Treatment of a genetic liver disease in mice through transient prime editor expression

Abstract

AbstractPrime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept forin vivoprime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed anin vivoprime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at theDnmt1locus using AAV doses of 2.5×1013vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5×1013vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 µmol/l to below the therapeutic threshold of 600 µmol/l. Our study demonstrates the feasibility ofin vivogene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.