Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability...

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 (fVIIIdelta756-1679) or Thr761 to Asn1639 (fVIIIdelta761-1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD-fVIII of the predicted size in both lysates and supernatants (Mr approximately 155 kD for fVIIIdelta756-1679 and Mr approximately 160 kD for fVIIIdelta761-1639) after transient transfection into COS-1 cells. Functional fVIII quantification appeared maximal using fVIIIdelta761-1639, as evaluated by Coatest and clotting assay (98+/-20mU/ml/1x10(6) cells and 118+/-29 mU/ml/1x10(6) respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIdelta761-1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1-1) promoter, and demonstrated VIII activity approximately 30% of that seen using CMV promoter. This BDD-fVIII (rAAV(pHU1-1) fVIIIdelta761-1639) 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 approximately 35 (Coatest 15 mU/ml; clotting assay 25+/-20 mU/ml/1x10(6) 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.