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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
Author(s) -
Gnatenko Dmitri,
Saenko Evgueni,
Jesty Jolyon,
Cao LiangXian,
Hearing Patrick,
Bahou Wadie
Publication year - 1999
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.1999.01137.x
Subject(s) - microbiology and biotechnology , transfection , recombinant dna , genetic enhancement , virology , virus , biology , parvovirus , gene , adeno associated virus , transgene , clotting factor , mutant , chemistry , vector (molecular biology) , medicine , genetics
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 Phe 756 to Ile 1679 (fVIIIΔ756–1679) or Thr 761 to Asn 1639 (fVIIIΔ761–1639). [ 35 S]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/1×10 6 cells and 118 ± 29 mU/ml/1×10 6 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/1×10 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.