Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.
Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.
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带有修饰的脂蛋白裂解位点的新因子VIII变体提高了基因疗法对血友病的疗效。
DOI：
10.1111/jth.13543
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发表时间：
2017-01
期刊：
影响因子：
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通讯作者：
Sabatino DE
中科院分区：
文献类型：
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作者：
Nguyen GN;George LA;Siner JI;Davidson RJ;Zander CB;Zheng XL;Arruda VR;Camire RM;Sabatino DE
因子 (F) VIII 是一种低效表达的蛋白质。使用体外和体内测定法纯化和表征弗林蛋白酶缺失 FVIII 变体。这些经过最小修改的新型 FVIII 变体具有增强的功能。这些变体提供了在血友病 A 基因治疗中增加 FVIII 表达的策略。开发 A 型血友病基因疗法的主要挑战是人因子 VIII (hFVIII) 具有导致生物合成效率低下的内在特性。在细胞内加工过程中，hFVIII 主要在配对的碱性氨基酸裂解酶 (PACE) 或弗林蛋白酶裂解位点处裂解，产生异二聚体，这是分泌蛋白的主要形式。先前对 B 结构域缺失 (BDD) 犬 FVIII 和 hFVIII-R1645H 的研究表明，这些蛋白主要以单多肽链 (SC) 形式分泌，并表现出增强的功能，两者与 hFVIII 的差异在于该位点的单个氨基酸。我们假设弗林蛋白酶位点的缺失可调节 FVIII 生物学并可能增强其功能。将一系列重组 hFVIII-弗林蛋白酶缺失变体引入 hFVIII-BDD [Δ1645、1645-46(Δ2)、1645-47(Δ3)、1645-48(Δ4) 或 Δ1648] 并进行表征。 在体外，重组纯化的 Δ3 和 Δ4 主要是 SC，有趣的是，与 FVIII-BDD 相比，其促凝血活性高出 2 倍。在体内，这些变体还具有改善的止血功能。腺相关病毒 (AAV) 载体递送后，这些变体的表达比 hFVIII-BDD 高 2-4 倍。对 hFVIII-BDD 耐受的小鼠中每种变体的蛋白质挑战均未显示出抗 FVIII 免疫反应。这些数据表明，弗林蛋白酶缺失的 hFVIII 变体优于 hFVIII-BDD，且免疫原性不增加。在基于基因的治疗中，这些新的变体提供了一种独特的策略来增加 FVIII 表达，从而降低载体剂量，这是血友病 A 基因治疗的关键因素。
Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. The major challenge for developing gene‐based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B‐domain‐deleted (BDD) canine FVIII and hFVIII‐R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. A series of recombinant hFVIII‐furin deletion variants were introduced into hFVIII‐BDD [Δ1645, 1645‐46(Δ2), 1645‐47(Δ3), 1645‐48(Δ4), or Δ1648] and characterized. In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2‐fold higher procoagulant activity compared with FVIII‐BDD. In vivo, the variants also have improved hemostatic function. After adeno‐associated viral (AAV) vector delivery, the expression of these variants is 2–4‐fold higher than hFVIII‐BDD. Protein challenges of each variant in mice tolerant to hFVIII‐BDD showed no anti‐FVIII immune response. These data suggest that the furin deletion hFVIII variants are superior to hFVIII‐BDD without increased immunogenicity. In the setting of gene‐based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.