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<h3>Iteration 1 - AVVs vs LNPs</h3>
<p>Initially, this project part started with a discussion with Kristian Müller, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential [1] and bigger loading capacity [2]. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs [3]. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, Dr. Marco Radukic form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from PlasmidFactory as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
<p>Initially, this project part started with a discussion with <aonClick={()=>goToPagesAndOpenTab('kristian','/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential [1] and bigger loading capacity [2]. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs [3]. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p><aonClick={()=>goToPagesAndOpenTab('weber','/human-practices')}>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink</a> from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, <aonClick={()=>goToPagesAndOpenTab('radukic','/human-practices')}>Dr. Marco Radukic </a>form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from <ahref="https://www.plasmidfactory.com/custom-dna/minicircle-dna/"title="PlasmidFactory">PlasmidFactory</a> as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
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<h3>Interation 3 - Corden LNP</h3>
In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2 of Corden Pharma</strong> [5], because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of Dr. Katharina Kolonko, it was determined that capsaicin was not suitable for our planned pulmonary application.
In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong> [5] of <aonClick={()=>goToPagesAndOpenTab('corden','/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <aonClick={()=>goToPagesAndOpenTab('kolonkofirst','/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable for our planned pulmonary application.
