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Human factor VIII can be packaged and functionally expressed in an adeno‐associated virus background: applicability to haemophilia A gene therapy

Adeno‐associated virus (AAV) is a single‐stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including non‐pathogenicity and potential for long‐term transgene expression from either integrated or episomal forms. We have generated and characterized two B‐domain‐deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIΔ756–1679) or Thr761 to Asn1639 (fVIIIΔ761–1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD‐fVIII of the predicted size in both lysates and supernatants (Mr ~ 155 kD for fVIIIΔ756–1679 and Mr ~ 160 kD for fVIIIΔ761–1639) after transient transfection into COS‐1 cells. Functional fVIII quantification appeared maximal using fVIIIΔ761–1639, as evaluated by Coatest and clotting assay (98 ± 20 mU/ml/1×106 cells and 118 ± 29 mU/ml/1×106 respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIΔ761–1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1‐1) promoter, and demonstrated fVIII activity ~30% of that seen using CMV promoter. This BDD‐fVIII (rAAV(pHU1‐1) fVIIIΔ761–1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS‐1 cells using a maximally‐achievable MOI ~35 (Coatest 15 mU/ml; clotting assay 25 ± 2.0 mU/ml/1×106 cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.