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bioRxiv · 10.1101/2025.02.24.639678

Enhancement of Therapeutic Transgene Insertion for Murine Phenylketonuria

Martinez, M.·Richards, D. Y.·Winn, S. R.·Baris, A. M.·Vonada, A.·Dudley, S.·Harper, L.·Harding, C. O.
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Abstract

Low in vivo transgene integration frequency limits the therapeutic efficacy of homology-directed repair (HDR)-mediated gene insertion as a treatment for Mendelian 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. Targeted integration of a Pah-expressing transgene into the hepatocytes of neonatal mice was enhanced with vanillin, a potent inhibitor of non-homologous end joining (NHEJ). This was further improved following combination of vanillin and novobiocin, an inhibitor of microhomology-mediated end joining (MMEJ). Combined NHEJ and MMEJ inhibition yielded PAH-expressing transgene insertions in approximately 10% of targeted alleles and was associated with a 70.6% decrease in serum phenylalanine. Demonstrating that pharmacologic inhibition of DNA repair pathways that compete with HDR can significantly enhance HDR-mediated transgene insertion in vivo.

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Martinez, M., Richards, D. Y., Winn, S. R., Baris, A. M., Vonada, A., Dudley, S., Harper, L., Harding, C. O.. 2025-02-28. Enhancement of Therapeutic Transgene Insertion for Murine Phenylketonuria. https://doi.org/10.1101/2025.02.24.639678

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