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Abstract

The rapid development of CRISPR/Cas9-based gene editing technologies has provided potential therapeutic strategies to permanently treat the underlying cause of the majority of previously untreatable genetic disorders. With the current suite of available gene editing tools, over 90% of disease-causing mutations can in principle be changed to a non-pathogenic form. However, delivery of these editing tools into body in a safe and effective manner presents the largest barrier for their clinical translation. Of available delivery methods, lipid nanoparticles (LNPs) that delivery gene editors in the form of RNA present one of the most appealing options for in vivo use. While LNPs have achieved preclinical successes with in vivo gene editing and are now entering clinical trials, there remains room for their improvement, with one area being lowering the dose needed to achieve therapeutic levels of gene editing. Recent research has demonstrated that formulating LNPs with a 300 mM Na-Citrate pH 4 buffer enhanced the functional delivery of RNA. The goal of this thesis sought to examine if the use of this same pH 4 buffer also improved gene editing efficiencies in the case of LNP-mediated base and prime editing in vivo. This was first tested in the case of base editing, where results indicated that the 300 mM Na-Citrate buffer gave no benefit to in vivo gene editing efficiency when using LNP systems with the most effective clinically approved ionizable lipids SM-102 and ALC-0315, however some benefit was seen with the previous generation ionizable lipids MC3 and KC2. In the case of prime editing, in vivo editing efficiencies were ultimately too low to draw any conclusions on LNP-RNA factors influencing editing, motivating optimization experiments for future testing in a more sensitive in vivo reporter system. Initial optimization experiments here resulted in an approximately 9-fold increase in editing efficiency, providing the foundation for future in vivo testing that may facilitate more rapid optimization of LNP-mediated prime editing.