Low-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response.

AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 −/−), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose (2 × 10⁹ vg/neonate and 1 × 10¹⁰ vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9^R338L variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases. Here, the authors develop an AAV-CRISPR mediated somatic knock-in. They apply this system to restore hemostasis in neonatal hemophilia B mice and show liver-specificity of the knock-in and low serum antibody production. [ABSTRACT FROM AUTHOR]