Treatment of a metabolic liver disease in mice with a transient prime editing approach

Prime editing is a versatile genome editing technology that circumvents the need for DNA double-strand break formation and homology-directed repair, making it particularly suitable for in vivo correction of pathogenic mutations. Here we developed liver-specific prime editing approaches with temporally restricted prime editor (PE) expression. We first established a dual-delivery approach where the prime editor guide RNA is continuously expressed from adeno-associated viral vectors and only the PE is transiently delivered as nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNP). This strategy achieved 26.2% editing with PEmax and 47.4% editing with PE7 at the Dnmt1 locus using a single 2 mg kg^-1 dose of mRNA-LNP. When targeting the pathogenic Pah^enu2 mutation in a phenylketonuria mouse model, gene correction rates reached 4.3% with PEmax and 20.7% with PE7 after three doses of 2 mg kg^-1 mRNA-LNP, effectively reducing blood L-phenylalanine levels from over 1,500 µmol l^-1 to below the therapeutic threshold of 360 µmol l^-1. Encouraged by the high efficiency of PE7, we next explored a simplified approach where PE7 mRNA was co-delivered with synthetic prime editor guide RNAs encapsulated in LNP. This strategy yielded 35.9% editing after two doses of RNA-LNP at the Dnmt1 locus and 8.0% editing after three doses of RNA-LNP at the Pah^enu2 locus, again reducing L-phenylalanine levels below 360 µmol l^-1. These findings highlight the therapeutic potential of mRNA-LNP-based prime editing for treating phenylketonuria and other genetic liver diseases, offering a scalable and efficient platform for future clinical translation.