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 nonpathogenicity 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 (M(r) ~ 155 kD for fVIIIΔ756-1679 and M(r) ~ 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/1x10⁶ cells and 118 ± 29 mU/ml/1x10⁶ 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 vital 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/1x10⁶ 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.