In Vivo Prime Editing by Lipid Nanoparticle Co-Delivery of Chemically Modified pegRNA and Prime Editor mRNA

Prime editing (PE) has gained significant attention as a next-generation gene editing technology, owing to its unique advantages. However, realizing its potential in vivo requires effective delivery strategies. While adeno-associated virus (AAV) has been employed for in vivo delivery 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 nonviral 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 for in vivo PE. 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 NOD scid gamma immunodeficient mouse model, the efficacy of LNP-delivered PE was enhanced by 2.8-fold. In addition, the nature of the ionizable lipids and phospholipids strongly influenced PE efficiency in vivo. Overall, these findings will greatly contribute to the future development of LNPs as a robust platform for delivering prime editors in vivo, fostering progress in PE research and therapeutic applications.