Alternative AAV gene therapy for hemophilia A using expression of Bi8, a novel single-chain FVIII-mimetic antibody

ABSTRACT: The recent approval of adeno-associated virus (AAV)-based gene therapies for hemophilia A (HA) represents a major advancement in the management of this X-linked bleeding disorder, offering multiyear bleed protection and improved quality of life over factor VIII (FVIII) replacement. However, challenges remain, including concerns over long-term durability of expression and the difficulty of packaging the oversized FVIII transgene into AAV vectors. To address these limitations, we developed AAV8-Bi8, a liver-directed gene therapy encoding Bi8, a novel 54.5-kilodalton FVIII-mimetic antibody. Bi8 is expressed as a compact, single-chain tandem, single-chain fragment variable, and is delivered via a 4.4-kilobase expression cassette packaged within AAV8 capsids, well within the vector packaging capacity. In vitro, Bi8 demonstrated FVIII-mimetic activity, and effectively corrected FVIII-deficient human plasma to levels comparable with emicizumab, the current market standard. In vivo, a single administration of AAV8-Bi8 in FVIII-deficient mice resulted in dose-dependent, durable expression of Bi8, complete phenotypic correction of bleeding, and therapeutic equivalence to both emicizumab-treated and wild-type animals. Importantly, no toxicity or antidrug antibody responses were observed. This approach, based on delivering FVIII-mimetic antibodies through AAV rather than truncated FVIII transgenes, could provide a more flexible and efficient platform for gene therapy in HA. AAV8-Bi8 has the potential to offer sustained, lifelong hemostatic control, including in patients who have developed inhibitors to FVIII.