Nguyen G N, George L A, Siner J I, Davidson R J, Zander C B, Zheng X L, Arruda V R, Camire R M, Sabatino D E
The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Department of Pediatrics, Division of Hematology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
J Thromb Haemost. 2017 Jan;15(1):110-121. doi: 10.1111/jth.13543. Epub 2016 Nov 25.
Essentials 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.
Background 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. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods 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. Results 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. Conclusions 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.
凝血因子（F）VIII是一种表达效率低下的蛋白质。使用体外和体内试验对弗林蛋白酶缺失的FVIII变体进行了纯化和表征。这些经过最小修饰的新型FVIII变体具有增强的功能。这些变体为在A型血友病基因治疗中提高FVIII表达提供了一种策略。
背景 开发基于基因的A型血友病疗法的主要挑战在于人凝血因子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主要在成对碱性氨基酸裂解酶（PACE）或弗林蛋白酶裂解位点被切割，产生一种异二聚体，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，这两种蛋白在此位点均与hFVIII相差一个氨基酸，它们主要以单链多肽（SC）形式分泌并表现出增强的功能。目的 我们假设弗林蛋白酶位点的缺失会调节FVIII生物学特性并可能增强其功能。方法 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。结果 在体外，重组纯化的Δ3和Δ4主要为SC，有趣的是，与FVIII-BDD相比，其促凝血活性高2倍。在体内，这些变体的止血功能也有所改善。腺相关病毒（AAV）载体递送后，这些变体的表达比hFVIII-BDD高2至4倍。在对hFVIII-BDD耐受的小鼠中对每个变体进行蛋白质激发试验，未显示抗FVIII免疫反应。结论 这些数据表明，弗林蛋白酶缺失的hFVIII变体优于hFVIII-BDD，且不会增加免疫原性。在基于基因的治疗中，这些新型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A型血友病基因治疗的关键因素。
