AAV-Mediated Expression of a Single-Chain FVIII Mimetic Antibody Efficiently Corrects Bleeding in Haemophilia a Mice
Authors: Muczynski Vincent , Bun Eric , Tanner Lewis , McIntosh Jenny H. , Christophe Olivier D. , Nathwani Amit C. 
Published: December 2023

Background:
The recent approval of gene therapy for haemophilia A (HA) using adeno-associated viruses (AAVs) marks an important and long-awaited landmark as it is associated with markedly reduced bleeding events whilst overcoming the demands of regular life-long administration of factor concentrates or antibodies with FVIII mimetic activity. However, the benefits of AAV gene therapy in severely affected HA patients are likely to be short-lived as clinical data from most of the advanced trials show a gradual decline in transgenic Factor VIII (FVIII) levels over time. The reasons for this decline in expression remain unexplained but may be due in part to the packaging of an oversized FVIII expression cassette into AAV vectors. This hypothesis is supported by the fact that such decline in transgene expression has not been observed in other gene therapy approaches in clinic where the transgene expression cassette is within the packaging limits of AAV vectors. Therefore, we explored an alternative therapy approach for HA that utilises an expression cassette encoding a novel FVIII mimetic bispecific antibody (Bi8) that remains within the packaging constrains of AAV vectors, thus potentially improving the prospects of long-term control of the bleeding diathesis in severely affected HA subjects. .
Methods:
Bi8, is a small 54kDa single-chain bispecific antibody designed as a tandem of single-chain fragment variables (scFvs) targeting Factors IXa (FIXa) and X (FX). FVIII mimetic activity of recombinant Bi8 protein was evaluated in vitro using (1) the catalytic conversion of purified FX into FXa in a chromogenic assay, and (2) factor XIa (FXIa)-triggered thrombin generation assay (TGA) and activated partial thromboplastin time (aPTT) using spiked human HA plasma. Bi8 sequences were cloned in an AAV expression cassette downstream of a strong liver-specific promoter, pseudotyped with AAV serotype 8 capsid and evaluated in vitro as well as in vivo, including in FVIIIKO mice to assess potency in a tail vein transection assay, kinetics of Bi8 expression and the occurrence of anti-drug antibodies (ADAs).
Results:
In preliminary experiments, we demonstrated that the single-chain format used for Bi8 retained haemostatic functions. When tested in a chromogenic assay, recombinant Bi8 protein showed kinetics of FXa formation identical to that of emicizumab. Furthermore, Bi8 spiked in human HA plasma at 350nM corrected the clotting time (17.2 sec) in an aPTT assay to levels observed with emicizumab (22.7 sec). TGA with recombinant Bi8 and emicizumab showed comparable efficacy with peaks of thrombin generation at 354.2 and 358.4nM respectively. Next, human hepatocyte HuH7 cells were transduced with AAV8 encoding Bi8 (AAV8_Bi8) vectors in vitro, leading to the expression of a fully functional bispecific antibody with an activity/antigen ratio of 0.99 when compared to the recombinant Bi8 protein.
Three doses of AAV8_Bi8 vector were then evaluated in 6-8 weeks old, male FVIIIKO mice, with a single intravenous infusion of 4e 11; 4e 12 and 1.2e 13 vector genome (vg) per kilo. Expression of the bispecific antibody was detectable in plasma for all 3 doses and peaked 2 weeks post-infusion with respective concentrations of 4.5; 47.4 and 178.8nM (Panel A). Transgenic expression was then stably maintained for 12 weeks and ADAs were not detected in any of the treated animals. When challenged in a bleeding assay, haemostasis was secured in all cohorts of mice infused with AAV-Bi8 vectors, including at the lowest dose of 4e 11 vg/kg where the average total blood loss was reduced to 401.2μL (median 406.2μL) compared to 760.4μL (median 773.1μL) in non-treated FVIIIKO mice (Panel B).
Conclusion:
This study establishes the feasibility of using AAV vectors to deliver an expression cassette encoding Bi8, a novel FVIII mimetic antibody formatted as a single-chain tandem-scFv, leading to circulating concentrations of bispecific antibody at levels that can secure haemostasis and reduce blood loss in a murine model of HA. This non FVIII-based expression cassette meets the packaging capacity of AAVs and therefore provides an alternative approach for AAV-mediated gene therapy for HA that may be better suited for stable long-term expression than FVIII transgenes.