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bioRxiv · 10.1101/2025.07.30.667771

Therapeutic Knock-in Genome Editing Using Single AAV Vectors in Mouse Models of Inherited Liver Disease

Batjargal, K.·Togashi, T.·Kashiwakura, Y.·Baatartsogt, N.·Tsuchida, K.·Sato, T.·Hayakawa, M.·Tsukida, K.·Muramatsu, K.·Hoshino, A.·Nureki, O.·Ohmori, T.
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Abstract

Gene knock-in therapy has the potential to cure inherited liver diseases but is limited by low efficiency and delivery complexity. Here, we developed a single adeno-associated virus (AAV) vector system comprising a compact CRISPR effector, enAsCas12f, a guide RNA, and a donor template to enable therapeutic genome editing via non-homologous end joining (NHEJ). We targeted the system to the murine Alb locus and applied it to mouse models of hemophilia B, protein C (PC) deficiency, and ornithine transcarbamylase (OTC) deficiency. NHEJ-mediated knock-in showed higher efficiency than homology-directed repair, with successful therapeutic gene insertion in both neonatal and adult mice. The strategy restored plasma factor IX activity in hemophilia B (F9-/-) mice, prolonged survival of PC-deficient (Proc-/-) mice, and prevented hyperammonemia and weight loss in OTC-deficient (Otcspf-ash) mice upon high protein challenge. Importantly, gene integration was restricted to the liver, with no evidence of germline transmission. This compact, all-in-one AAV knock-in platform simplifies vector production, enables efficient delivery, and achieves reliable transgene expression in vivo. Our findings highlight the potential of liver-targeted knock-in genome editing as a transplant-independent treatment for neonatal-onset metabolic diseases, offering a clinically feasible path towards curative gene therapies for a wide range of monogenic liver disorders.

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Batjargal, K., Togashi, T., Kashiwakura, Y., Baatartsogt, N., Tsuchida, K., Sato, T., Hayakawa, M., Tsukida, K., Muramatsu, K., Hoshino, A., Nureki, O., Ohmori, T.. 2025-07-30. Therapeutic Knock-in Genome Editing Using Single AAV Vectors in Mouse Models of Inherited Liver Disease. https://doi.org/10.1101/2025.07.30.667771

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