RNA-LNP-mediated in vivo prime editing corrects disease phenotypes in a mouse model of citrullinemia type I.

Abstract

Citrullinemia type I (CTLN1) is a severe urea cycle disorder caused by pathogenic variants in the(argininosuccinate synthetase 1) gene, for which liver transplantation remains the only curative option. Here, we used prime editing to correct themouse model of CTLN1. Adeno-associated virus (AAV)-mediated delivery of the PE7 prime editor with an optimized prime editing guide RNA (pegRNA) achieved 71 and 54% correction of the pathogenicmutation in hepatocytes of neonates and juveniles, respectively. Delivery of mRNA-encoded PE7 and synthetic pegRNA via lipid nanoparticles (LNPs) resulted in 24% correction after a single 3 mg kgdose in neonates and 13% after three 4 mg kgdoses in juveniles. All treated groups showed full normalization of survival and of blood citrulline and ammonia concentrations, with restored urea cycle function and correction of natural behavior defects. Consistent with these findings, immunostaining demonstrated restoration of wild-type-like ASS1 protein localization in functionally relevant periportal and intermediate-zone hepatocytes. Editing was confined to the liver, with minimal indel formation and off-target activity and only transient elevations in liver enzymes. In a cellular reporter system, 6 of 15 recurrent human pathogenicmutations studied, including the most commonvariant, were corrected with similar or higher efficiencies than. These findings highlight prime editing as a precise and potentially curative treatment strategy for individuals with CTLN1 and other genetic liver diseases.