AAV-Mediated Expression of a Single-Chain FVIII Mimetic Antibody Efficiently Corrects Bleeding in Haemophilia a Mice

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.482-482
• Main Authors: Muczynski, Vincent, Bun, Eric, Tanner, Lewis, McIntosh, Jenny H., Christophe, Olivier D., Nathwani, Amit C.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-186285

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 obse