Therapeutic base editing to generate a gain-of-function F9 variant for hemophilia B

Developing variant-specific therapies for genetic diseases is not scalable, particularly for disorders with extensive allelic heterogeneity such as hemophilia B, caused by variants in the F9 gene encoding coagulation factor IX (FIX). Instead of correcting individual variants, we investigated a gain-of-function strategy using CRISPR–Cas9–based cytosine base editing to introduce the hyperactive R338Q (Shanghai) variant (c.1151G>A) into the endogenous F9 locus. This substitution enhances intrinsic tenase activity without increasing protein expression. An optimized base editor delivered via adeno-associated virus (AAV) achieved >60% target-site conversion in human cells and knock-in mouse models, resulting in increased FIX activity across multiple patient-derived F9 variants in knock-in models. Lipid nanoparticle (LNP) delivery also enabled efficient in vivo editing, while supporting repeat dosing and controlled editor exposure. These approaches resulted in a three- to sixfold increase in circulating FIX activity. Together, this gain-of-function genome-editing strategy provides a scalable and physiologically regulated therapeutic framework for hemophilia B.