Nonviral delivery of a base editor enables personalized correction of hemophilia B nonsense variants in a mouse model.

BACKGROUNDHemophilia B is caused by loss-of-function variants in the F9 gene encoding factor IX (FIX). Although adeno-associated virus (AAV) vector-based gene therapy can restore FIX expression for over a decade, it does not restore the pathogenic variants and faces limitations related to immunogenicity and re-dosing.

OBJECTIVESTo develop a variant-specific, nonviral base editing strategy using lipid nanoparticles (LNPs) for the precise personalized correction of pathogenic nonsense variations in F9.

METHODSEditing efficiency was evaluated in HEK293 cells harboring F9 variants treatable by A•T to G•C base editing. Functional and molecular restoration in vivo was assessed in hemophilia B model mice created with AAV8 vectors expressing F9 variants. LNPs harboring adenine base editor (Cas9 nickase conjugated with ABE8e) mRNA and the corresponding guide RNA were intravenously injected into the mice.

RESULTSWe created HEK293 stably expressing 6 correctable nonsense variants: c.1067G>A, c.1068G>A, c.1222C>T, c.1292G>A, c.1358G>A, and c.1359G>A. Transfection of the cells with a base editor consisting of SpRY, an engineered SpCas9 with broader protospacer adjacent motif compatibility, and ABE8e, together with the guide RNA sequence, successfully induced A>G conversion at all target sites. The administration of LNPs harboring the base editor mRNA and guide RNA to the hemophilia B model restored the pathological variants (62.8% ± 14.4% for c.1068G>A, 35.9% ± 3.8% for c.1222C>T, and 70.6% ± 2.7% for c.1292G>A) and increased plasma FIX activity.

CONCLUSIONThis variant-specific, nonviral base editing platform represents a truly curative intervention for severe hemophilia B caused by single-nucleotide variants.