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<p>Our project has evolved through a deeply collaborative and human-centered approach, integrating diverse feedback from patients, clinicians, researchers, and industry experts. These insights shaped not only the technical aspects of our gene therapy for cystic fibrosis (CF) but also our commitment to addressing real-world patient needs, ethical considerations, and the disparities in CF treatment worldwide. From <a onClick={() => goToPagesAndOpenTab('maxfirst', '')}>Max Beckmann’s</a> patient perspective to expert guidance on technical and ethical issues, each stakeholder contributed to refining our solution, ensuring it is both innovative and empathetic. Our focus on gene therapy targeting CF’s complex mutations, integrating physiotherapy, and ensuring global accessibility demonstrates our holistic and inclusive vision for this project. Importantly, the collaboration with researchers in nanoparticle stability and gene therapy, along with the development of bilingual surveys and outreach materials, highlights our efforts to make science more accessible and transparent, bridging gaps in knowledge and care. </p>
<H5 text="Human Practices Integration "/>
<p>From the start, we prioritized engaging with CF patients, making sure that our project aligned with both their needs and scientific expectations. Early input from Max Beckmann, a CF patient and friend of our team, guided key design decisions, such as our focus on lung-targeted gene therapy. His insights also shaped aspects like hygiene protocols for immunocompromised patients and the portrayal of CF in our outreach materials. Max’s ongoing feedback provided invaluable emotional insight, helping us ground the project in the real-world experiences of CF patients.</p>
<H5 text="Stakeholder Engagement "/>
<p>We consulted with medical professionals like <a onClick={() => goToPagesAndOpenTab('olariu', '')}>Dr. Olariu </a> who emphasized the importance of early diagnosis and mental health support in CF treatment. These insights led us to integrate mental health considerations into our therapy design and focus on reducing racial and global disparities in CF care. Physiotherapist <a onClick={() => goToPagesAndOpenTab('westhoffinv', '')}>Katrin Westhoff</a> highlighted the need for a user-friendly inhalation-based therapy for younger patients, validating our direction towards creating accessible treatments. </p>
<H5 text="Ethical, Legal, and Regulatory Considerations "/>
<p>Collaboration with regulatory experts, such as <a onClick={() => goToPagesAndOpenTab('berens', '')}> Dr. Eva-Maria Berens </a> , ensured our work adhered to ethical and legal standards, particularly in patient consent and biosafety protocols. Through feedback from legal and bioethics committees, we refined our consent processes and improved our management of personal data, ensuring our project complied with both German and international regulations. </p>
<p>We continuously integrated expert technical feedback into the project. Contributions from <a onClick={() => goToPagesAndOpenTab('rnhale', '')}>Dr. Benjamin Winkeljann</a> and <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a> helped us improve the stability and scalability of our spray-dried lipid nanoparticles (LNPs), which are essential for RNA delivery. This practical focus on scalable solutions allowed us to address global challenges, such as the need for transportable therapies. Collaborating with yeast cultivation experts like <a onClick={() => goToPagesAndOpenTab('nberelsmann', '')}>Nils Berelsmann</a>, we also optimized <a onClick={() => goToPageAndScroll ('lnps', '/materials-methods')}> RNA delivery Systems </a> to ensure effective lung penetration. </p>
<p>Recognizing the disparities in CF care across different regions, particularly in underrepresented areas like Asia, we adjusted our approach to create a more inclusive therapy. With feedback from stakeholders like <a onClick={() => goToPagesAndOpenTab('joshua', '')}>Joshua Bauder</a> from CF Vest International and <a onClick={() => goToPagesAndOpenTab('sriram', '/human-practices')}>Dr. Sriram Vaidyanathan</a>, we ensured our therapy addressed a wider range of CF mutations. This global focus led to bilingual surveys and expanded outreach efforts to raise awareness about CF and gene therapy. </p>
<H5 text="Feedback Loops and Project Evolution "/>
<p>We employed a structured feedback cycle based on Gibbs' Reflection Cycle, ensuring our design continuously evolved with stakeholder input. From initial design to public outreach, every phase of PreCyse was shaped by the feedback we received, allowing us to develop a patient-centered and globally relevant solution. </p>
<H5 text="Documentation and Future Guidance "/>
<p>We are committed to transparency and open science. Our detailed documentation of stakeholder interactions and methods—including protocols for working with <a onClick={() => goToPageAndScroll ('Cell Culture3', '/materials-methods')}> human nasal epithelial cells </a>—provides a foundation for future iGEM teams. Our outreach efforts, including educational materials and public engagement campaigns, help foster a broader understanding of CF and gene therapy, reflecting iGEM’s vision of responsible and impactful scientific practices. </p>
<li><p><b>Advanced Testing and Preclinical Trials:</b></p> <p>Following the successful in vitro experiments, our next major milestone is the initiation of animal studies to evaluate the safety and efficacy of our gene therapy approach, particularly in CF lung models.</p></li>
<li><p><b>Enhancing RNA Delivery Systems:</b></p> <p>We will continue optimizing our lipid nanoparticle (LNP) formulations, particularly exploring chitosan integration, to improve RNA stability, lung penetration, and scalability for broader global applications.</p></li>
<li><p><b>Clinical Partnerships:</b></p> <p>Establish stronger ties with clinical institutions to move towards human trials, ensuring that our therapy is aligned with clinical needs and regulatory standards.</p></li>
<li><p><b>Expanded Global Outreach:</b></p> <p>Increase awareness and education on CF and gene therapy through multilingual platforms and collaboration with international CF communities, particularly in underrepresented regions.</p></li>
<li><p><b>Ethical and Legal Considerations:</b></p> <p>Finalize all bioethical protocols for patient sampling and data management to pave the way for safe, compliant future research, including the development of guidelines for future teams.</p></li>
<p>By continuing this patient-driven, scientifically rigorous, and globally conscious trajectory, we aim to advance cystic fibrosis care and make a lasting impact on patient lives, ensuring that our project remains poised for clinical success.</p>
return(
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<AnalyseMax/>
<AalyseOlariu/>
<AnalyseWesthoff/>
<AnalyseJoshua/>
<AnalyseRnhale/>
<AnalyseKolonko/>
<AnalyseMoor/>
<AnalyseWeber/>
<AnalyseBerens/>
<AnalyseMichaela/>
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<AnalyseWischmeyer/>
<AnalyseIgnatova/>
<AnalyseBharti/>
<AnalyseSriram/>
<AnalyseKristian/>
<AnalyseSaito/>
<AnalyseNils/>
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}
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<b>Figure x.</b> Figure Caption
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<figcaption><b>Figure x.</b> Caption </figcaption>
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return(
<Collapsible title="Nils Berelsmann & Hakan Soytürk - Specialists in yeast cultivation " id="nilsanalyseC">
<p>Nils Berelsmann and Hakan Soytürk, specialists in yeast cultivation, played pivotal roles in advancing our project. Nils provided us with a yeast strain compatible with <a onClick={() => goToPagesAndOpenTab('saito', '/human-practices')}>Dr. Saito's</a> recommendations, particularly one that doesn’t express proteases, which would degrade our nickase candidates. This strain was essential for maintaining the integrity of our engineered enzymes. </p>
<p>Hakan equipped us with the methodological expertise needed to work with yeast, guiding us through the complexities of yeast cultivation. With their support, we adapted our engineering design specifically for yeast expression. Nils also supplied us with a target vector optimized for yeast expression, which we successfully used to integrate a CasX nickase candidate. This proved especially valuable as our Spu candidates were difficult to integrate due to the use of overhangs. We are still working on this challenge, staying in close contact with the experts to refine our process. </p>
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<p><strong>Yeast strain provision:</strong> Nils provided a yeast strain compatible with Dr. Saito's recommendations, which does not express proteases that would degrade our nickase candidates, maintaining the integrity of our engineered enzymes.</p>
<p><strong>Methodological guidance:</strong> Hakan provided expertise in yeast cultivation, guiding us through the complexities of working with yeast.</p>
<p><strong>Engineering adaptation for yeast:</strong> With their support, we adapted our engineering design specifically for yeast expression.</p>
<p><strong>Target vector for yeast:</strong> Nils supplied an optimized target vector for yeast expression, which allowed successful integration of a CasX nickase candidate.</p>
<p><strong>Challenge with Spu candidates:</strong> Integration of Spu candidates proved difficult due to overhangs; we are still working on this challenge and are staying in close contact with the experts to refine the process.</p>
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function AnalyseSaito(){
return(
<Collapsible title="Dr. Makoto Saito – Leading Research Expert for FANZOR" id="saitoanalyseC">
<p>Early in our project design, we decided to optimize the Prime Editing technology. From the literature, we identified FANZOR, a small, eukaryotic RNA-binding DNA endonuclease, as a potential alternative to Cas9 for our approach. Through collaboration with the enzyme’s discoverer, Dr. Makoto Saito, we were able to refine our engineering strategy. Dr. Saito encouraged us to modify the TNB domain of FANZOR using site-directed mutagenesis, ensuring the non-coding strand is not positioned in the active site of the RuvC domain. This approach mirrors the nickase strategy used in Cas12 variants. </p>
<p>Dr. Saito also motivated us to express our potential nickase variants in <i>Saccharomyces cerevisiae </i> to better characterize their functionality. He played a key role in supporting our decision to explore additional Cas endonuclease candidates, such as CasX, which is more closely related to Cas9 and Cas12, providing additional flexibility for our gene-editing goals. </p>
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<p><strong>FANZOR as Cas9 alternative:</strong> Identified FANZOR, a small eukaryotic RNA-binding DNA endonuclease, as a promising alternative to Cas9 for Prime Editing.</p>
<p><strong>Collaboration with Dr. Saito:</strong> Worked closely with the enzyme’s discoverer, Dr. Makoto Saito, to refine our engineering strategy for FANZOR.</p>
<p><strong>TNB domain modification:</strong> Dr. Saito suggested modifying the TNB domain via site-directed mutagenesis to prevent the non-coding strand from entering the RuvC active site, following the nickase strategy used in Cas12 variants.</p>
<p><strong>Yeast expression for functionality:</strong> Recommended expressing nickase variants in Saccharomyces cerevisiae to assess their functionality more effectively.</p>
<p><strong>Exploration of other endonucleases:</strong> Supported the exploration of additional endonucleases like CasX, offering greater flexibility and adaptability in our gene-editing approach.</p>
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}
function AnalyseKristian(){
return(
<Collapsible title="Prof. Dr. Kristian Müller – Expert for Synthetic Biology, Genetic design, cloning strategies, molecular and cellular biotechnology " id="kristiananalyseC">
<p>In close collaboration with Prof. Müller’s lab, we learned both basic and advanced cloning strategies within synthetic biology. Early on, Prof. Müller suggested making our Prime Editing complex smaller and more stable, enhancing its suitability for various therapeutic applications. His continuous support was crucial in guiding us through the cloning of all constructs, and as an expert in gene therapy, he provided ongoing feedback on the safety aspects of our research. </p>
<p>With access to a BSL2 laboratory provided by Müller’s team, we were able to successfully cultivate primary cell cultures. To ensure safe work conditions, we underwent training for biosafety procedures and routinely tested for HPV, HIV, HBV, and Mycoplasma contamination. Thanks to Müller’s guidance, we contributed a composite part to the iGEM registry. Our PreCyse cassette allows for modular Prime Editing systems that are particularly efficient, stable, and safe based on our research. </p>
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<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>
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<p><strong>Cloning strategies:</strong> Provided both basic and advanced cloning training, crucial for developing our Prime Editing complex within synthetic biology.</p>
<p><strong>Optimization of Prime Editing complex:</strong> Suggested making the Prime Editing complex smaller and more stable, enhancing its suitability for therapeutic applications.</p>
<p><strong>Ongoing feedback on safety:</strong> As a gene therapy expert, Prof. Müller gave continuous feedback on the safety aspects of our research.</p>
<p><strong>Access to BSL2 lab:</strong> Enabled us to successfully cultivate primary cell cultures in a BSL2 lab, with training in biosafety procedures.</p>
<p><strong>Contamination testing:</strong> Regularly tested for HPV, HIV, HBV, and Mycoplasma contamination to ensure safe working conditions.</p>
<p><strong>Contribution to iGEM registry:</strong> With Prof. Müller's guidance, we developed and contributed the PreCyse cassette, a modular and efficient Prime Editing system, enhancing stability and safety based on our findings.</p>
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}
function AnalyseSriram(){
return(
<Collapsible title="Dr. Sriram Vaidyanathan – Expert on Pediatric medicine and CF in Asia" id="sriramanalyseC">
<p>In our discussions with Dr. Sriram Vaidyanathan, we deepened our understanding of the genetic diversity of CF mutations, especially in Asian populations. He highlighted that while the F508del mutation is common worldwide, many rare CFTR mutations complicate diagnosis and treatment in specific regions.</p>
<p>This insight pushed us to adapt our gene therapy approach to be more inclusive. We adjusted our strategy to ensure our therapy could target a broader range of CF mutations, making it more globally applicable. Dr. Vaidyanathan’s input shaped our efforts to create a treatment that addresses CF’s diverse genetic landscape. </p>
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<p><strong>Contamination challenge:</strong> Helped us address a major issue with fungal contamination in our CF cell cultures, which were unstable and prone to infection.</p>
<p><strong>Antibiotic solution:</strong> Recommended a specific antibiotic composition effective against fungal infections.</p>
<p><strong>Successful implementation:</strong> Applied the antibiotic mixture, which resolved the contamination problem, allowing us to maintain stable CF cell lines.</p>
<p><strong>Project advancement:</strong> This breakthrough enabled us to proceed with testing our prime editing approach effectively, advancing our project to the final stages of in vitro testing.</p>
<p><strong>Next steps:</strong> Thanks to Dr. Bharti’s insights, we are now positioned to consider potential animal studies as the next phase of our research.</p>
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function AnalyseBharti(){
return(
<Collapsible title="Dr. Nikhil Bharti – Expert in Primary culture " id="bhartianalyseC">
<p>Dr. Nikhil Bharti's expertise helped us overcome a major challenge with our CF cell cultures, which were consistently becoming contaminated with fungi due to their instability. He recommended a specific antibiotic composition effective against fungal infections. </p>
<p>We immediately applied this antibiotic mixture to our cell cultures, which resolved the contamination issue and allowed us to maintain stable CF cell lines. This breakthrough enabled us to proceed with testing our prime editing approach effectively. Thanks to Dr. Bharti's insights, we advanced our project to a stage where we reached the limit of in vitro testing, and the next step would be potential animal studies. </p>
<p><strong>Contamination challenge:</strong> Helped us address a major issue with fungal contamination in our CF cell cultures, which were unstable and prone to infection.</p>
<p><strong>Antibiotic solution:</strong> Recommended a specific antibiotic composition effective against fungal infections.</p>
<p><strong>Successful implementation:</strong> Applied the antibiotic mixture, which resolved the contamination problem, allowing us to maintain stable CF cell lines.</p>
<p><strong>Project advancement:</strong> This breakthrough enabled us to proceed with testing our prime editing approach effectively, advancing our project to the final stages of in vitro testing.</p>
<p><strong>Next steps:</strong> Thanks to Dr. Bharti’s insights, we are now positioned to consider potential animal studies as the next phase of our research.</p>
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)
}
function AnalyseIgnatova(){
return(
<Collapsible title="Prof. Dr. Ignatova – CF Expert & Researcher" id="ignatovaanalyseC">
<p>Through our collaboration with <a href="https://2024.igem.wiki/hamburg/" title="iGEM Hamburg" > iGEM Hamburg</a>, we were introduced to Prof. Ignatova, a leading expert in cystic fibrosis (CF) research. Initially, we consulted her to gain a deeper understanding of CF. Later, when the HEK cells from Leuven proved unsuitable for our tests, we reached out again to explore alternative cell models. </p>
<p>Prof. Ignatova provided access to the CFBE41o- cell line, immortalized CF cells derived from a CF patient, which we obtained with permission from Prof. Karl Kunzelmann at the University of Regensburg. This cell line offered us a new, reliable testing system, and we successfully cultivated the cells in our lab, although they required significant time to acclimate and grow. </p>
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function AnalyseWischmeyer(){
return(
<Collapsible title="Prof Dr. Eberhard Wischmeyer - Academic Expert on Patch-Clamp Techniques" id="wischmeyeranalyseC">
<p>Prof. Dr. Erhard Wischmeyer provided critical guidance on the patch-clamp technique for measuring CFTR functionality. His expertise, alongside the hands-on support of Dr. Oliver Dräger from his lab, helped us optimize the patch-clamp experiments using HEK cells provided by Uni Leuven. Together, we conducted electrophysiological measurements to characterize the functionality of CFTR mutations, specifically comparing the delta 508 CFTR mutant with overexpressed healthy CFTR cells. </p>
<p>The experiments showed significant differences between CFTR-deficient cells and healthy controls, but we began questioning the suitability of the delta 508 overexpression cells as a functional test system. After adjusting the culture conditions in consultation with Leuven, we repeated the measurements but still found no meaningful differences. </p>
<p>Ultimately, the feedback cycle with both Leuven and Wischmeyer's lab confirmed that patch-clamp was insufficient for our purposes, leading us to consider Ussing chamber measurements as a more suitable method for assessing CFTR function. </p>
<p><strong>Patch-clamp technique guidance:</strong> Provided critical guidance on using the patch-clamp technique to measure CFTR functionality, optimizing our experiments.</p>
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<figcaption>Figure. Patch-clamp measurement.</figcaption>
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<p><strong>Support from Dr. Oliver Dräger:</strong> Hands-on support from Dr. Dräger helped us perform patch-clamp experiments using HEK cells provided by Uni Leuven.</p>
<p><strong>CFTR functionality assessment:</strong> Conducted electrophysiological measurements to compare delta 508 CFTR mutants with overexpressed healthy CFTR cells, revealing significant differences between CFTR-deficient cells and healthy controls.</p>
<p><strong>Suitability concerns:</strong> Despite adjustments to culture conditions, repeated measurements raised doubts about the delta 508 overexpression cells as a functional test system.</p>
<p><strong>Feedback cycle with Leuven and Wischmeyer:</strong> Confirmed that patch-clamp was not suitable for our needs, prompting us to explore Ussing chamber measurements as a more appropriate method for assessing CFTR function.</p>
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}
function AnalyseMattijs(){
const {goToPageAndScroll} = useNavigation();
return(
<Collapsible title="Mattjis Bulcaen – University of Leuven, CF and Prime Editing Expert" id="mattijsanalyseC">
<p>Mattijs Bulcaen, being a researcher working on a topic very close to ours, provided invaluable guidance in the early stages of our project. He gave us an insight into the current advances in the field that we that we were able to make use of later on. He reviewed with us our own ideas and considerations, such as the use of the PEAR reporter system. Following our interview with Mattijs we integrated the structural motif trevopreQ1 into the pegRNA, which enhanced the prime editing efficiency - a critical improvement we successfully tested and demonstrated in our results. </p>
<p>Our decision of first testing prime editing in HEK293 cells instead of other cell lines or primary cells was also based on his statement that HEK cells are by far the easiest to archieve editing in. Mattijs additionally recommended the use of <a onClick={() => goToPageAndScroll ('Cell Culture', '/materials-methods')}> HEK293T cell lines </a> overexpressing CFTR and CFTR F508del. However, after conducting patch clamp measurements, we found that the HEK cells were unsuitable for our needs, requiring us to adjust our testing system. As a result, we switched to <a onClick={() => goToPagesAndOpenTab('ignatova', '/human-practices')}>Ignatova's</a> cells from Hamburg for further experiments. </p>
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<figcaption>Figure. Validation of different pegRNAs efficiency via Patch Clamp via FACS analysis. Portion of Living fluorescent cells against internal control, suggesting high efficient pegRNAs.
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function AnalyseMichaela(){
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<Collapsible title="Michaela Bienert – Scientific Sales Representative at Stemcell Technologies" id="michaelaanalyseC">
<p>Through hands-on experience with Michaela Bienert and Julie Watson, we gained valuable insights into the cultivation methods of Air-Liquid Interface (ALI) and apical-out organoids. We evaluated the advantages and limitations of different culturing techniques, which enabled us to make informed decisions about their implementation in our research. With the protocols and resources provided, we successfully generated ALI cultures and organoids from primary cells of CF patients and healthy control donors, which we were able to test throughout the course of our project. </p>
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<p><strong>Hands-on experience:</strong> Gained practical insights from Michaela Bienert and Julie Watson into the cultivation methods for Air-Liquid Interface (ALI) and apical-out organoids.</p>
<p><strong>Evaluation of culturing techniques:</strong> Assessed the advantages and limitations of different cultivation techniques, allowing us to make informed decisions about which methods to use in our research.</p>
<p><strong>Successful generation of cultures:</strong> Using the protocols and resources provided, we successfully generated ALI cultures and organoids from primary cells of CF patients and healthy control donors.</p>
<p><strong>Testing and experimentation:</strong> These cultures and organoids were tested throughout the project, contributing significantly to our research outcomes.</p>
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}
function AnalyseBerens(){
return(
<Collapsible title="Dr. Eva-Maria Berens – Head of ethics committee at University of Bielefeld" id="berensanalyseC">
<p>In our pursuit of generating primary cultures from human nasal epithelial cells for testing, we sought advice from the Biosafety and Security Committee of iGEM, as well as consult with Dr. Eva-Maria Berens, our ethics officer. Through in-depth discussions with Dr. Berens, we carefully assessed the risks and legal obligations associated with our project. We learned that, given the minimal invasiveness of the procedure, no formal bioethics application was necessary. However, we recognized the importance of developing a legally sound informed consent form for participants, ensuring that we adhered to the relevant legal frameworks. </p>
<p>In collaboration with various legal institutions, we drafted a comprehensive informed consent form that complies with both national regulations in Germany and the specific policies of Bielefeld University. As the first iGEM team to tackle the complex cultivation of human primary nasal epithelial cells, we were committed to paving the way for future teams. To support this, we created a guideline documenting the proper handling of human biomaterial obtained through these types of sample collection. </p>
<p>Additionally, we worked with Mr. <a onClick={() => goToPagesAndOpenTab('timm', '/human-practices')}> Timm Weber </a> , who is working for the local biobank OWL, to establish protocols for the management and storage of sensitive, personally identifiable data. While no special procedures were required for our specific project, we nonetheless made it a priority to implement anonymized methods to protect participant privacy. </p>
<p>The input and feedback from Dr. Berens and Mr. Weber formed the basis of our ethical consideration of our project. With their help, we were able to improve our technology on primary cultures with a higher level of safety and an adapted guidline. In addition, our biosafety contributions were significantly improved as we were able to ensure compliance with all legal and ethical standards.. </p>
<p>Having established the necessary ethical and legal groundwork, we turned our focus to the practical aspects of handling and cultivating patient samples. This required specialized expertise, which led us to seek assistance from <a onClick={() => goToPagesAndOpenTab('michaela', '/human-practices')}>Stemcell Technologies</a> ensure the successful cultivation and maintenance of the nasal epithelial cells. Their support was essential in enabling us to push forward with our testing and bring our project closer to real-world applications. </p>
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<p><strong>Ethical consultation:</strong> Consulted with Dr. Eva-Maria Berens, our ethics officer, to assess the risks and legal obligations of generating primary cultures from human nasal epithelial cells. It was determined that no formal bioethics application was required due to the minimal invasiveness of the procedure.</p>
<p><strong>Informed consent:</strong> Collaborated with legal institutions to draft a comprehensive informed consent form that complies with German national regulations and Bielefeld University policies.</p>
<p><strong>Guideline creation for future teams:</strong> As the first iGEM team to work with human primary nasal epithelial cells, we developed a guideline for handling human biomaterial to aid future teams in similar projects.</p>
<p><strong>Data privacy protocols:</strong> Worked with Ms. Gabriele Anton to establish protocols for managing and storing sensitive personal data, prioritizing anonymization to protect participant privacy.</p>
<p><strong>Ethical and legal foundation:</strong> Input from Dr. Berens and Ms. Anton was crucial in building the ethical and legal foundation of our project, ensuring compliance with all standards and enabling us to proceed with official approval.</p>
<p><strong>Practical expertise:</strong> After securing the ethical and legal framework, we sought assistance from Stemcell Technologies for practical guidance in handling and cultivating patient samples, which was essential for advancing our testing and pushing the project toward real-world applications.</p>
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}
function AnalyseWeber(){
return(
<Collapsible title="Prof. Dr. Weber and Dr. Große-Onnebrink " id="weberanalyseC">
<p>Prof. Weber and Dr. Große-Onnebrink emphasized the potential of targeting ionocytes due to their crucial role in CFTR expression while highlighting the challenges of accessing these cells and penetrating the mucus barrier. Additionally, they suggested using an Ussing chamber to assess CFTR function, although they noted its limitations, and recommended exploring chitosan-based nanoparticles as a safer alternative to PEG-lipid systems. </p>
<p>In response to their insights, we continued to explore ionocytes but expanded our focus to include other cell types to enhance testing flexibility. We further investigated chitosan-based nanoparticles and optimized their size for better lung penetration. We plan to utilize the Ussing chamber for CFTR measurements and are also considering patch clamping for detailed transfection analysis. </p>
<p>Prof. Weber highlighted the innovative aspects of our project, particularly regarding cell culture methods. He advised us to consider the ethical and legal implications, which led us to consult with the head of the ethics committee at the University of Bielefeld, <a onClick={() => goToPagesAndOpenTab('berens', '/human-practices')}>Dr. Eva-Maria Berens</a>. Due to legal concerns, the committee is unable to support our project directly, as they have an interest in developing their own Chitosan LNPs, which conflicts with the open-source nature of the iGEM competition. Nevertheless, they provided us with valuable information up to that point, guiding our understanding and approach to the project. </p>
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<img src="https://static.igem.wiki/teams/5247/integrated-human-practices/orientierung.webp" alt=""/>
<p><strong>Targeting ionocytes:</strong> Emphasized the potential of targeting ionocytes due to their critical role in CFTR expression, while also highlighting the challenges of accessing these cells and penetrating the mucus barrier in CF patients.</p>
<p><strong>Ussing chamber recommendation:</strong> Suggested using the Ussing chamber to assess CFTR function, noting its limitations, and recommended exploring patch clamping for more detailed transfection analysis.</p>
<p><strong>Chitosan-based nanoparticles:</strong> Encouraged investigating chitosan-based nanoparticles as a safer alternative to PEG-lipid systems, which led us to optimize chitosan nanoparticle size for better lung penetration.</p>
<p><strong>Expanded cell targeting:</strong> Based on their insights, we expanded our focus beyond ionocytes to include other cell types to increase flexibility in testing.</p>
<p><strong>Ethical and legal guidance:</strong> Prof. Weber advised us to consider ethical and legal concerns, leading to consultations with Dr. Berens, head of the ethics committee at the University of Bielefeld. Legal concerns around conflicting interests (the university's own chitosan LNP developments) limited direct support, but the committee provided valuable guidance.</p>
<p><strong>Innovative cell culture methods:</strong> Prof. Weber praised the innovative aspects of our project, particularly our approach to cell culture, further validating our project’s scientific direction.</p>
</Collapsible>
)
}
function AnalyseMoor(){
return(
<Collapsible title="Benjamin Willem Moorlach – Chitosan Expert " id="moorlachanalyseC">
<p>We gained valuable insights into the unique properties of chitosan, a cationic polymer with significant potential to stabilize RNA in our lipid nanoparticle (LNP) formulations. Chitosan offers robust protection against RNases and exhibits heat stability, making it suitable for processing methods like spray drying. Additionally, its mucoadhesive properties enable optimal choice as LNP component. </p>
<p>A critical insight was the necessity for chitosan to be in an acidic environment (pH 4-6) to maintain its positive charge, which is essential for effective RNA interaction. While it cannot replace PEG due to its hydrophilic nature, chitosan is ideal for forming RNA-chitosan complexes, which can then be encapsulated within LNPs. This approach significantly enhances RNA stability during spray drying, a method we intend to further test in collaboration with <a href="https://rnhale.com/">Rnhale</a>. </p>
<p> In terms of implementation, Benjamin educated us on the chemical and structural properties of chitosan, reinforcing our approach to improve stability, particularly against heat, in our LNP formulations. He provided guidance on formulating chitosan-RNA complexes and developed a protocol for integrating them into our LNP formulation without affecting the charge of the nanoparticles. Additionally, he supplied us with chitosan in various sizes, enabling us to test different chitosan complexes for optimal results. </p>
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<img src="https://static.igem.wiki/teams/5247/integrated-human-practices/chitosan.webp" style={{height:"50%", width:"auto"}} alt=""/>
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<p><strong>Chitosan properties:</strong> Provided valuable insights into chitosan, a cationic polymer with strong potential to stabilize RNA in lipid nanoparticle (LNP) formulations due to its robust protection against RNases and heat stability.</p>
<p><strong>Mucoadhesive properties:</strong> Highlighted chitosan's mucoadhesive characteristics, making it an ideal component for LNPs in terms of RNA delivery.</p>
<p><strong>Acidic environment requirement:</strong> Emphasized the need for chitosan to maintain a positive charge in an acidic environment (pH 4-6) for effective RNA interaction.</p>
<p><strong>Chitosan vs. PEG:</strong> While chitosan cannot replace PEG due to its hydrophilic nature, it is optimal for forming RNA-chitosan complexes, which can be encapsulated within LNPs.</p>
<p><strong>RNA stability in spray drying:</strong> Chitosan's use in RNA-chitosan complexes significantly enhances RNA stability during spray drying, a method we will continue to test with RNhale.</p>
<p><strong>Implementation guidance:</strong> Benjamin provided chemical and structural knowledge of chitosan, guiding us on how to improve LNP stability, especially against heat, and formulating chitosan-RNA complexes. He also supplied various sizes of chitosan, allowing us to test for optimal results in our formulations.</p>
</Collapsible>
)
}
function AnalyseKolonko(){
return(
<Collapsible title="Dr. Katharina Kolonko – Nanoparticle Researcher " id="kolonkoanalyseC">
<p>Dr. Kolonko's expertise in nanoparticle stability was invaluable in refining our delivery system. Her recommendation to utilize chitosan for greater stability influenced our formulation choice and we further investigated how to integrate <a onClick={() => goToPagesAndOpenTab('moorlach', '/human practices')}> chitosan in our LNP </a>. Additionally, her insights on cytotoxicity testing, particularly the MTT assay, and medium conditions such as using OptiMEM for transfection, greatly improved our experimental design. We explored the potential and importance of chitosan in lipid nanoparticles, learned various techniques for characterizing LNPs, and implemented effective cultivation techniques and tips for cell culture, with a focus on contamination risks and the use of OptiMEM medium. Overall, Dr. Kolonko’s guidance has been instrumental in shaping the safety and efficacy of our project. </p>
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<img src="https://static.igem.wiki/teams/5247/fanzor/sort-mtt.webp " alt="" />
<figcaption>Figure. MTT Assay of LNPs from all iterations performed on HEK293 including Triton as negative control and untreated cells as positive control. Mean +/- SEM for n=6. For statistics one-way ANOVA was performed</figcaption>
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<img src="https://static.igem.wiki/teams/5247/photos/optimem-1.webp" style={{height:"55%", width:"auto"}} alt=""/>
<p><strong>Expertise in nanoparticle stability:</strong> Provided invaluable insights that significantly refined our delivery system.</p>
<p><strong>Chitosan recommendation:</strong> Suggested the use of chitosan for increased stability in lipid nanoparticles (LNPs), influencing our formulation choice and leading to further investigations into integrating chitosan in our LNP system.</p>
<p><strong>Cytotoxicity testing improvements:</strong> Her guidance on cytotoxicity testing, particularly the MTT assay, greatly enhanced the reliability of our experiments.</p>
<p><strong>Optimized transfection conditions:</strong> Introduced us to the use of OptiMEM for transfection, improving our medium conditions and experimental design.</p>
<p><strong>Characterization and cultivation techniques:</strong> Helped us explore techniques for characterizing LNPs and implementing effective cultivation methods, focusing on contamination risks and optimal cell culture practices.</p>
<p><strong>Overall impact:</strong> Dr. Kolonko's contributions were instrumental in shaping the safety and efficacy of our project, improving both the stability of the formulation and the quality of our experimental processes.</p>
</Collapsible>
)
}
function AnalyseRnhale(){
return(
<Collapsible title="Dr. Benjamin Winkeljann – RNhale, Industry Expert & Researcher " id="rnhaleanalyseC">
<p>Dr. Benjamin Winkeljann from <a href="https://rnhale.com/" >Rnhale</a> provided crucial technical guidance that significantly enhanced our project. His expertise in spray-dried lipid nanoparticles (LNPs) allowed us to improve the shelf-life and scalability of our formulations. We learned about the limitations of CF therapies regarding international access, cost, and availability, which highlighted the need for sustainable solutions. </p>
<p>We discovered that spray drying is an effective method for stabilizing LNPs, enabling transport without refrigeration — a more environmentally friendly approach that reduces energy consumption. </p>
<p>Our close collaboration with RNhale will continue even after the wiki freeze, focusing on producing spray-dried LNPs and testing them on our primary cultures. We are also investigating how to enhance the stability of LNPs, particularly in protecting RNA from heat damage, and are seeking experts in chitosan for further support. </p>
<p>During discussions with a young startup entrepreneur, we explored the possibility of pursuing an <a onClick={() => goToPageAndScroll ('Further Engagement3H', '/human-practices')}> entrepeneuship-oriented </a> project but ultimately decided to focus on a human-centered approach that prioritizes the needs of CF patients over cost-driven industrial pathways. </p>
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<img src="https://static.igem.wiki/teams/5247/photos/hp/hp-rnhale-zoom.png" alt=""/>
<figcaption>Figure. Delivery Team Zoom call with RNhale </figcaption>
</figure>
<p><strong>Expertise in spray-dried LNPs:</strong> Provided critical technical guidance, enhancing the shelf-life and scalability of our formulations.</p>
<p><strong>Sustainable solutions:</strong> Highlighted the limitations of CF therapies regarding international access, cost, and availability, emphasizing the need for sustainable alternatives.</p>
<p><strong>Spray drying benefits:</strong> Introduced us to spray drying as an effective method for stabilizing LNPs, enabling transport without refrigeration and reducing energy consumption—an environmentally friendly solution.</p>
<p><strong>Ongoing collaboration:</strong> Continued collaboration with RNhale, focusing on producing and testing spray-dried LNPs on primary cultures, while investigating ways to improve RNA stability and protection from heat damage.</p>
<p><strong>Exploration of entrepreneurship:</strong> Discussed the potential for an entrepreneurship-oriented project, but ultimately prioritized a human-centered approach that focuses on the needs of CF patients over industrial cost-driven strategies.</p>
</Collapsible>
)
}
function AnalyseJoshua(){
const {goToPagesAndOpenTab} = useNavigation();
const {goToPageAndScroll} = useNavigation();
return(
<Collapsible title="Joshua – Vice president of CF Vest international, Father of a CF child" id="joshuaanalyseC">
<p>Through discussions with <a onClick={() => goToPagesAndOpenTab ('joshua', '/human-practices')}> Joshua </a>, we learned that CF statistics are inadequate, primarily representing the white population, which skews understanding of the disease's prevalence. We discovered that in Asian countries like Thailand, CF is underrepresented, leading to insufficient access to therapies and medications. </p>
<p>This awareness sharpened our focus on the need for improved science communication and highlighted the ongoing issues of racism and discrimination within scientific research. In response, we are committed to enhancing the data landscape in Germany by creating <a onClick={() => goToPageAndScroll ('our-surveys-on-cystic-fibrosis-and-gene-therapy', '/human-practices')}> surveys </a> in both German and English to gather broader insights and increase outreach. </p>
<img src="https://static.igem.wiki/teams/5247/photos/hp/joshua-zoom.webp" alt=""/>
</figure>
<p><strong>Inadequate CF statistics:</strong> Highlighted that current CF data mainly represents the white population, skewing understanding of the disease’s true prevalence.</p>
<p><strong>Underrepresentation in Asia:</strong> Revealed that CF is underreported in Asian countries like Thailand, leading to limited access to therapies and medications.</p>
<p><strong>Focus on science communication:</strong> This awareness shifted our focus to the importance of improving science communication to address these disparities.</p>
<p><strong>Racism and discrimination in research:</strong> Brought attention to the ongoing issues of racial bias and discrimination within scientific research, prompting us to take action.</p>
<p><strong>Commitment to improve data landscape:</strong> In response, we are developing bilingual (German and English) surveys to gather more inclusive data and expand our outreach efforts, ensuring a broader understanding of CF prevalence and care needs.</p>
</Collapsible>
)
}
function AnalyseWesthoff(){
const {goToPagesAndOpenTab} = useNavigation();
return(
<Collapsible title="Katrin Westhoff – Physiotherapist at local community " id="westhoffanalyseC">
<p><a onClick={() => goToPagesAndOpenTab ('westhoffinv', '/human-practices')}> Kathrin Westhoff expertise </a> in CF physiotherapy reinforced our approach to develop an inhalation-based therapy, especially for young patients. Her experience with children highlighted the need for a simple, accessible treatment, as younger patients may struggle with responsibility and consistency in therapy. This validated our decision to focus on inhalation delivery for ease of use. </p>
<p>Additionally, we recognized through her insights that gene therapy, while impactful, is not a complete solution. Physiotherapy remains crucial in CF care, and our approach now integrates this understanding into a more holistic treatment plan. </p>
<div><img src="https://static.igem.wiki/teams/5247/photos/hp/besuch-westhoff/untitled-design.png" style={{height:"50%", width:"50%"}}/></div>
<p><strong>Expertise in CF physiotherapy:</strong> Reinforced the importance of developing an inhalation-based therapy, especially for young CF patients.</p>
<p><strong>Focus on accessibility:</strong> Her experience with children highlighted the need for a simple, easy-to-use treatment, validating our decision to focus on inhalation delivery.</p>
<p><strong>Therapy adherence:</strong> Addressed the challenges younger patients face with responsibility and consistency in therapy, influencing our design for a more accessible solution.</p>
<p><strong>Gene therapy limitations:</strong> Her insights helped us realize that while gene therapy is impactful, it alone is not enough.</p>
<p><strong>Holistic approach:</strong> Integrated physiotherapy into our treatment plan, ensuring a more comprehensive and patient-centered approach to CF care.</p>
</Collapsible>
)
}
function AalyseOlariu(){
const {goToPagesAndOpenTab} = useNavigation();
return(
<Collapsible title="Prof. Dr. Olariu – Physician and Clinical CF Expert " id="olariuanalyseC">
<p>Prof. Olariou's clinical insights shaped our project by emphasizing the complexities of CF treatment and the emotional burden on patients and families. His focus on early diagnosis, infection risks, and psychosocial impacts led us to design a solution that reduces the long-term care burden</p>
<p>Following his advice, we consulted <a onClick={() => goToPagesAndOpenTab ('psychol', '/human-practices')}> psychologists </a> to integrate mental health considerations and tailored our therapy to address different disease severities. Prof. Olariu also highlighted racial and global disparities in CF care, prompting us to explore these issues through further research and interviews. </p>
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<iframe title="Bielefeld-CeBiTec: Interview with Dr. Cristian-Gabriel Olariu (2024) [German]" height="310px" width="500px" src="https://video.igem.org/videos/embed/0ea76b9c-9a0e-4f58-8230-31ab6b0e08d9" sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
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<p>His guidance on balancing treatment effectiveness with cost and overmedication concerns ensured our project remained patient-centered and practical. We also expanded our outreach efforts, raising CF awareness and collecting public data to align our project with community needs. </p>
<p><strong>Complexities of CF treatment:</strong> Emphasized the multifaceted nature of CF care, including the emotional burden on patients and families.</p>
<p><strong>Early diagnosis & infection risks:</strong> Focused on the importance of early detection and managing infection risks, which shaped our solution to reduce the long-term care burden.</p>
<p><strong>Mental health integration:</strong> Recommended consulting psychologists, leading us to incorporate mental health considerations in our therapy and design it to address varying disease severities.</p>
<p><strong>Racial and global disparities:</strong> Highlighted inequities in CF care across different populations, prompting us to explore these issues through further research and interviews.</p>
<p><strong>Balance between effectiveness, cost, and overmedication:</strong> Guided us in creating a treatment that balances efficacy with practical considerations like cost and avoiding overmedication.</p>
<p><strong>Patient-centered focus:</strong> His feedback ensured that our project remained grounded in real-world patient needs, making it both practical and accessible.</p>
<p><strong>Expanded outreach efforts:</strong> Encouraged us to raise awareness and collect public data, helping align our project with the broader CF community’s needs.</p>
</Collapsible>
)
}
function AnalyseMax(){
const {goToPagesAndOpenTab} = useNavigation();
return(
<Collapsible title="Max Beckmann – CF Patient and fellow student" id="maxanalyseC">
<p>Max's input as a cystic fibrosis (CF) patient directly influenced several key aspects of our project. After learning about the daily challenges of living with CF, we adapted our gene therapy approach to target the lungs, aligning our treatment with patient needs. His insights on the shortcomings of existing therapies strengthened our focus on developing a more effective solution.</p>
<p>Following Max's feedback, we implemented changes to our <a onClick={() => goToPagesAndOpenTab ('Patient MattersH', '/contribution')}> hygiene plan</a>, ensuring it meets the needs of immunocompromised individuals which we later presented to <a onClick={() => goToPagesAndOpenTab ('johannfunke', '/human-practices')}> Mr. Johannfunke</a>, contact person for students with disabilities and impairments at the university of bielefeld, and checked its feasibility. Max’s perspective also shaped the content of our outreach materials, helping us portray CF in a more realistic and respectful way during the whole project. </p>
<p>Additionally, Max contributed to the project by donating cells for our experiments, which allowed us to test our model systems effectively. Our ongoing communication with him has ensured that we stay patient-focused throughout, continually refining our approach based on his experiences. </p>
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<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={true} sandbox="allow-same-origin allow-scripts allow-popups allow-forms"></iframe>
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<p><strong>Invaluable first-hand insights:</strong> Provided direct experience of living with CF, shaping our project.</p>
<p><strong>Hygiene and medical needs:</strong> Highlighted the importance of constant hygiene, frequent medical checkups, and the variability in CF symptoms.</p>
<p><strong>Need for flexible solutions:</strong> Stressed the need for patient-centered, adaptable therapies.</p>
<p><strong>Gene therapy focus:</strong> Supported our focus on gene therapy targeting the lungs due to current treatment inadequacies.</p>
<p><strong>Outreach influence:</strong> Continually provided feedback on how CF is portrayed in outreach materials, influencing our hygiene plan and presentation video.</p>
<p><strong>Deepened emotional understanding:</strong> Helped us develop emotional intelligence regarding the burdens CF patients face, enhancing our science communication efforts.</p>
<p><strong>Therapy alignment:</strong> Guided us in focusing our therapy on specific CF mutations, ensuring a human-centered approach.</p>
<p><strong>Community access:</strong> Provided access to various CF communities, influencing the direction of our project.</p>
<p><strong>Cell donation:</strong> Donated cells for crucial model experiments; we successfully cultured these cells for testing.</p>
<p><strong>Ongoing collaboration:</strong> Regular feedback, including on aspects like hygiene protocols, ensuring the project remains grounded in real-world patient needs.</p>
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</Collapsible>
)
}