title: Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.
abstract: Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.
authors: University College London Cancer Institute, London, United Kingdom.
authors: Jenny
authors: J
authors: McIntosh
authors: Peter J
authors: PJ
authors: Lenting
authors: Cecilia
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authors: Rosales
authors: Doyoung
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authors: Lee
authors: Samira
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authors: Rabbanian
authors: Deepak
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authors: Cancio
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authors: Zhou
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mesh_terms: D000818:Animals; D015153:Blotting, Western; D000229:Dependovirus / Q000235:genetics*; D005169:Factor VIII / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D015316:Genetic Therapy; D014644:Genetic Variation / Q000235:genetics*; D005822:Genetic Vectors / Q000008:administration & dosage*; D006031:Glycosylation; D006467:Hemophilia A / Q000235:genetics / Q000628:therapy*; D006801:Humans; D007108:Immune Tolerance; D008099:Liver / Q000378:metabolism; D008253:Macaca mulatta; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D010446:Peptide Fragments; C078147:F8 protein, human; D005169:Factor VIII