背景:血友病A(HA)是由内源性凝血级联中因子VIII(FVIII)基因缺乏引起的X连锁单基因疾病。目前HA的蛋白质替代疗法(PRT)有许多局限性,包括短期有效性,高成本,和终身治疗要求。基因治疗已成为HA的一种有希望的治疗方法。原位功能性FVIII生物合成对其凝血活性至关重要。
方法:为了研究FVIII的靶向表达,我们开发了一系列先进的慢病毒载体(LV)携带通用启动子(EF1α)或多种组织特异性启动子,包括内皮特异性(VEC),内皮和上皮特异性(KDR),和巨核细胞特异性(Gp和ITGA)启动子。
结果:为了检查组织特异性,在人内皮细胞和巨核细胞系中测试B结构域缺失的人F8(F8BDD)基因的表达。功能测定表明LV-VEC-F8BDD和LV-ITGA-F8BDD在转导的内皮细胞和巨核细胞的治疗范围内的FVIII活性,分别。在F8敲除小鼠(F8KO小鼠,F8null小鼠),静脉内(iv)注射LV说明了不同载体的不同程度的表型校正以及抗FVIII免疫应答。LV-VEC-F8BDD和LV-Gp-F8BDD的静脉给药在180天内实现了80%和15%的治疗性FVIII活性,分别。与其他LV结构不同,LV-VEC-F8BDD在处理的F8null小鼠中显示出低FVIII抑制反应。
结论:LV-VEC-F8BDD具有较高的LV包装和交付效率,在F8null小鼠中具有内皮特异性和低免疫原性,具有很大的临床应用潜力。
Hemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities.
To investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters.
To examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice.
The LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.