. For our planned set of 12 samples, it was recommended to use three different PBS lengths (differing by +/- 1nt from that close to 37°C) in combination with four reverse transcriptase template (RTTs) to achieve the best result. Another important finding was the use of non-annotated regions with overhangs for cloning, which could give better results in our experiments. However, we also encountered concerns that circRNA, a covalently closed circular RNA molecule, might be sterically hindered by Cas9, which we need to investigate further. When discussing cloning overhangs, we learned that a base-pair length close to 60°C is optimal. However, the use of a 15nt PBS was not recommended as it has a lower temperature range which could affect performance. Although we still need to confirm the oligonucleotide delivery time, these findings will help us to refine our cloning strategy, optimize PBS selection and improve our overall approach to primer editing, especially in terms of the pegRNA design.",
implementation: "We incorporated the lessons learned from our discussions on prime editing and silent editing directly into our project by refining our approach to gene editing. Based on feedback about the optimal length of primer binding sequences (PBS) and RTTs, we adjusted the design of our pegRNAs to ensure greater precision and efficiency in our experiments. In particular, we learned that using PBS lengths close to 37°C melting temperatures (e.g. 16-17 nucleotides) increased stability, which led us to fine-tune these sequences for improved editing results. The concept of silent editing became an integral part of our safety strategy[Link Biosafety], allowing us to make changes to the DNA more precise. We also revised our cloning strategies by considering the appropriate overhang length, targeting a base pair length near the melting temperature of 60°C to improve cloning efficiency. We also reassessed the practicality of ordering shorter PBS sequences, concluding that lengths shorter than 15 nt were less advantageous due to reduced efficiency. By integrating these findings, we optimised our experimental workflow and made informed decisions about the tools and methods for our prime editing experiments. ",
summary: "We engaged in a valuable discussion with Jan-Phillip Gerhards regarding prime editing and pegRNAs, leveraging his internship experience with these technologies. Key insights included the effectiveness of silent edits, which can enhance the safety of our PrimeGuide by modifying DNA sequences without altering the resultant protein, thereby preventing pegRNA rebinding. We also learned the importance of optimizing the primer binding site (PBS) length to achieve ideal temperatures close to 37°C, recommending lengths of 16-17 nucleotides. Additionally, we discovered the potential benefits of using non-annotated regions with overhangs for cloning, while also recognizing concerns about circRNA steric hindrance by Cas9. These insights directly informed our project, allowing us to refine our pegRNA design and cloning strategies, ultimately enhancing the precision and efficiency of our gene editing approach.",
months: "May"
job: "PhD Researcher at Laboratory for Molecular Virology & Gene Therapy",
affiliation: "KU Leuven",
pictureurl: pics['mattijs'],
tag: "Academia",
heading: "Discussion with a Prime Editing Expert on Similar Approaches for Different Mutations",
interviewtabid: "mattijsinv",
quoteNachname: "Bulcaen, CF Research Expert and Gene Therapy Specialist",
quoteVorname: "Mattijs",
quote: "[…] Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure.",
aimofcontact: [<p>Shortly after we decided to use prime editing as the gene editing method for our CF therapy, Mattijs Bulcaen from the Laboratory of Molecular Virology and Gene Therapy at KU Leuven and his colleagues published a paper directly related to our research <TabScrollLink tab="mattijsinv" scrollId="desc-1" num="1" />. In contrast to our approach, Bulcaen et al. 2024 targeted other, less common but drug-refractory CFTR-specific mutations (L227R- and N1303K). </p>],
insights: [<p>The insights gained from this interview proved extremely valuable in shaping our subsequent mechanistic approaches. At that point in the project we were starting to design the components of our prime editor, but we were lacking a broader overview over the state of the field. Mattijs gave us this insight, mentioning techniques like PE3b systems, dsgRNAs and a talk given by <HPLinktoOtherHPTab tab="liu" text="David Liu" />, the principal
investigator behind prime editing that helped us to consider further novel advancements in in Prime Editing and include them into our project. He discussed with us the difficulties that might await us when targeting the CFTR F508del deletion and mentioned that insertions of all the edits possible with prime editing are the hardest to make, the recognition of edits in the region might attract mismatch repair systems and the chromatin organization might negatively impact prime editing efficiency. Also, we learned a
lot about how to design our pegRNAs, with important inputs being the 3’ stem loop motif trevopreQ1 used by Mattijs in his publication and the suggestion to use prediction tools to evaluate sgRNA spacer cutting efficiency. We reviewed our approach of testing pegRNAs using the PEAR reporter system and Mattjis recommended to use HEK cell lines for screening because of their easy handling and naturally impaired mismatch repair system. </p>],
implementation: [<p>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.</p>],
<QaBox q="We have prepared some questions for you. The first question is: You mentioned that it was quite challenging to target the F508 delta mutation. Could you provide more detailed reasons for why this is the case or explain why this mutation is particularly difficult to target compared to others?"
a="Yes, that's the million-dollar question. First of all, let me clarify: our group has never directly worked on that mutation because we immediately focused on the drug-refractory mutations, such as nonsense mutations where the protein is not formed, indel mutations, or severe missense mutations that do not respond to modulator therapies. Of course, we know several groups in the field who either work on gene editing or focus on cystic fibrosis (CF). We've heard from some of them who attempted to target the F508 delta mutation. For example, some collaborators really tried to design different guides but were unable to find anything above the detection limit.
F508del is probably one of the most logical mutations to try to correct, not just for CF but for the entire gene-editing field. If you look at the frequencies of mutations that cause genetic diseases, the F508 delta mutation is by far the most common deletion mutation causing a severe disease. This is because CF, along with sickle cell disease, is one of the most common deadly inherited diseases, and it's overrepresented within CF. So, it makes sense that they would have been trying to target it from the beginning.
Interestingly, if you read the Prime Editing paper by Anzalone, F508 delta is mentioned in the introduction in connection with CF. 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.
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However, last weekend, there was an online seminar where David Liu gave a talk, and he showed some unpublished data indicating that they managed to achieve quite good Prime Editing efficiency on F508 delta. It's worth noting that David Liu rarely presents unpublished data unless the publication is either accepted or very close to acceptance. So, we all kind of expect that the paper will be published soon, perhaps within the next week or at least within a month. From what I saw, it appears they leveraged many of the approaches available today to enhance Prime Editing.
Now, regarding your question about why this mutation is so difficult to target with Prime Editing, I can't provide an exact answer. However, I can list some potential difficulties associated with the mutation, and it’s likely that F508 delta is challenging for several of these reasons. For instance, it could be related to the genomic region itself. Writing insertions can be more difficult; the easiest edits are single-point mutations, followed by deletions, and the most challenging are insertions. This difficulty arises because it involves writing a third strand and then relying on DNA damage repair mechanisms to fix it.
It could also be that the region around the F508 delta mutation is challenging due to flap equilibration or that it attracts pathways such as mismatch repair that negatively impact Prime Editing. Additionally, the chromatin organization around that region could play a role. Over the past year, we’ve gathered clues that chromatin organization significantly affects Prime Editing capability, while this is much less of an issue for Cas9 and base editors.
Studying this is not straightforward; you would need to conduct experiments like ATAC-seq to determine the chromatin organization around the mutation and how it might interfere. I also noticed on a slide that dsgRNAs were mentioned, though David Liu didn't discuss them in his talk. After looking them up online, I found that this technique, published a few years ago by other researchers, is specifically designed to open up chromatin. It seems they use different guides, without the three-prime extension, to open up the chromatin, which could be one way to overcome the limitations in Prime Editing efficiency.
There could be other factors as well, and it’s often difficult to predict what will work and what won't. We have prediction tools for Prime Editing guides that work to some extent, but they are not as effective as the prediction tools available for regular CRISPR guide RNAs. This suggests that the Prime Editing system is more complex than the canonical CRISPR systems, with more variables that can influence success or failure. I hope this answers your question somewhat. " />
<QaBox q="That has already been very helpful, thank you for that. We'll consider this and might look into it a bit more.
Perhaps we could quickly discuss which part of the prime editing complex you think plays the most significant role in making insertions much more challenging compared to deletions. Is it the reverse transcriptase or the RNA? "
a="I don't think it's primarily the reverse transcriptase that's the issue. People have shown that longer insertions are definitely possible. I believe the challenge lies in the process when your cell has to repair the new DNA strand, which is generated and exists as a three-stranded intermediate. We don’t directly intervene in this process; it entirely depends on the cell and the DNA damage repair pathways active in those cells. Through expression of dominant negative DNA damage repair effectors, or by nicking the non-edited strand, the outcome can be steered to some extent.
When you perform an insertion, the new strand must hybridize with the bottom strand, which remains intact. This creates a small loop that needs to be incorporated. At this point, the cell faces two options: it can either revert to the original state or incorporate the edit you’re trying to introduce. In certain circumstances, perhaps due to how the new DNA strand folds or the sequence context of the region of interest, the cell might heavily favor reverting to the original state, resulting in the absence of the intended edit.
This process is extremely difficult to predict, but there are several indications pointing in this direction. For example, in the case of point mutations, it has been shown that it’s easier to convert a C to a G rather than the reverse, simply due to how these mismatches are recognized by the DNA damage repair mechanisms. This area is very complex, and I don’t think anyone fully understands it yet. It’s also difficult to study.
I don't believe the rate of reverse transcription is the limiting factor here, although it could play a role for long or structured pegRNAs. You might have already come across this, but the PE6 generation of Prime Editors, which were released about half a year ago, involve engineered or evolved reverse transcriptases that are more processive and can more easily synthesize longer transcripts.
Another factor that could play a role is the secondary structure of the guide RNA. Each prime editing guide RNA faces a common problem: it has a spacer that binds the bottom strand and a three-prime extension that binds the top strand. Since these two parts of the RNA bind complementary strands, they are also complementary to each other, meaning every prime editing guide has some tendency to bind itself. If the Gibbs free energy is too high, the guide RNA may fold in on itself, preventing it from binding to the prime editor, which then inhibits prime editing.
Additionally, the three-prime extension itself can fold independently. I haven’t specifically examined this for the F508 delta guides, but it is something that can be predicted. There are tools available that can predict the secondary structure of an RNA sequence, and if there’s a significant hairpin structure, it might mean the three-prime extension remains closed, preventing the reverse transcriptase from using it as a template. The PE6 prime editors have been engineered to be more effective in such scenarios, being less affected by secondary structures and better able to read through them. " />
<QaBox q="Yes, exactly, we noticed the same thing when predicting the secondary structure of our guide RNA. As you mentioned, the spacer and the binding site are complementary, so we end up with a really long complementary strand that binds to itself. We were also unsure whether it would open up or remain bound together."
a="I think the Liu lab mentioned in the PE6 paper a threshold for the free energy of the guide RNA structure. They suggest that every guide will behave differently, but there’s often a more complex interaction at play than just a simple threshold. If the free energy is not too low, the guide RNA may still function efficiently and be incorporated into the prime editor, with everything remaining in equilibrium. However, if the free energy is too low, meaning high propensity for self-folding, it can cause problems.
I also recently came across a paper from the group of Keith Joung, another prominent CRISPR scientist from the U.S., where they demonstrated that applying a heat shock to the guide RNA can help it refold. This is particularly relevant if you’re using RNP or mRNA with synthetic guide RNA. They linked this specifically to the self-binding capacity of the guide RNA, suggesting that heat shock can mitigate the issues caused by self-binding. " />
<QaBox q="What would be the application? Would you administer the heat shock in vivo?"
a="I believe they used it to engineer zebrafish embryos or something along those lines. It’s quite specific, of course. If you plan to deliver your guide RNA through a viral vector or similar method for human therapy, the application would differ significantly. You obviously can't administer a heat shock to humans, so it really depends on the context of your application." />
<QaBox q="Okay, that's interesting. Given the time constraints, let's move on to the next question. Due to our limited resources, we are targeting a PE2 system, and we'd like to ask if you see any chances of success with this system. If so, how high do you think the chances of success are? We understand that the PE3 system, as shown in your paper, is much more advanced and performs significantly better. But given our situation, do you think our PE2 system could still be effective, or would you suggest that it only makes sense to use something like PE3?"
a="PE2 can work, but it really depends on your application and the methods you have to assess the editing efficiency. If you can use NGS (Next-Generation Sequencing) for everything, you'll be able to detect edits even with PE2 systems. However, I would generally expect the efficiency to be low. Whenever possible, I would always recommend trying the PE3 system. Could you share what your specific application is, or is that confidential?" />
<QaBox q="So our goal is to eventually use it in vivo, but for now, we're focusing on trying to correct the mutation first in regular cell cultures and then later in primary cells."
a="Is your focus specifically on the F508 delta mutation? If so, we could potentially help you get you started, as we already have constructs and cells with that mutation. We would need to discuss the financial aspects, but we might be able to assist. However, are you fully committed to targeting F508, or are you also considering other diseases or mutations?" />
<QaBox q="The timeframe of the project, combined with the fact that we’re all studying on the side, limits us to a certain scope. Since this is our first time tackling a project like this, it makes sense to stick to something more manageable. So, we're somewhat committed to focusing on F508 due to these constraints."
a="That's understandable. It can be really tough to juggle a project like this along with exams and studies, especially if you're also involved in competitions. But it's definitely worth the effort, even if you don't achieve huge results right away. The experience and learning, as well as the connections you make, are incredibly valuable. I'm a big supporter of such projects. So, what resources do you currently have? Do you already have cells with the F508 delta mutation, or...? " />
<QaBox q="We have one patient who is willing to provide us with cells, but we don't have them yet. "
a="It sounds like you're aware of the challenges, and I don't want to discourage you, but just to be realistic, working with primary cells and getting everything ready could be tricky, especially considering the competition is in October. Experiments in human cells can take time, especially if you need to do multiple iterations or clone constructs—it could easily take a week or more per experiment.
Regarding the cells we have, as mentioned in our paper, we screened all our guides on HEK cells with an integrated copy of the CFTR cDNA. HEK cells are easy to work with, but they don't naturally express CFTR, even though the gene is present in their genome. So, we introduced the mutation of interest into these cells, making it easier to screen.
I'm not entirely sure if we can send over the cells due to ethical regulations, which can be complex and time-consuming to navigate. However, there's an alternative approach that might help you. Early on, we found that it's actually quite easy to screen guides using what we call a 'transient target'. In this method, you would transfect all your prime editing plasmids into HEK cells, along with a plasmid containing the CFTR cDNA with the mutation of interest. While this approach isn’t as physiological as editing the chromosome directly, our side-by-side comparisons showed almost equal efficiencies between transient and chromosomal targets. It's much easier and faster than working with patient-derived cells. I can definitely send you the plasmid, which would save you a lot of time and effort. This method is much simpler and could be a practical solution for your project. " />
<QaBox q="Our initial plan is to work with a reporter plasmid that expresses eGFP, where we've removed a splice site, until we have patient cells or cell lines with CFTR mutations. This will allow us to screen easily without needing to sequence everything. Do you maybe have any suggestions or advice on this approach? "
a="Is that the PEAR system? No, it’s a different one, but we also have a similar system. The advantage of this approach is that you can very easily see if it works, and it’s very sensitive—much easier than extracting and sequencing DNA. The downside, however, is that… actually, I’m not familiar with the 'flu PEAR system.'
Actually, we use the exact same system in our lab. It’s very useful for optimizing delivery strategies because it’s easy to see results. The downside, of course, is that the guides you’re using for that system aren’t specific to the F508 delta mutation, right? So, these are scientific trade-offs. You could, for example, design a reporter that uses your F508 delta guide and also results in fluorescence, but you would need to design the reporter first. It’s challenging to prove that it works because you might not have a perfect guide for F508 delta.
It really depends on what you want to achieve. If your goal is to first check if you can successfully perform prime editing, then using the reporter is definitely a good first step." />
<QaBox q="We will edit the plasmid, specifically the vector, so that we have almost the same pegRNA. The only difference will be downstream, behind the edit."
a="Is this approach based on a paper from the Netherlands, or is it something you came up with yourself? " />
<QaBox q="Based on a paper. "
a="Yeah, that sounds like a very good way to start. Do you already have the reporter plasmid ready? " />
<QaBox q="Yeah, we bought the reporter, and now we’re making the necessary edits so we can use it. "
a="Okay, so do you also already have guides targeting F508 right now? " />
<QaBox q="We’ve designed some guides, but we haven’t tested them yet. That’s one of our next steps. So, at the moment, we’re just in the design phase, or we have already designed them, and..." a="Yeah, okay, cool. Good luck with that! And I suppose you’re starting off with HEK cells as well, right?"/>
<QaBox q="We have HEK and HeLa cells, but we haven't decided yet which ones we'll use." a="I would start off in HEK cells because, by total accident or coincidence, they are much easier to achieve prime editing in. This is because the MLH1 gene, which negatively impacts prime editing outcomes, is naturally disabled in these cells—they don't produce the MLH1 protein. Of all cell lines available, HEK cells are the easiest to achieve editing with, so I would definitely recommend starting there. In terms of transfection, HEK cells are also very easily transfected. If I can offer another piece of advice, always include GFP controls—plasmids that simply express GFP without requiring editing—and use them to determine your transfection efficiency. It's crucial to have a very high transfection efficiency because you'll be working with a three-component system: your reporter, your prime editor, and your guides. All three plasmids need to be present in the same cell for the editing to occur, so you should aim for at least 70% transfection efficiency, preferably 80% or higher. I don't know what transfection method you're planning to use, but we've always used Lipofectamine 3000. It’s expensive, but it works very well. However, if you're looking for more cost-effective options, we recently discovered two other transfection reagents, Jet Optimus and Jet Prime, which are much cheaper and also work quite well. That said, I would advise against starting with any of the cheaper transfection reagents; you really need to aim for high transfection efficiency. Always make sure to measure and report transfection efficiency for every experiment because if it's low, the experiment might not yield useful results. If you have the funds or resources, I would also recommend designing P3 or even P3b guides, as they might offer better efficiency. When it comes to designing P3b guides, if you're primarily focused on P2 right now, there are some specific considerations to keep in mind. I'll provide you with a site that can help with this, and I'll give you the link in just a moment. So, it's very advisable to check the Doench score. Do you know what it is?"/>
<QaBox q="No, not really." a="There are papers by John Doench, an American researcher, from quite a while ago that, in my opinion, are some of the best around. He developed a comprehensive scoring matrix specifically for regular Cas9 that can evaluate the quality of the spacer in your guide RNA. This is important because Cas9 tends to prefer certain sequences over others. For instance, a good spacer should have an appropriate GC content and should avoid hairpins that might cause it to fold in on itself, which would prevent it from functioning properly. You can use this matrix to give a score for the quality of a guide RNA. I’m going to pull up an example here. The site from Synthego, a commercial provider of CRISPR reagents, allows you to check the quality of your guide. When you validate it, the site gives a score based on various factors, including off-target effects, although that might not be your primary concern at the moment. If you hover over a specific area, it will show you the Doench Score, which is crucial. Ideally, you want a guide with a good Doench Score. A good score starts at around 0.4, indicated by a green check mark for good efficiency. If the score is very low, it means that the guide likely has low CRISPR-Cas9 activity and may not be very efficient. When designing prime editing guides, RNA, we always check the spacer for a good Doench Score. If we are designing nicking guides for a PE3 or PE3b strategy, we also ensure that they have a good score. This is one of the easiest tools to check for that. Whenever possible, try using PE3. In some cases, PE3 performs better than PE2, though not always. PE3b might not always work either, but for many mutations, we have seen significant increases in editing efficiency by including the PE3 guide."/>
<QaBox q="Okay, yeah, that was quite clear from your results; the diagram illustrated that very well. Are there more off-target effects when using PE3 since you have to make another cut?" a="If you decide to use PE3, it's important to be aware that while it's not exactly an off-target issue, there is a risk of an undesired on-target outcome. The concern with regular PE3 is that both strands of DNA can be nicked simultaneously, which can lead to a staggered double-strand break. This can result in the formation of indels (insertions or deletions). In your case, this means that if the region around the F508 delta mutation is broken, the prime editor might not be able to repair it properly, leading to additional base pairs being removed or added, and thus, the sequence might be altered in an unintended way. The risk of on-target indels is definitely higher with PE3 compared to PE2. However, this risk is reduced when using PE3b, which employs sequential nicking. The PE3b nicking guides are designed to recognize the wild-type sequence, and they can only nick the opposite strand if the correction has already been made on the top strand. This sequential action helps to avoid the generation of indels. Introducing a second guide into the system also brings the possibility of off-target editing by that guide however, since only a Cas9 nickase is used, off-target indels should be limited."/>
<QaBox q="Yes, okay, thank you. Do you have time left, or are we out of time?"a=" It's fine."/>
<QaBox q="We have more or less one last question. If it’s not possible, that’s completely fine. We just wanted to ask if you could possibly forward the contact details for the Ussing chamber setup in Paris that you mentioned in your email. Would that be possible?" a="You can certainly try to contact them, but I actually know that there are quite good labs in Germany that work on similar things.
One major drawback for you might be the time it takes to differentiate cells. If you harvest stem cells or basal cells from patients, they will have the CFTR gene, but they don’t express it immediately. It takes about four weeks for them to differentiate and start producing the CFTR protein. Without this differentiation, you can't measure the currents, which could slow you down significantly. I'm not sure if you have that kind of time.
If I can give you one piece of advice: it’s less physiological, but it’s still an accepted assay—try it on organoids. We could actually perform both assays here. If you find guides that work really well, we could consider doing those tests here. Someone could come over, or we could do the experiments if they’re not too expensive and have a good chance of working. I think we wouldn’t mind adding the F508 delta mutation to our list of editable mutations.
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. "/>
<QaBox q="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."a="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.
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. On top of that, the cells need to be differentiated properly, and you have to know how to handle them correctly. The medium required is very expensive, and working with these cells is almost more of an art than a science. You have to know when the cells look 'happy' or not because you don't want to waste time on cells that aren't in good condition. I've run quite a few of these assays myself, and while they are great for CF work and provide results that are relevant to patient outcomes, they are technically challenging and very demanding. If you want a functional output to show that the CFTR protein is working again, I would recommend starting with one of the easier models, like organoids. We also have in our lab 16HBE cells with a YFP sensor. I don't know if you've heard or read about that. These cells express YFP, which is sensitive to halide ions, including chloride and iodide. When you add a buffer containing these ions to the cells, the YFP intensity quenches. This is something we typically use in our experiments. For wild-type cells, you see a rapid and dramatic quenching because CFTR allows these ions to enter the cells. In cells with the mutation, there’s no quenching because the channel isn’t working. While it’s less relevant because these aren't patient cells, it’s closer to reality. The 16HBE cell line is an airway epithelial line, and the expression of CFTR is endogenous, so it’s not at the exaggerated levels you might see in more artificial models like HEK cells. Using the YFP assay could be a good alternative or a Plan B for getting a functional readout. This assay is medium to high throughput—you can run entire 96-well plates in about half an hour. All you need for this is the cells and a plate reader that can measure fluorescence and inject the buffer. If you don’t have a plate reader with an injection system, you can also manually add the buffer and quickly place the plate in the machine. "/>
<QaBox q="Yes, that sounds quite good. I think we’ll definitely consider that as a method.
If you have a little more time, I wanted to ask about the pegRNA. You stabilized it with a stem loop or some kind of motif in the paper, like the trevopreQ1. Did you test other motifs as well, or...? " a="Yeah, there was actually one published before the one from the Liu lab, but we tried that one—I can't remember the exact name, maybe CSX4 or something like that. It didn’t even make it into the main papers, just the supplementary material. The principle was similar, and according to the paper, it should have worked. I tested it on two or three guides, locations, or mutations, but it didn’t work. However, the trevopreQ1 one definitely works.
Also, something to note is that these motifs become more important when you move to primary cells, where you have fewer guides. In transfection experiments, where you’re essentially flooding the cells with plasmids that transcribe large amounts of these guides, the protection of the three-prime end isn’t as critical. But in primary cells, where there are more nucleases and you likely have less pegRNA due to your delivery method, this protection becomes more important. The paper does discuss the trevopreQ1 one and tMPK knot, but due to time constraints, we only tested one and didn’t look into the other.
I think the advantage of the one we included in the paper was that they also investigated whether it was necessary to include a linker between the motif—like the trevopreQ1—and the pegRNA with the three-prime extension. For the motif we chose, they found that the linker wasn’t really necessary, whereas it was for the other one. To keep it simple, we chose the one without the need for a linker because, for many sites, the efficiencies were comparable.
If we had infinite time and resources, it would definitely be worthwhile to test both motifs. The paper did show some examples where one motif was clearly better than the other. But no, we didn’t have the time to explore that further. "/>
<QaBox q="Okay. Thank you. But like it's shown in the paper, it worked quite well. Right. Yeah. " a="There was definitely added value. When we moved to organoids, the effects of adding that motif were quite dramatic and very clear. "/>
<QaBox q="Yes, that was our last question. Thank you so much again for having me. We really appreciate the time you took for us, and also for your very detailed answers and your help." a="No problem. My pleasure."/>
<QaBox q="Thank you so much. We will definitely keep you updated on how it goes. Thank you again! "a="It was a pleasure"/>
</>,
references: <MattijsInterviewSources />,
summary: "Our discussion with Mattijs Bulcaen from KU Leuven provided critical insights into the complexities of using prime editing for CF therapy. As we began designing our prime editor, Mattijs highlighted challenges specific to targeting the CFTR F508del deletion, including the influence of mismatch repair systems and chromatin organization on editing efficiency. He introduced us to advanced techniques, such as PE3b systems and dsgRNAs, and recommended using the 3’ stem loop motif from his research to enhance our pegRNA design. Additionally, he advised utilizing HEK cell lines for screening due to their ease of handling and reduced mismatch repair activity. These insights directly influenced our design choices and helped refine our approach to developing an effective prime editing strategy.",
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
tag: "Milestone",
heading: "From Insights to Impact: Enhancing Awareness and Prime Editing for Cystic Fibrosis",
interviewtabid: "inisghts",
cardtext: "",
quoteNachname: "Köhler, Teammember",
quoteVorname: "Vera",
quote: "I’m excited about our partnership with Mukoviszidose e.V. Deutschland to raise awareness in our community. Educating people about cystic fibrosis and gene therapy is essential, and I believe our scientific advancements will have a broader impact beyond just this condition.",
summary: [<p>
During the early stages of our project, we discovered through our survey that while many participants were open to trying gene therapies, they lacked adequate knowledge about them. Additionally, most respondents were unfamiliar with cystic fibrosis, highlighting the need for greater awareness. Driven by a desire to educate the people of Bielefeld, we collaborated with Mukoviszidose e.V. Deutschland to support the Muko Move campaign, a successful initiative aimed at raising awareness about cystic fibrosis. On the scientific front, we elevated our project to a new level. With valuable feedback from Mattijs Bulcaen at the University of Leuven, we incorporated a novel RNA structural element into our Prime Editing complex, significantly improving its efficiency. After successfully optimizing the pegRNA, we moved forward with enhancing the Prime Editing protein complex. Our goal is to make Prime Editing not only safer but also easier to apply, so that our research can benefit more than just cystic fibrosis patients, ultimately broadening the impact of our work.
</p>,
<ul>
<li>
<strong>Survey Insights:</strong> Many participants were motivated to try gene therapies but lacked knowledge about them and cystic fibrosis, highlighting the need for better public education.
</li>
<li>
<strong>Awareness Campaign:</strong> Partnered with Mukoviszidose e.V. Deutschland to support the Muko Move campaign, raising awareness about cystic fibrosis in the local community.
</li>
<li>
<strong>Scientific Advancements:</strong> Improved the efficiency of Prime Editing by incorporating a novel RNA structural element, with further efforts to optimize the Prime Editing protein complex for broader applicability beyond cystic fibrosis.
</li>
</ul>],
{
vorname: "'Der Teuto ruft'",
nachnname: "",
pictureurl: pics['teuto'],
tag: "Education",
heading: "Educational city tour for young and old",
interviewtabid: "teuto",
type: "meta",
cardtext: "",
quoteNachname: "Sahami Moghaddam, Teammember",
quoteVorname: "Asal",
quote: "I had a great time educating the kids about gene therapy and CF in a playful way, and there were some very interesting conversations with the parents.",
summary: [<p>"Der Teuto ruft!"[LInk zu Teuto unten] is a community event in Bielefeld where iGEM Bielefeld participated to raise awareness about cystic fibrosis (CF) and gene therapy. They engaged children through interactive experiments, such as creating lung models and simulating mucus to help them understand the challenges faced by CF patients. Adults were informed about their gene therapy project and had meaningful discussions about the implications of CF treatment. Collaborating with other institutions like the life science student initiative btS, the team expanded their outreach. Despite changeable weather, the event was a success in educating the public and improving science communication.</p>],
{
vorname: "Julia",
nachnname: "",
job: "parent",
affiliation: "independent",
pictureurl: pics['julia'],
tag: "Patient",
heading: "Interview with a CF Parent about their experience and treatment needs",
interviewtabid: "julia",
cardtext: "",
language: "de",
quoteNachname: ", Mother of a CF patient",
quoteVorname: "Julia",
quote: "At first, our world fell apart. I still remember the conversation with the doctor. ",
aimofcontact: [<p>We learned from our discussion with <HPLinktoOtherHPTab tab="maxfirst" text="Max" /> that CF has a profound impact on the whole family – not just the patient. In order to gain further insight into this subject, we sought to engage with the next of kin of CF patients.
We were able to make contact with Julia through the self-help group of <a href="https://www.muko.info/ " >Mukviszidose e.V. </a> of which Max is a member. She subsequently reached out to us following Max's request for potential candidates for an interview with a patient group.
She and her husband have a six-year-old daughter carrying the F508del mutation in the CFTR gene and a toddler without CF. </p>],
insights: [<p> The interview with Julia shifted our focus to a new group of stakeholders: The patient’s support systems. Most people do not get genetically tested before having children and due to that, many people could get in the position of having a loved one with CF.
We considered the societal impacts, such as the rising health care costs, which Nicole Friedlein emphasized during our interview. She explained how the long-term nature of treatment, frequent hospital visits, and the need for specialized medications place a significant
financial burden on both patients and the health care system. This insight shaped our understanding of the broader economic challenges faced by families and institutions involved in managing chronic illnesses. Meanwhile, Julia brought attention to the psychological impact,
stressing the emotional strain that accompanies not only the illness itself but also the financial pressures. She also showed us more perspectives on parenting of children with CF, than we heard before, and told us about the way from the first diagnosis to growing accustomed
to and living with a child with CF. Julia also confirmed that most children will have no issue using an inhalative therapy like we envision our gene therapy to be and shone light onto the comparatively very good situation for CF patients in Germany. </p>],
implementation: [<p> This interview helped us confirm the delivery method we planned to use as we were previously concerned how and if children would be able to use the inhalative therapy. Besides that, Julia gave us further insights into the emotional side of
dealing with CF and we were able to discuss the situation for patients in Germany in comparison to other countries better in later <HPLinktoOtherHPTab tab="joshua" text="interviews" />. </p>],
interview: <>
<QaBox q="Can you tell us a bit about your family? How old are your children and yourselves?" a="I’m 37, my husband is 44, and our daughter is six, turning seven soon. We also have a son who’s about a year and a half." />
<QaBox q="Does your son also have cystic fibrosis?" a="No, he doesn’t." />
<QaBox q="When was your daughter diagnosed with cystic fibrosis?" a="Right after birth. She was transferred to a bigger hospital due to an intestinal blockage and had surgery. After about two to three weeks in intensive care, the cystic fibrosis diagnosis came through newborn screening. At that time, the results took longer to process than they do now." />
<QaBox q="That intestinal issue can happen for many reasons, right?" a="Yes, it was all new to us. The beginning was difficult, but things have gotten better since then, and we’re very grateful." />
<QaBox q="How did you feel when you first heard the diagnosis?" a="It felt like our world was falling apart. I still remember the moment—it was like being in a movie. We were told in a separate room, and it felt overwhelming. One doctor even suggested we go home to think about it in peace, but all I could think about was returning to my child. It was a lot to take in, especially thinking about how we’d tell our family." />
<QaBox q="That sounds incredibly hard. How did you handle it as time passed?" a="It was tough, but we were fortunate to have a doctor who really understood what we were going through, as he had a disabled child himself. He never scared us unnecessarily and guided us step by step, which made a big difference. We know many families who live in constant fear, but since those first months, we’ve learned to manage the situation without being overwhelmed by fear." />
<QaBox q="Did any particular support help your family adjust to the diagnosis?" a="Yes, the rehab program we attended was a huge help. It was a family-oriented program, so my husband could be there too, which was important since I manage most things day-to-day. It really helped our daughter realize she’s not alone—she met other kids with similar conditions, which was a huge comfort." />
<QaBox q="How did you explain the illness to your daughter?" a="We try to give it as little attention as possible in daily life. She’s been inhaling medication since she was eight weeks old, and it’s just part of her routine now. Thankfully, she doesn’t fight it or question it much, and her school and kindergarten haven’t made a big deal of it either, which is what we wanted." />
<QaBox q="Does she ever ask about her illness compared to her younger brother, who doesn’t have cystic fibrosis?" a="She does sometimes ask why she’s sick and he’s not, but she’s not upset by it. We’ve made sure not to give her any special treatment because of her illness, which can be hard at times, but we want her to understand that her illness doesn’t define her." />
<QaBox q="That sounds like a good balance. What about medications—did she start on any special treatments?" a="Yes, she started on Orkambi at around three years old but had to stop briefly due to high liver values. Now she’s on Kaftrio, which she started shortly before her sixth birthday, and it’s been going well." />
<QaBox q="Did you face any issues with the health insurance for covering these medications?" a="Fortunately, no. We have statutory health insurance, and they’ve covered everything without any issues. We’ve heard it can be more complicated for those with private insurance." />
<QaBox q="Have you ever had difficulties with access to medication?" a="Yes, there have been times when we’ve had to wait a few days for certain medications, like Kreon or antibiotics, especially in the winter. But we always plan ahead and keep a buffer, so we’ve never been without what we need." />
<QaBox q="What would you say has been the most affected area for your daughter?" a="Her intestines are the most affected. Before she started Kaftrio, she had fatty stools and frequent bowel movements, even with the right Kreon dosage. Since starting Kaftrio, this has improved significantly." />
<QaBox q="What kind of support would you have liked to receive earlier?" a="We wish we had been given more information about available services early on. We found out about Mukoviszidose e.V. from another family, not from our doctor. It would have been helpful to know about these resources right from the start." />
<QaBox q="How about psychosocial support?" a="Initially, we didn’t have any psychological support—our doctor took care of everything. Now, where we live, there are more resources, and we think it’s a good thing. The rehab helped a lot in coming to terms with everything. We wish we had known about such services sooner." />
<QaBox q="Does your daughter do physiotherapy?" a="Yes, once a week for about an hour. She’s been going since she was discharged from the hospital, and she has a close bond with her physiotherapist. They’ve been working together since she was a baby, and she goes by herself now." />
<QaBox q="Are there any restrictions for her in terms of physical activities?" a="No, not really. She does dancing once a week, physiotherapy, and she’s even done a swimming course without any problems." />
<QaBox q="How do you handle communicating about her illness?" a="We try not to make a big deal of it. When I looked for information, I found what we needed. There’s nothing we’ve really felt was missing." />
</>,
summary: "Julia's insights shifted our focus to the support systems surrounding CF patients. She highlighted the societal implications of CF, including rising healthcare costs due to the long-term nature of treatment and the financial burdens faced by families. Additionally, Julia emphasized the emotional strain that accompanies the illness, alongside the complexities of parenting a child with CF. Importantly, she affirmed that most children adapt well to inhalative therapies, reinforcing our planned delivery method for gene therapy. This interview enriched our understanding of the challenges faced by families and enabled us to better compare the experiences of CF patients in Germany to those in other countries.",
months: "june"
},
{
title: "Prof. Dr.",
vorname: "David",
nachnname: "Liu",
job: " Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare",
affiliation: "vice chair of the faculty at the Broad Institute of MIT and Harvard",
pictureurl: pics['david'],
tag: "Academia",
language: "en",
heading: "Influence of research by David Liu on our design decisions ",
interviewtabid: "liu",
cardtext: "",
quote: "X",
aimofcontact: [<p>David Liu is the principal investigator responsible for the development of the prime editing systems and his laboratory is actively working on improving prime editors, also for application in CFTR mutation F508del. </p>],
insights: [<p>The talk and papers from David Liu not only introduced advances for prime editors, but also included valuable data for us to decide on which method might work best for our project. Because of this we decided to use an advanced system based on the PE6c and PE4 systems, both of which were published by the laboratory of David Liu. </p>,
<p>As the principal investigator responsible for the development of the prime editing systems. The initial prime editing system was developed in his laboratory
<TabScrollLink tab="liu" scrollId="desc-1" num="1" />
and since then a number of papers on this topic have been published under his supervision. The research conducted by David Liu and his laboratory was the inspiration for our gene editing approach and thus absolutely crucial in the development of PreCyse. Unfortunately, we were not able to talk to David Liu himself or one of his coworkers, but the impact of their publications can be seen in a big part of the mechanism engineering timeline. When speaking to
<HPLinktoOtherHPTab tab="mattijsinv
" text="Mattijs Bulcaen" />
in June, he mentioned that David Liu had recently given a talk on recent advances in the prime editing field at the online conference <a href="https://www.genengnews.com/multimedia/the-state-of-crispr-and-gene-editing-2024/" >'The State of CRISPR and Gene Editing 2024'</a> hosted by Genetic Engineering & Biotechnology News.
In his talk, David Liu presented both published as well as unpublished work. One of the most significant takeaways for us were the use of evolved improved and smaller reverse transcriptases in PE6 prime editing systems
<TabScrollLink tab="liu" scrollId="desc-2" num="2" />
. We were also very interested in the protein evolution strategies used to create these new enzymes called PACE and PANCE (phage assisted (non-)continuous evolution <TabScrollLink tab="liu" scrollId="desc-3" num="3" /> and the idea of using this system to optimize our planned new endonuclease domains for the prime editors. We eventually discarded this idea because of time constraints. Apart from that, David Liu presented results from his at this point unpublished paper about CFTR F508del correction using prime editing, which was especially interesting for us considering the goal of our project. When the paper was published on the 10th of July 2024 <TabScrollLink tab="liu" scrollId="desc-4" num="4" />, it actually supported some of our already made pegRNA design decisions, such as the choice of the targeted protospacer, the introduction of silent edits and the use of the trevopreQ1 stem loop. Additionally, it offered novel insights into prime editing systems suited for F508del correction, such as PE6, and optimized primer binding site as well as reverse transcriptase template lengths. </p>
],
implementation: "x",
summary: " David Liu, the principal investigator behind prime editing systems, has significantly contributed to the development of advanced gene editing techniques, including applications for the CFTR mutation F508del. His research provided valuable insights for our project, leading us to adopt a system based on the PE6c and PE4 prime editors. Although we couldn't directly engage with Liu or his team, the impact of his work is evident in our gene editing approach for PreCyse. Notably, Liu's recent talk at the 'State of CRISPR and Gene Editing 2024' conference highlighted advancements such as improved reverse transcriptases and their applications in prime editing. The publication of his findings on CFTR F508del correction in July 2024 further validated our design choices for pegRNA and offered new strategies for optimizing our approach.",
tag: "Academia",
heading: "Discussion on how health insurance companies manage cystic fibrosis patients and gene therapy treatments",
interviewtabid: "nicole",
quote: "Public health insurance operates under an economic efficiency principle, meaning the most cost-effective treatments are preferred. But if gene therapies become the only treatment option for certain conditions, they will likely have to be included in the coverage, which could be a challenge for the system.",
aimofcontact: "The main objective of the contact was to learn from the discussion on issues related to cystic fibrosis (CF), gene therapy, health insurance processes and regulatory pathways. In particular, we wanted to understand the real-world challenges and technical aspects of gene editing, especially prime editing, as well as the complexities of approval and reimbursement of gene therapies for CF patients.",
insights: "The regulatory approval process, particularly by the European Medicines Agency (EMA) for advanced medical devices, has highlighted the bureaucratic hurdles that gene therapies must overcome. We learned that such therapies for cystic fibrosis have to navigate complex European and German regulatory systems. The discussion on the AMNOG process was crucial. We learnt that the additional benefit of a therapy is assessed for reimbursement by the statutory health insurance funds. We implemented this insight in our project by considering the long-term regulatory and economic effects as important milestones for therapy development. We also gained insight into how public and private health insurers may differ in their reimbursement of such therapies. Public insurers have stricter guidelines, while private insurers can be more flexible, but both require strict justification, especially for rare diseases such as cystic fibrosis. Information on newborn screening and genetic counselling covered by public health insurance was crucial to understanding how preventive measures for CF are managed. This underlines the importance of early intervention and diagnosis in our project. Atypical forms of CF, where health insurance companies do not cover treatment due to non-standardised test results, were identified as a key problem. This helped us to recognise the need for more adaptable insurance policies and clearer pathways for the treatment of atypical cases in our project plans. The debate about whether healthcare systems can afford the high costs of gene therapies highlighted an important issue in the current medical landscape. We have incorporated this insight into our project by discussing possible cost-effective alternatives and the need for thorough cost-benefit analysis in the development of treatments.",
implementation: [<p>After the interview, we further tailored our project to focus on a simple delivery method to reduce the therapeutic effort. To gain an overview of the regulatory requirements and to better deliver the project, one of our team members attended a <HPLinktoOtherHPTab tab="gxpcourse" text="GxP course"/> to ensure we met all the necessary standards. To deepen our knowledge of entrepreneurship, we conducted further interviews with start-ups and <HPgoToPageAndScroll id="Further Engagement3H" path="\human-practices" text="industrial companies" />, which gave us important insights into practical implementation. These steps ensure that our project is not only based on scientific research, but also takes into account the practical, regulatory and social aspects that are crucial to bringing new CF therapies to the market. We are currently developing strategies to successfully implement our ideas and the project in the future.</p>],
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<QaBox q="To start with this interview. Do you have any questions about this project?"
a="Are you writing a paper on this, or are you conducting actual laboratory research? Or is it primarily literature review? How does your work look?" />
<QaBox q="It’s not just literature review, though we do start with that. We have a lot of lab work ahead of us. Ideally, we would have a finished construct to present at the end, maybe even a functional gene therapy, though that’s quite ambitious and probably not possible in the short time frame. We’re working on various gene-editing approaches and testing plasmids in HEK cells. We are also exploring Prime Editing and trying to improve its efficiency using different reverse transcriptase enzymes. So, it’s a mix of lab work, literature research, and preparing for a presentation at a competition."
a="Are you writing a formal paper?" />
<QaBox q="We’re not writing a formal text-based paper, but everything will be available on a website. We will document most of our work on the website, with sub-pages detailing lab work, interviews, and research."
a="What exactly is Prime Editing, and how does it differ from altering the germline? Where in the genome does this therapy act?" />
<QaBox q="Our current plan is to deliver the therapy via a lipid nanoparticle system, which will be inhaled and go into the lungs. While cystic fibrosis (CF) affects all mucus membranes, the lungs are the most critical area, so we’re focusing on that. The therapy will only target surface cells in the lungs, not the basal cells responsible for producing new lung cells."
a="Thank you for giving me insights into your project." />
<QaBox q="Do you know how cystic fibrosis (CF) approval works in terms of health insurance and regulatory processes?"
a="The approval process for gene therapies is primarily done through the EMA (European Medicines Agency) under specific EU regulations for Advanced Medical Products, including gene therapies. There is also a national approval process in Germany for individualized treatments, but large-scale therapies must go through the EU process." />
<QaBox q="Can you share more about the approval and reimbursement processes for CF treatment?"
a="The approval process is separate from reimbursement by public health insurance. CF is considered a rare disease if it affects fewer than five out of 10,000 people, and treatments for rare diseases often face special reimbursement challenges. If more than five out of 10,000 people are affected, the disease is relatively common, and approval and reimbursement go through a different procedure known as the AMNOG process. For more common diseases, an additional benefit (Zusatznutzen) must be demonstrated during the approval process." />
<QaBox q="Have you heard about issues with reimbursement from private insurance companies?"
a="We’ve heard that private insurance companies can make it difficult to get treatments reimbursed, especially experimental ones. One of our colleagues almost had to go to court to get his treatment reimbursed by his private insurer, which was quite expensive. Eventually, he switched to public insurance, but the situation was difficult." />
<QaBox q="Why did your colleague have issues with private insurance?"
a="He was privately insured, but the treatment was very expensive, around €16,000 per month, and the insurance company was reluctant to cover it." />
<QaBox q="Do you need legal information for your project?"
a="Both. We want to be well-informed to identify potential obstacles early on, such as legal restrictions or bans on altering certain chromosomes. Although we won’t be running clinical trials, understanding the regulatory landscape is crucial for our future planning." />
<QaBox q="How does genetic counseling and testing work for CF?"
a="Genetic counseling and testing are usually covered by health insurance if there’s a medical reason, such as a family history or suspicion that the parents might be carriers. However, if both parents are healthy and there’s no family history of CF, insurance might not cover the tests." />
<QaBox q="Are there differences between public and private insurers for genetic tests?"
a="Public insurance has different regulations than private insurance, but I’m not entirely sure if that leads to different decisions regarding genetic testing. I can look into the public insurance regulations if that would be helpful." />
<QaBox q="Is newborn screening for CF covered by health insurance?"
a="Yes, newborn screening is part of a set of health examinations for children and adolescents, regulated under §26 SGB V (Social Security Code). Since it’s part of the regular screening process, it’s covered by health insurance without additional requirements." />
<QaBox q="How does public insurance handle CF treatment when a test comes back negative?"
a="Public health insurance works with standardized guidelines, and if a test comes back negative, it may no longer meet the criteria for coverage. However, if a doctor reconfirms the diagnosis, the treatment should continue to be covered." />
<QaBox q="Is there no rule that says genetic diseases, once diagnosed, should remain covered since genetics don’t change?"
a="In theory, yes. But the guidelines are usually based on medical evidence at the time, and re-testing can sometimes lead to complications in terms of coverage if the result differs. However, with proper medical documentation, it should be possible to maintain coverage." />
<QaBox q="Have recent changes in gene therapy costs impacted public health insurance?"
a="Not much has changed. It’s a political and societal question—how willing are we to finance these expensive therapies? Right now, public health insurance operates under an economic efficiency principle, meaning the most cost-effective treatments are preferred. But if gene therapies become the only treatment option for certain conditions, they will likely have to be included in the coverage, and it could be a challenge for the system. There are also ongoing price negotiations between insurers and manufacturers." />
<QaBox q="Do patents play a significant role in keeping gene therapy costs high?"
a="Yes, patents certainly influence the price, but the production of gene therapies is inherently expensive due to the complex research and manufacturing process." />
<QaBox q="Would private supplemental insurance be an option for covering expensive gene therapies?"
a="It’s possible that private supplemental insurance could cover these therapies if public health insurance doesn’t. However, this raises concerns about equity and accessibility. If public insurance doesn’t cover it, the burden might fall on private insurance, which could create disparities in access to treatment." />
<QaBox q="Is gene therapy research driven more by biology or medicine?"
a="It’s definitely interdisciplinary. Both biologists and medical professionals contribute. For example, at our university, the medical and biology faculties collaborate closely. Biologists usually handle the research, while medical professionals focus more on clinical applications." />
<QaBox q="Do biologists or medical professionals develop gene therapies?"
a="In terms of development, it’s mainly biologists and biotechnologists. Medical professionals get involved primarily in clinical trials. Some doctors do research, but they’re often needed in hospitals, so hands-on development is mostly handled by molecular biologists or biotechnologists." />
<QaBox q="Does research in genome medicine and gene therapies come from biology, medicine, or both?"
a="It’s mainly interdisciplinary. A lot of funding comes from industry, like BioNTech, or foundations like Mukoviszidose e.V., which funds research on cystic fibrosis. But in terms of practical research, it’s usually biologists or biotechnologists. Without industry support, research can struggle due to a lack of funding, so having backing is essential." />
</>,
summary: "Our discussion addressed the complexities of cystic fibrosis (CF) treatments, focusing on gene therapy and health insurance processes. We learned about the regulatory challenges gene therapies face, particularly regarding the European Medicines Agency (EMA) and the AMNOG process for reimbursement assessments. Public insurers impose stricter guidelines than private insurers, emphasizing the importance of early intervention in CF and the need for adaptable policies for atypical cases. We recognized the high costs associated with gene therapies and incorporated cost-benefit analysis into our project planning. Following the interview, we refined our approach to include straightforward delivery methods and attended a GxP course for regulatory compliance. Engaging with start-ups further informed our practical implementation strategies, ensuring our project aligns with both scientific and regulatory needs.",
months: "june",
pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/zoom-nicole.webp",
vorname: "Visiting Achema ",
nachnname: "in Frankfurt",
pictureurl: pics['frankfurtmesse'],
job: "Team iGEM",
affiliation: "Bielfeld CeBiTec 2024",
heading: "Some of our team members attended the fair in Frankfurt to network with industry leaders",
quote: "Achema was a great experience. I got to meet a lot of interesting people and learn about their projects. It really motivated me for my own work.",
quoteNachname:"Wiesner, Teammember",
quoteVorname: "Lisa",
aimofcontact: [<p>At Achema 2024, we aimed to connect with industry leaders and innovators, to explore potential collaborations and gather insights on laboratory practices. Our goal was to enhance our understanding of cutting-edge technologies and resources in the life sciences sector.</p>],
insights: [<p>During our time at Achema, we gained valuable insights into life sciences, with a strong focus on LNP production and lab best practices. Our discussions with experts, including Jutta from a life science company, provided us with innovative solutions and cutting-edge techniques. Her insights into current trends helped us better understand how to advance our project.</p>,
<p>Through various expert interactions, we deepened our knowledge of LNP production, learning key manufacturing techniques essential for developing effective therapeutics. These insights allowed us to optimize our production methods, ensuring more efficient processes moving forward.</p>,
<p>Understanding rigorous quality control processes was another key takeaway, enabling us to implement checks that will boost the reliability and safety of our therapeutics. These comprehensive insights have not only supported our current goals but also prepared us for future challenges, equipping us with the tools to navigate the complexities of research and therapeutic development.</p>],
implementation: [<p>Discussions with industry experts have provided invaluable insights into the production of LNPs[LINK LNPcycle], a crucial aspect of our project. We have identified innovative manufacturing techniques and quality control procedures that we can integrate into our processes. This knowledge not only supports our immediate objectives but also positions us to overcome future challenges in developing effective therapies.</p>],
summary: [<p>During discussions with industry experts at Achema 2024, we gained valuable insights that will have a significant impact on our project and provide us with innovative manufacturing techniques and quality control procedures that we are now integrating into our workflows. These insights will help us to optimise our production processes to achieve more efficient and reliable results in therapeutic development. The new knowledge supports our current goals and prepares us for future challenges in the development of effective therapies</p>],
months: "june",
pictureurl_aim:"https://static.igem.wiki/teams/5247/photos/hp/achema1.webp",
pictureurl_implementation: "https://static.igem.wiki/teams/5247/photos/hp/achema2.webp",
pictureurl_interview: "https://static.igem.wiki/teams/5247/photos/hp/achema3.webp",
more_pictures: ["https://static.igem.wiki/teams/5247/photos/hp/achema4.webp,"],
pictureurl: pics['westhoff'],
tag: "Medical Professional",
heading: "In-Depth Visit to Specialized Physiotherapist for CF Breathing Therapy",
interviewtabid: "westhoffvisit",
quote: "Children are the world's best “mucus hiders”.",
aimofcontact: [<a>During the last interview with <HPLinktoOtherHPTab tab="westhoffinv" text="Katrin Westhoff" />, she invited us to join a few physiotherapy sessions – not just as spectators but as participants. We gladly accepted and visited her in her practice. Over a few hours, we took part in four sessions with different children – not all of them CF patients. </a>],
insights: "During the sessions, we could ask Katrin as well as the respective parents and children questions. We learned that breathing therapy is also useful for other illnesses and that you can easily do some of the exercises yourself. Despite having cystic fibrosis, the children were better at the breathing exercises than we and Katrin were! The sessions take 30 to 60 minutes and include both manual therapy and playful elements to help engage the children. Most older children range from mildly unhappy to enthusiastic, but babies often cry during the treatments as it feels uncomfortable. This is often hard on the parents even though the treatment brings good results. A lot of children tend to hide that they have mucus sitting in their lungs by suppressing coughs. Especially with young children, it is important to stay on top of it and do regular breathing therapy even if it seems like it is currently not necessary. We also learned about the various informational material aimed at children to help explain therapies and symptoms to them and what accessories for breathing therapy there are. For example, a flutter is to train breathing out forcefully by breathing against a small weight and a binder can be worn at night to promote deep breathing. ",
implementation: "The most important thing was that both Katrin and the parents agreed that the children were able to inhale at an early age and that there were generally no physical problems with inhalation in general. This reinforced our decision to work towards delivery by inhalation. It was very interesting to see the different ways children deal with their exercises and hear about the progress they made. ",
<p>Robin will soon start 4th grade and takes modulators. Since taking them, many problems have subsided. No regular pneumonia with long hospital stays and the mucus comes out easier. Nevertheless, Robin still goes to physiotherapy regularly to do manual breathing therapy to get the mucus out. Katrin tells us how the mucus changes color the longer it stays in the lungs. The new mucus is white, and the older mucus gets yellow first and then gets darker with time until it reaches a black color. Nowadays, Robin rarely has dark mucus or clumps, but we can still hear the rustling as Katrin starts the autogenous drainage (Autogene Drainage) by pressing on Robin's chest. The goal is to get out the mucus deep in the lungs. To do that, Robin must repeat the routine – breathing in deeply, holding, breathing out – multiple times and then cough and spit the mucus out. Sometimes it works, but other times the mucus does not come out easily. While according to Katrin the autogenous drainage is the gold standard, they do other useful exercises, too. For example, pressing the Vojta points (which the children call “the magic points”) on the chest to activate a deep breathing reflex and get air into parts of the lungs that may not have been used previously. Or physical activity such as climbing a few steps on a climbing ladder and hanging on it to stretch the thorax muscles.</p>
<p>Sam and Alex are siblings and do not have CF but another affliction that causes a persistent cough. They come together with a parent twice a week and do hanging exercises from the ceiling, nasal showers with needleless syringes, and the “magic points.” Katrin also checks their lungs for mucus in a similar manner to autogenous drainage. We, too, tried to do the nasal shower, and being a grown-up really does not guarantee being able to do that properly! This highlighted that the children know all their exercises by heart at a young age. On request, their parent told us that the physiotherapy made a big difference for both of them.</p>
<p>Toni has a light version of CF and has been doing physiotherapy with Katrin since shortly after birth. In contrast to most children we met or talked about, Toni refuses medication. Modulators are a possibility, but them and 'everything stinky' is a no-go, even though inhaling would be very beneficial due to the mucus buildup. Most exercises result in crying and screaming, which is very exhausting for the child. Due to the light nature of Toni's variant, they are not in danger, but a permanent therapy would be very beneficial.</p>
<p>Chrissi takes modulators and will soon take a trip to a water park with some friends. Katrin teaches us that when the children do not breathe out properly, air stays in the lungs and causes hyperinflation – with which it is actually harder to float in water! After the manual drainage, Katrin gets all of us glasses with water and dish soap and straws. Blowing bubbles is a playful way to train how to properly breathe out by either trying to blow bubbles as long as possible or trying to make an existing bubble as big as possible!</p>
summary: "In the visit with Katrin Westhoff, we participated in physiotherapy sessions for children, including those with cystic fibrosis (CF). We observed that breathing therapy is beneficial for various illnesses and learned techniques that can be practiced at home. Sessions last 30 to 60 minutes, combining manual therapy with playful elements. While older children engaged well, infants often found the exercises uncomfortable. Importantly, both Katrin and parents noted that children could inhale without issues from an early age, reinforcing our focus on inhalation delivery methods for therapies.",
pictureurl_interview:"https://static.igem.wiki/teams/5247/photos/hp/besuch-westhoff/untitled-design.png",
title: "Dr.",
vorname: "Marco",
nachnname: "Radukic",
job: "Postdoc at AG Cellular and Molecular Biotechnology",
affiliation: "University Bielefeld",
pictureurl: pics['marco'],
tag: "Academia",
heading: "Optimizing LNP Transfection: Insights into working with LNP Kits",
interviewtabid: "radukic",
language:"de",
quote: "After the interview with Dr Marco Radukic, we were able to produce LNPs for the first time.",