Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.
Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency.
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DOI：
10.1016/j.omtm.2020.10.013
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发表时间：
2020-12-11
期刊：
影响因子：
--
通讯作者：
Xiao W
中科院分区：
文献类型：
--
作者：
Cao W;Dong B;Horling F;Firrman JA;Lengler J;Klugmann M;de la Rosa M;Wu W;Wang Q;Wei H;Moore AR;Roberts SA;Booth CJ;Hoellriegl W;Li D;Konkle B;Miao C;Reipert BM;Scheiflinger F;Rottensteiner H;Xiao W
在血友病A基因治疗中实现人类第八因子(FVIII)治疗性表达的一个重要限制是FVIII蛋白的低效分泌。用相应的猪FVIII残基取代人FVIII A1结构域中的五个氨基酸，产生了一个称为B域缺失(BDD)-FVIII-X5的分泌增强型人FVIII变异体，导致体外细胞检测系统上清液中的FVIII活性水平比未修饰的人BDD-FVIII高8倍。对纯化的重组BDD-FVIII-X5和BDD-FVIII的分析表明，这两种蛋白的特异性活性相似，表明X5的改变仅限于增加FVIII的分泌。在FVIII缺陷小鼠中静脉注射旨在表达BDD-FVIII-X5或BDD-FVIII的肝靶向腺相关病毒(AAV)载体，即使使用了高效的密码子优化的F8核苷酸序列，也能显著提高BDD-FVIII-X5的血浆FVIII活性水平。使用体外刺激试验和各种体内血友病A临床前模型进行的综合免疫原性评估表明，与BDD-FVIII相比，BDD-FVIII-X5变体没有表现出更高的免疫原性风险。总之，BDD-FVIII-X5是一种有效的FVIII变异体分子，可以进一步开发用于血友病A患者的基于基因和蛋白质的治疗方法。限制第八因子的分泌仍然是血友病A基因治疗的障碍。人类凝血因子VIII的微小变化导致了分泌增强的突变体BDD-FVIII-X5，它改善了表达，但仍保持了正常的特定活性和免疫原性特征，与未经修饰的FVIII相似，支持其进一步发展为治疗血友病A患者的药物。
One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A. Limited secretion of factor VIII remains a hurdle in hemophilia A gene therapy. Minimal alterations in human factor VIII resulted in the secretion-enhanced variant BDD-FVIII-X5 that improved expression yet retained normal specific activity and an immunogenicity profile similar to that of unmodified FVIII, supporting its further development as therapeutics for hemophilia A patients.
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