Regulations on genetic engineering. In addition to the general safety briefing, specific instructions for the safe operation of each device were provided. The Safety and Security Officer within the laboratory highlighted the potential hazards and necessary precautionary measures. We have focused on planning our laboratory activities to minimize risk for safer practices. This ensures not only the safe and proper use of equipment but also the generation of reliable data. To meet all safety requirements, additional safety protocols have been put in place for all targeted areas of the laboratory equipment.
<Subesctiontitle="Safety aspects of our Airbuddy"id="Biosafety2">
<H4text="SORT LNP and Cytotoxicity"></H4>
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We have carefully considered the biosafety aspects of our delivery system, starting with the decision between Adeno-associated viruses (AAV) or lipid nanoparticles (LNPs) as delivery systems. Our comparison revealed that the biocompatibility and safety of LNPs are paramount for our approach. That is why we chose selective organ-targeting (SORT) lipid nanoparticles (LNPs) [1] in the context of targeted pulmonary mRNA delivery. One of our primary concerns with the LNP was the potential cytotoxicity of polyethylene glycol (PEG), a common stabilizing agent in LNP formulations. Aware of the immune responses PEG can trigger, potentially leading to cytotoxicity [2], we aimed at optimizing its concentration in our SORT LNPs to minimize such reactions while maintaining therapeutic efficacy. By the use of low molecular weight PEG, we addressed this problem. To test weather our approach succeeded, we conducted MTT and proliferation assays to ensure that our LNP posed no cytotoxicity risks.
We have carefully considered the biosafety aspects of our delivery system, starting with the decision between Adeno-associated viruses (AAV) or LNPs as delivery systems. Our comparison revealed that the biocompatibility and safety of LNPs are paramount for our approach. That is why we chose selective organ-targeting (SORT) lipid nanoparticles (LNPs) [1] in the context of targeted pulmonary mRNA delivery. One of our primary concerns with the LNP was the potential cytotoxicity of polyethylene glycol (PEG), a common stabilizing agent in LNP formulations. Aware of the immune responses PEG can trigger, potentially leading to cytotoxicity [2], we aimed at optimizing its concentration in our SORT LNPs to minimize such reactions while maintaining therapeutic efficacy. By the use of low molecular weight PEG, we addressed this problem. To test weather our approach succeeded, we conducted MTT and proliferation assays to ensure that our LNP posed no cytotoxicity risks.
Our project focuses on the genetic disease cystic fibrosis, specifically targeting the Delta-508 mutation. The aim is to correct this mutation using Prime Editing, a precise genome-editing technique. We have explored different strategies to optimize the Prime Editing complex for this specific application.
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The Prime Editing complex consists of a nickase, a reverse transcriptase, a pegRNA. The pegRNA guides the editing process by directing the complex to the target DNA sequence, allowing for precise genetic modifications. For targeted delivery, we selected lipid nanoparticles (LNPs) to introduce the mRNA encoding the Prime Editing components specifically into lung epithelial cells, where the CFTR protein is highly expressed. Additionally, we investigated alternatives to the conventional Cas9 nickase, such as the smaller CasX and Fanzor, aiming to reduce the overall size of the Prime Editing complex. In our optimization efforts, we also explored smaller reverse transcriptases to enhance the efficiency of the system in human cells.
The Prime Editing complex consists of a nickase, a reverse transcriptase, a pegRNA. The pegRNA guides the editing process by directing the complex to the target DNA sequence, allowing for precise genetic modifications. For targeted delivery, we selected LNPs to introduce the mRNA encoding the Prime Editing components specifically into lung epithelial cells, where the CFTR protein is highly expressed. Additionally, we investigated alternatives to the conventional Cas9 nickase, such as the smaller CasX and Fanzor, aiming to reduce the overall size of the Prime Editing complex. In our optimization efforts, we also explored smaller reverse transcriptases to enhance the efficiency of the system in human cells.
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Furthermore, we have developed a modular plasmid that contains the backbone of our Prime Editing complex. The individual components can be cloned individually into the backbone. This plasmid allows us to either deliver the construct directly into target cells or transcribe the plasmid into RNA, enabling the delivery of the Prime Editing complex in the form of mRNA. The modularity of the plasmid is a key feature; specific restriction sites are included to facilitate the easy exchange of the complex's components. This design makes it straightforward to adapt the Prime Editing complex for various use cases and therapeutic requirements.
