Prime editing has gained significant attention as a next-generation gene editing technology, owing to its unique advantages. However, realizing its potentialrequires effective delivery strategies. While adeno-associated virus (AAV) has been employed fordelivery of prime editors in research settings, it presents inherent limitations related to vector size, ongoing expression, and inability to re-dose patients. Conversely, lipid nanoparticles (LNPs) do not face these limitations and are emerging as a leading non-viral approach for the delivery of gene editors. In this study, we demonstrate successful co-delivery of chemically modified pegRNA and prime editor mRNA using LNPs forprime editing. We investigate the impact of pegRNA chemical modifications on editing efficiency and explore different re-dosing regimens. In a daily-repeat dose regimen, we saw striking liver toxicity and no increase in editing; by contrast, weekly-repeat dosing was well tolerated and enabled 1.8-fold increase in editing efficacy. Furthermore, in the NSG immunodeficient mouse model, the efficacy of LNP-delivered prime editing was enhanced by 2.8-fold. In addition, the nature of the ionizable lipids and phospholipids strongly influenced prime editing efficiency. Overall, these findings will greatly contribute to the future development of LNPs as a robust platform for delivering prime editors, fostering progress in prime editing research and therapeutic applications. in vivo in vivo in vivo in vivo in vivo
