Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

• Published in: Blood Vol. 121; no. 17; pp. 3335 - 3344
• Main Authors: McIntosh, Jenny, Lenting, Peter J., Rosales, Cecilia, Lee, Doyoung, Rabbanian, Samira, Raj, Deepak, Patel, Nishil, Tuddenham, Edward G.D., Christophe, Olivier D., McVey, John H., Waddington, Simon, Nienhuis, Arthur W., Gray, John T., Fagone, Paolo, Mingozzi, Federico, Zhou, Shang-Zhen, High, Katherine A., Cancio, Maria, Ng, Catherine Y.C., Zhou, Junfang, Morton, Christopher L., Davidoff, Andrew M., Nathwani, Amit C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.04.2013; American Society of Hematology
• Subjects: Animals; Blotting, Western; Dependovirus - genetics; Factor VIII - genetics; Factor VIII - immunology; Factor VIII - pharmacology; Gene Therapy; Genetic Therapy; Genetic Variation - genetics; Genetic Vectors - administration & dosage; Glycosylation; Hemophilia A - genetics; Hemophilia A - therapy; Humans; Immune Tolerance; Liver - metabolism; Macaca mulatta; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptide Fragments - genetics; Peptide Fragments - metabolism; Promoter Regions, Genetic - genetics
• ISSN: 0006-4971; 1528-0020; 1528-0020
• DOI: 10.1182/blood-2012-10-462200

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 × 1012 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. •Novel, more potent codon-optimized human FVIII variant (codop-hFVIII-V3).•Codop-hFVIII-V3 is safe and efficacious in mice and nonhuman primates, thus improving the prospects of gene therapy for hemophilia A.