Re-engineering of Dendrimer-Based Lipid Nanoparticles for Efficient and Precise HDR-Mediated Gene Editing


August 2021


Farbiak, Lukas John

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CRISPR/Cas gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via Homology Directed Repair (HDR) that requires careful orchestration of complex steps. Rather, many reports of in vivo gene editing rely on an error-prone mechanism called Non-Homologous End Joining (NHEJ). While this pathway is effective for elimination of protein function via the introduction of insertions and deletions (Indels) into the genome, it presents little to no utility for correcting disease-causing mutations in DNA. As such, there is a pressing need to develop effective non-viral carriers capable of replacing the mutated DNA sequence with the corrected sequence via HDR. Currently, non-viral, in vivo gene editing techniques have been limited in their capacity to precisely correct mutations in DNA, with most examples yielding correction rates of less than 1%. Additionally, many delivery systems aimed at inducing HDR have consisted of multiple transfection vehicles, including virus, due to the diverse set of nucleic acid cargoes required for this process. However, techniques relying on viral vectors and/or separate carriers are nonoptimal due to potential immunogenicity and process dependence all three nucleic acids. This dissertation details the development of dendrimer-based lipid nanoparticles (dLNPs) for the encapsulation and delivery of multiple nucleic acids necessary for HDR: Cas9 mRNA, sgRNA, and a donor DNA template containing the correct nucleic acid sequence, as well as the optimization of intra-particle nucleic acid ratios for efficient induction of HDR in vivo. To assess in vivo HDR efficiency, we employed xenograft tumors consisting of BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation. Through systematically adjusting the individual internal ratios of Cas9 mRNA, sgRNA, and donor ssDNA, an optimal balance of components resulted in a HDR rate of greater than 20% in vivo. This is the first report of a completely non-viral, LNP-based, fully nucleic acid-mediated delivery system capable of inducing HDR. Due to the all-in-one simplicity and high efficacy, HDR dLNPs provide a route forward towards correcting DNA mutations responsible for genetic disease.

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CRISPR-Cas Systems, Dendrimers, DNA End-Joining Repair, Gene Editing


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