期刊
BLOOD
卷 121, 期 17, 页码 3335-3344出版社
AMER SOC HEMATOLOGY
DOI: 10.1182/blood-2012-10-462200
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资金
- Medical Research Council
- National Institute for Health [RP-PG-0310-1001]
- French National Agency for Research [ANR-CEXC-08-018-0]
- ASSISI Foundation of Memphis
- American Lebanese Syrian Associated Charities
- Howard Hughes Medical Institute
- National Heart, Lung, and Blood Institute [HL094396]
- MRC [G0902219] Funding Source: UKRI
- Medical Research Council [G0902219] Funding Source: researchfish
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 knockout mice following administration of 2 x 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.
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