Genome editing-mediated knock-in of therapeutic genes ameliorates the disease phenotype in a model of hemophilia.

Recently, clinical trials of adeno-associated virus-mediated replacement therapy have suggested long-term therapeutic effects for several genetic diseases of the liver, including hemophilia. However, there remain concerns regarding decreased therapeutic effects when the liver is regenerated or when physiological proliferation occurs. Although genome editing using the clustered regularly interspaced short palindromic repeats/Cas9 system provides an opportunity to solve this problem, low knock-in efficiency may limit its application for therapeutically relevant expression. Here, we identified a novel gene, , in which a strong promoter facilitated the expression of knocked-in genes in hepatocytes. We also investigated the effects of editing using a small Cas9 protein derived from (CjCas9) in a hemophilic model. We demonstrated that adeno-associated virus-mediated delivery of CjCas9 and donor led to moderate levels of human factor 9 expression in APOC3-humanized mice. Moreover, knock-in-driven expression induced substantial recovery of clotting function in mice with hemophilia B. There was no evidence of off-target editing or . Collectively, our findings demonstrated therapeutically relevant expression using a precise and efficient -editing platform, providing insights into the development of further long-term therapeutics for diverse monogenic liver diseases.