From cf6eefca90050e734ac3609195618e371c1a6988 Mon Sep 17 00:00:00 2001
From: Kaya <kaya.lange@uni-bielefeld.de>
Date: Wed, 4 Dec 2024 00:20:28 +0100
Subject: [PATCH] hp timeline
---
src/data/hptimelinedata.tsx | 34 +++++++++++++++++-----------------
1 file changed, 17 insertions(+), 17 deletions(-)
diff --git a/src/data/hptimelinedata.tsx b/src/data/hptimelinedata.tsx
index 226c248..9b54d45 100644
--- a/src/data/hptimelinedata.tsx
+++ b/src/data/hptimelinedata.tsx
@@ -329,7 +329,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
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 <OurLink path='engineering' tabId="tab-delivery" scrollToId='del1head' text="exploring LNPs"/> {/*KAYA!*/}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>],
+ 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 <OurLink path='engineering' tabId="tab-delivery" scrollToId='del1head' text="exploring LNPs"/> 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: [<p>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 <OurLink path='engineering'tabId='delivery' scrollToId='del1head' text="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.</p>],
months: "apr",
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></>,
@@ -416,7 +416,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
implementation: [<p>These insights helped refine our iGEM project in several key ways:
<ul>
<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><OurLink path='engineering' tabId="tab-delivery" scrollToId='del1head' text="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>
</ul></p>],
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.",
@@ -661,7 +661,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
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>],
+ 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 <OurLink path='engineering' tabId='reporter' scrollToId='rep1head' text="our design"/>.</p>],
interview: <>
<QaBox q="We have prepared some questions for you. The first question is: You mentioned that it was quite challenging to target the F508 delta mutation. Could you provide more detailed reasons for why this is the case or explain why this mutation is particularly difficult to target compared to others?"
a="Yes, that's the million-dollar question. First of all, let me clarify: our group has never directly worked on that mutation because we immediately focused on the drug-refractory mutations, such as nonsense mutations where the protein is not formed, indel mutations, or severe missense mutations that do not respond to modulator therapies. Of course, we know several groups in the field who either work on gene editing or focus on Cystic Fibrosis (CF). We've heard from some of them who attempted to target the F508 delta mutation. For example, some collaborators really tried to design different guides but were unable to find anything above the detection limit.
@@ -868,7 +868,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
language: "de",
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.",
+ aimofcontact: "The main objective of the contact was to learn from the discussion on issues related to 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 <OurLink tabId="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>],
interview: <>
@@ -878,9 +878,9 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
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."
+ <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 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?"
+ <QaBox q="Do you know how 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 distinct from the question of reimbursement by public health insurance and is not part of the AMNOG process. In Germany, CF is considered a rare disease if it affects fewer than five out of 10,000 people, and treatments for such rare diseases often face unique reimbursement challenges. If the prevalence exceeds this threshold, the disease is categorized as relatively common. In such cases, the AMNOG process evaluates the additional benefit (Zusatznutzen) of the treatment after it has already been approved, determining whether and to what extent costs will be reimbursed by statutory health insurance. This separation between approval and reimbursement highlights the different procedural and regulatory considerations for rare versus more common diseases.Have you heard about issues with reimbursement from private insurance companies?"/>
@@ -895,7 +895,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<QaBox q="Do private and public insurance differ in covering gene therapies? "
a="Private insurance can sometimes be more flexible, but it varies case by case. In the example we discussed, the individual had to fight for coverage, even though private insurance is often seen as more lenient. Statutory insurance tends to have clearer guidelines but can also be restrictive. "/>
<QaBox q="What happens when someone with Cystic Fibrosis has an atypical form of the disease?"
- a="Atypical cases can present significant challenges, particularly when test results don't align with standard expectations. For example, in cases like the one we encountered, a patient diagnosed with cystic fibrosis (CF) no longer met the clinical criteria for treatment reimbursement after a negative sweat test. Despite still having the disease, the medical indication for CF treatment was considered absent, leading to the partial or complete withdrawal of therapy coverage by the statutory health insurance (GKV). This highlights critical gaps in how the healthcare system addresses such cases, where rigid adherence to clinical guidelines may fail to accommodate the complexities of individual medical needs."/>
+ a="Atypical cases can present significant challenges, particularly when test results don't align with standard expectations. For example, in cases like the one we encountered, a patient diagnosed with CF no longer met the clinical criteria for treatment reimbursement after a negative sweat test. Despite still having the disease, the medical indication for CF treatment was considered absent, leading to the partial or complete withdrawal of therapy coverage by the statutory health insurance (GKV). This highlights critical gaps in how the healthcare system addresses such cases, where rigid adherence to clinical guidelines may fail to accommodate the complexities of individual medical needs."/>
<QaBox q="Is there a mechanism within the health insurance system for dealing with atypical cases"
a="Yes, but it can be tricky. The statutory insurance system works based on established guidelines, so when someone doesn't fit those guidelines, they may struggle to receive coverage. There are no clear pathways for addressing atypical cases, which highlights the need for greater awareness in the medical community about these variations. "/>
<QaBox q="Is newborn screening for CF covered by health insurance?"
@@ -1005,7 +1005,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
In addition, he suggested quality control measures such as fluorescence testing, zeta potential, and light scattering analyses help ensure our LNP is stable. Additionally, spray drying was evaluated for potential use in long-term LNP stabilization. </p>],
implementation: [<p>Incorporating the insights Dr. Radukic and advice from recent consultations, we adjusted the lipid-to-nucleic acid ratio from 22:1 to 10:1 to enhance efficiency and modified the pH and concentration of sodium acetate solutions for better packaging.
Storage conditions were strictly managed at 4 °C, and pipetting/mixing techniques were refined to ensure quality consistency. Quality control was expanded to include fluorescence testing, zeta potential measurements, and light scattering, alongside cytotoxicity tests.
- These improvements not only address transfection challenges but also strengthen our foundation for future LNP applications. </p>],
+ These improvements not only address transfection challenges but also strengthen our foundation for future <OurLink path='engineering' tabId="tab-delivery" scrollToId='del1head' text="LNP applications"/>. </p>],
summary: "The aim of the contact was to address challenges in LNP transfection and improve formulation protocols for enhanced gene delivery. Dr. Radukic highlighted the importance of lipid-to-nucleic acid ratios, recommending adjustments like 10:1 for better transfection efficiency. He also emphasized optimizing pH and buffer composition, as well as strict storage and mixing practices. Additionally, quality control measures such as fluorescence testing and zeta potential analysis were suggested to ensure LNP stability. These insights were implemented into the project, improving transfection efficiency and paving the way for future LNP applications.",
months: "jun"
},
@@ -1222,7 +1222,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
<li><b>Collaboration Opportunities:</b> Corden Pharma is open to offering discounts or forming partnerships, with recognition in publications or acknowledgments.</li>
</ul>
],
- implementation: [<p>The insights from Corden Pharma had a major impact on our project, especially in selecting lipids critical for LNP stability and optimizing gene therapy applications. Initially, we used the Cayman kit, but it was suboptimal for delivering our Primeguide. After receiving feedback, we switched to Corden Pharma’s kit #2, which includes advanced lipid components like cationic lipids that improve cellular uptake and enhance LNP stability. This shift has significantly boosted the efficiency and robustness of our formulations.
+ implementation: [<p>The insights from Corden Pharma had a major impact on our project, especially in selecting lipids critical for LNP stability and optimizing gene therapy applications. Initially, we used the Cayman kit, but it was suboptimal for delivering our Primeguide. After receiving feedback, we switched to <OurLink path='engineering' tabId="tab-delivery" scrollToId='del3head' text="Corden Pharma’s kit #2"/>, which includes advanced lipid components like cationic lipids that improve cellular uptake and enhance LNP stability. This shift has significantly boosted the efficiency and robustness of our formulations.
Additionally, Corden Pharma's guidance on lipid modifications and antibody incorporation opened new possibilities for targeted therapies. These insights not only improved our technical approach but also paved the way for potential collaborations, offering cost benefits and increased scientific recognition. The feedback will continue to shape our testing process and improve therapeutic delivery. </p>],
interview: <>
<QaBox q="Is it possible to dry the LNPs designed by Corden Pharma, such as through spray drying?" a="It has not been confirmed whether LNPs have been successfully dried using spray drying. Further investigation or consultation with a specialized company would be required to determine feasibility."/>
@@ -1534,7 +1534,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
extensive wet lab work.</p>],
implementation: [
<p>The interview with Dr. Benjamin Winkeljann from RNhale provided invaluable insights that will significantly enhance our project
- focused on mRNA delivery to the lungs using spray-dried LNPs. By seeking to integrate their proven techniques and innovative approach
+ focused on mRNA delivery to the lungs using <OurLink path='engineering' tabId="tab-delivery" scrollToId='del5head' text="spray-dried LNPs"/>. By seeking to integrate their proven techniques and innovative approach
to spray-dry LNPs, we are optimistic about achieving superior stability, efficacy, and scalability in our therapeutic delivery systems. </p>
],
pictureurl_aim: "https://static.igem.wiki/teams/5247/photos/hp/hp-rnhale-zoom.png",
@@ -1596,7 +1596,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
</p>],
insights: [<p>Dr. Kolonko provided us with valuable insights into working with nanoparticles, particularly emphasizing the advantages of nanocapsules. She highlighted that nanocapsules are more stable than nano-complexes, which is crucial for experiments involving complex environments and high shear forces. Additionally, her use of capsaicin wasn’t aimed at improving transfection efficiency but was part of a broader strategy targeting specific channels. She also shared practical methods for measuring particle stability and cytotoxicity, giving us clear guidance on tools and techniques that we can apply to our project.
Furthermore, Dr. Kolonko discussed the use of chitosan as a component in nanoparticle formulations. Chitosan, with its positive charge, can interact with mRNA, potentially enhancing the stability of the cargo. As an outlook, we plan to explore modifications using chitosan to improve the stability and performance of our mRNA delivery system. This approach may provide a more robust solution for optimizing nanoparticle formulations in future experiments. </p>],
- implementation:[<p>We directly applied Katharina’s insights to improve our nanoparticle design and testing methods. Her recommendations on using nano-capsules and OptiMEM as the transfection medium helped refine our experimental approach. She emphasized using a medium with fewer additives, like OptiMEM, and suggested removing antibiotics 24 hours before transfection to avoid interference, ensuring more controlled and effective conditions. We also explored new stability testing ideas, including nasal spray solutions and dry powder formulations.
+ implementation:[<p>We directly applied Katharina’s insights to improve <OurLink path='engineering' tabId="tab-delivery" scrollToId='del4head' text="our nanoparticle design "/>and testing methods. Her recommendations on using nano-capsules and OptiMEM as the transfection medium helped refine our experimental approach. She emphasized using a medium with fewer additives, like OptiMEM, and suggested removing antibiotics 24 hours before transfection to avoid interference, ensuring more controlled and effective conditions. We also explored new stability testing ideas, including nasal spray solutions and dry powder formulations.
We incorporated her insights as follows: </p>,
<ul>
<li><b>Nano-Capsules Focus:</b> Based on Katharina’s advice, we prioritized nano-capsules for their enhanced stability over nano-complexes.</li>
@@ -1819,7 +1819,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
quote: "Precision is key to minimizing side effects and ensuring the safety of your therapy.",
aimofcontact: "We conducted the interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti from the Institute of Biochemistry and Molecular Biology at the University of Hamburg, seeking to deepen our understanding of their research on Cystic Fibrosis (CF) and explore additional CF mutations, as well as to learn more about cell culture techniques specific to CF research, since they send us the CFBE41o- cell line. Our aim was also to gather more information about their approaches in CF research, particularly their focus on treating genetic mutations like nonsense mutations, which are highly prevalent in CF.",
insights: [<p>We were struck by Ignatova’s story about founding the iGEM team in Hamburg. Her passion for fostering creativity and innovation in science was inspiring. On a technical level, their advice on cell culture was incredibly practical and immediately useful. Dr. Nikhil Bharti explained how they handle CFBE41o- cells and ALI (air-liquid interface) cell culture. This advice directly addressed the challenges we’ve faced in our own lab, giving us a method to improve our cell culture success rates. During our interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti, their innovative approach to Cystic Fibrosis (CF) therapy, particularly "read-through" and tRNA-based therapies, stood out. "Read-through" therapies aim to bypass premature stop codons that prevent full protein production, offering a way to restore the function of critical proteins like CFTR in CF. This approach has the potential to treat a broad range of genetic diseases caused by similar mutations. The tRNA-based therapy is even more precise, targeting mRNA to correct faulty codons without altering the DNA, making it safer for long-term use. This flexibility, along with the ability to apply these therapies beyond CF, broadened our understanding of how such strategies can revolutionize treatments for genetic disorders.Ignatova highlighted, that using cell models closely resembling properties of the cell types targeted by our therapy is important for ensuring applicability of our approach to patient cells and its safety. A key focus throughout the discussion was safety. Prof. Ignatova emphasized the importance of ensuring that the therapies are highly specific, targeting only the defective codons while avoiding natural stop codons to prevent uncontrolled protein production. Moreover, their careful consideration of delivery systems further demonstrated their commitment to minimizing risks like toxicity in unintended organs. Their meticulous approach to safety has influenced how we think about developing and applying these therapies, making it clear that ensuring patient safety is as critical as achieving therapeutic success.</p>],
- implementation: "Prof. Ignatova's practical advice on cell culture had a transformative impact on our project. By adopting her method for CFBE41o- cells and improving our lab's sterilization protocols, we successfully established the cell line and significantly reduced the risk of contamination. In addition, her emphasis on safety in gene therapy guided us to review our Prime Editing construct and lipid nanoparticle (LNP) design. We focused on minimizing toxicity and off-target effects while ensuring precise targeting of lung tissues and the F508del mutation of the CFTR gene, making our approach safer and more efficient",
+ implementation: [<p>Prof. Ignatova's practical advice on cell culture had a transformative impact on our project. By adopting her method for <OurLink path='engineering' tabId="reporter" scrollToId='rep6head' text="CFBE41o- cells"/> and improving our lab's sterilization protocols, we successfully established the cell line and significantly reduced the risk of contamination. In addition, her emphasis on safety in gene therapy guided us to review our Prime Editing construct and LNP design. We focused on minimizing toxicity and off-target effects while ensuring precise targeting of lung tissues and the F508del mutation of the CFTR gene, making our approach safer and more efficient</p>],
summary: "In our interview with Prof. Dr. Zoya Ignatova and Dr. Nikhil Bharti from the Institute of Biochemistry and Molecular Biology at the University of Hamburg, we aimed to deepen our understanding of their research on Cystic Fibrosis (CF) and explore additional mutations. Their innovative approaches, particularly tRNA-based therapies, aim to restore the function of proteins like CFTR by bypassing premature stop codons, presenting potential treatments for various genetic diseases. They highlighted the importance of precision in therapy to minimize side effects and enhance safety, emphasizing careful delivery systems to avoid toxicity. Prof. Ignatova's practical insights into cell culture techniques significantly improved our lab's success with CFBE41o- cells, guiding us to enhance sterilization protocols and refine our Prime Editing constructs. This transformative exchange underscored the critical balance between achieving therapeutic success and ensuring patient safety.",
language: "en",
interview: <>
@@ -2049,10 +2049,10 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
quote: "It was a great connection and a tremendous help in analyzing the LNP. They provided us with insights into some truly exciting methods.",
quoteNachname: "Baack, Teammember",
quoteVorname: "Anna",
- aimofcontact: [<p>For our project, we collaborated closely with the Physical Chemistry workgroup to properly categorize our lipid nanoparticles (LNPs). We reached out to them to leverage their expertise and ensure that our characterization was thorough and precise. Marco, Uwe, and Yvonne were instrumental in this effort, not only advising us on appropriate characterization methods but also actively assisting us during the experimental process and data analysis. </p>],
- insights: [<p>We employed several analytical techniques, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential analysis. TEM and SEM allowed us to visualize the structural morphology of the LNPs, providing detailed images to understand their size and shape on the nanometer scale. DLS was used to measure the size distribution of the particles in solution, while the zeta potential analysis gave us insight into the surface charge, which is crucial for understanding stability in suspension. </p>],
- implementation: [<p>Thanks to the guidance and hands-on support of the Physical Chemistry team, we successfully completed these tests, gaining detailed insights into our LNPs that will be crucial for our project's further development. Their expertise not only streamlined the process but also ensured the reliability and accuracy of our results. Here a sneak peak of the results – take a look at the image of our SORT LNP taken via TEM. </p>],
- summary: "We collaborated with the Physical Chemistry workgroup to accurately characterize our lipid nanoparticles (LNPs). Their expertise, particularly from Marco, Uwe, and Yvonne, was invaluable in selecting and applying various analytical techniques, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential analysis. This collaboration not only enhanced our understanding of the LNPs' size, shape, and stability but also ensured the reliability of our results. With their guidance, we successfully completed our tests, providing crucial insights for the project's advancement.",
+ aimofcontact: [<p>For our project, we collaborated closely with the Physical Chemistry workgroup from Prof. Dr. Thomas Hellweg to properly categorize our LNPs. We reached out to them to leverage their expertise and ensure that our characterization was thorough and precise. Marco Annegarn, Uwe Güth, and Dr. Yvonne Hannappel were instrumental in this effort, not only advising us on appropriate characterization methods but also actively assisting us during the experimental process and data analysis. </p>],
+ insights: [<p>We employed several analytical techniques, including TEM, SEM, DLS, and zeta potential analysis. TEM and SEM allowed us to visualize the structural morphology of the LNPs, providing detailed images to understand their size and shape on the nanometer scale. DLS was used to measure the size distribution of the particles in solution, while the zeta potential analysis gave us insight into the surface charge, which is crucial for understanding stability in suspension. </p>],
+ implementation: [<p>Thanks to the guidance and hands-on support of the Physical Chemistry team, we successfully completed these tests, gaining detailed insights into our LNPs that will be crucial for our project's further development. Their expertise not only streamlined the process but also ensured the reliability and accuracy of our results. Here a sneak peak of the results – take a look at the image of <OurLink path='engineering' tabId="tab-delivery" scrollToId='del4head' text="our SORT LNP"/> taken via TEM. </p>],
+ summary: "We collaborated with the Physical Chemistry workgroup from Prof. Dr. Thomas Hellweg to accurately characterize our lipid nanoparticles (LNPs). Their expertise, particularly from Marco Annegarn, Uwe Güth, and Dr. Yvonne Hannappel were invaluable in selecting and applying various analytical techniques, including Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), and zeta potential analysis. This collaboration not only enhanced our understanding of the LNPs' size, shape, and stability but also ensured the reliability of our results. With their guidance, we successfully completed our tests, providing crucial insights for the project's advancement.",
months: "sep",
pictureurl_interview: "https://static.igem.wiki/teams/5247/delivery/cryo-em.webp",
pictureurl_implementation: " https://static.igem.wiki/teams/5247/delivery/tem-sort-lnp1.webp",
@@ -2096,7 +2096,7 @@ export const timelinedata: Array<TimelineDatenpunkt> = [
insights: [<p>From our discussion, we gained valuable insights into the unique properties of Chitosan, a cationic polymer with significant potential to stabilize RNA. Notably, Chitosan offers strong protection against RNases, making it highly beneficial for formulations like lipid-based nanoparticles (LNPs). Another key feature is its heat stability, withstanding temperatures up to 121°C, which makes it suitable for processing methods such as spray drying. However, at higher concentrations (0.5% or more), Chitosan can become toxic, suffocating cells and displaying antimicrobial properties. While it differs from PEG and cannot serve as a direct alternative, Chitosan can be a valuable complement, especially in stabilizing RNA within LNPs.
A critical point Benjamin emphasized is that Chitosan must be in an acidic environment, typically with a pH range of 4 to 6, to remain positively charged. This positive charge is essential for its effective interaction with RNA and successful integration into the LNP system.
One of the most important attributes of Chitosan is its ability to form complexes with RNA, offering a high degree of protection, which is crucial for the stability of LNP formulations. This characteristic makes Chitosan particularly advantageous in enhancing RNA stability during processes like spray drying. However, incorporating Chitosan directly into the lipid shell of LNPs poses challenges due to its hydrophilic nature and incompatible charge ratios, which prevent its use as an external coating on LNPs. Instead, it is more suitable for forming stable RNA-Chitosan complexes that can be encapsulated within the LNP structure, ensuring improved stability and protection.</p>],
- implementation: [<p>We have integrated the information by primarily using Chitosan as an RNA stabilizer, rather than embedding it directly into the LNP lipid shell. Benjamin suggested forming Chitosan-RNA complexes first and then encapsulating them within LNPs to ensure the RNA remains stable and functional. For this, Chitosan with a low molecular weight (around 5 kDa) is ideal, as it helps produce smaller particles that can be efficiently encapsulated.
+ implementation: [<p>We have integrated the information by primarily using Chitosan as an RNA stabilizer, rather than embedding it directly into the LNP lipid shell. Benjamin suggested <OurLink path='engineering' tabId="tab-delivery" scrollToId='del5head' text="forming Chitosan-RNA complexes"/> first and then encapsulating them within LNPs to ensure the RNA remains stable and functional. For this, Chitosan with a low molecular weight (around 5 kDa) is ideal, as it helps produce smaller particles that can be efficiently encapsulated.
Additionally, Benjamin recommended starting with small-scale tests (about 100 µL) before moving to larger formulations. The ratio of RNA to Chitosan is key to creating negatively charged particles, and a 2:1 ratio should be maintained. We will verify successful encapsulation using microscopic analysis and gel electrophoresis.
This knowledge has directly shaped our approach to using Chitosan. Our focus is now on forming stable RNA-Chitosan complexes, which can be encapsulated in LNPs. We’ve also learned the importance of optimizing concentrations to prevent aggregation or toxicity while ensuring the particles stay within the desired nanometer range. Microscopy and electrophoresis will now be key methods in our protocol to confirm complete RNA encapsulation within the LNPs. </p>],
summary: "In summary, the insights from Benjamin’s expertise were crucial in shaping our understanding of how to integrate Chitosan into our LNP formulations. Chitosan’s protective abilities for RNA, along with its heat stability, make it a valuable component in our project. However, its hydrophilic and cationic nature presents challenges for direct integration into LNP lipid shells, so we are focusing on its use as an encapsulation for the RNA. Benjamin’s advice on concentrations, molecular weight, and complex formation gave us a clear path forward, which will be validated through experimental testing. ",
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