Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant
Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant
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DOI：
10.1182/blood-2012-10-462200
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发表时间：
2013-04-25
期刊：
影响因子：
20.3
通讯作者：
Nathwani, Amit C.
中科院分区：
文献类型：
--
作者：
McIntosh, Jenny;Lenting, Peter J.;Nathwani, Amit C.
对编码人因子VIII （hFVIII）的重组腺相关病毒（rAAV）载体在血友病A （HA）基因治疗中的应用进行了系统评价。一个5.7 kb的raav表达盒（raav -HLP- codopa -hFVIII- n6）含有密码子优化的hFVIII cDNA，其中226个氨基酸（aa） B结构域间隔子取代了整个B结构域，混合肝脏特异性启动子（HLP）介导的小鼠hFVIII水平比非密码子优化的变体高10倍。通过用一个新的17-aa肽（V3）取代226-aa的N6间隔序列，进一步提高了两倍的效力，其中来自B结构域的6个糖基化三联体并置。由此产生的5.2 kb raav - hhp - codopo -hFVIII- v3盒式更有效地包装在AAV病毒粒子中，并在给药2 × 10（12）个载体基因组/kg后介导HA敲除小鼠的超生理hFVIII表达（正常的732 +/- 162%），该载体剂量在b型血友病受试者中被证明是安全的。在该剂量下，非人灵长类动物的hFVIII表达稳定在正常的15 +/- 4%。在接受较高剂量的该载体或N6变体的3只猕猴中，观察到hFVIII表达超过100%。这些动物产生了中和性的抗fviii抗体，这些抗体被短暂的免疫抑制所消除。因此，raav - hlp - codopa - hfviii - v3极大地改善了有效HA基因治疗的前景。
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.