Enhancement of therapeutic transgene insertion for treatment of murine phenylketonuria.

Low in vivo transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR) as a treatment for genetic disorders. This study demonstrates improved efficacy of HDR-mediated gene insertion for the treatment of murine phenylalanine hydroxylase (PAH) deficiency, a model of human phenylketonuria (PKU), through pharmacologic inhibition of competing DNA repair pathways. The targeted integration of a Pah-expressing transgene was enhanced with vanillin, a potent inhibitor of non-homologous end joining (NHEJ), reducing mean serum phenylalanine concentrations by 56.8% in treated mice. This was further improved following co-inhibition of NHEJ and microhomology-mediated end joining (MMEJ), yielding transgene insertions in approximately 10% of genomes with an associated 70.6% decrease in serum phenylalanine. Phenylalanine concentrations were further reduced to 392 μM after oral administration of sapropterin dihydrochloride, a pharmacologic cofactor of PAH. Separately, we demonstrate that rare hepatocytes harboring transgene insertions were successfully expanded to a therapeutically relevant population through selection based upon resistance to acetaminophen toxicity, but this method was hampered by adverse effects upon AAV vector production and the neurologic function of treated neonatal mice related to the presence of shRNA sequences in the AAV vector. These results demonstrate that pharmacologic inhibition of alternative DNA repair pathways can significantly enhance HDR-mediated transgene insertion in vivo.