In vivo lentiviral transduction of bone marrow cells for hemophilia gene therapy

项目总结 这项建议的目标是开发一种有效的基因治疗策略，通过以下方式治疗血友病A(HEMA) 将编码因子VIII(FVIII)基因的慢病毒载体直接导入骨髓细胞。目前的治疗方法 对HEMA患者的治疗涉及反复输注FVIII蛋白，这既昂贵又不方便。在……里面 此外，大约25%的接受治疗的患者产生了抗FVIII的免疫反应。基因治疗 可以实现长期表型纠正，而不会出现抗FVIII抗体形成的并发症 非常受欢迎。骨髓中的造血干细胞(HSCs)是基因和细胞的理想靶点。 遗传性疾病的基础治疗，因为它们是自我更新的，可以分化为成熟的血细胞。 骨髓内注射慢病毒载体被证明能有效地转导骨髓细胞。 未经预适应的小鼠。这种方法避免了体外HSC基因转移所遇到的困难。 例如干细胞特性的维持，细胞转移后植入潜力的丧失，以及 细胞因子刺激。此外，干细胞的体外操作和受试者的预适应将不会 使用这种方法是必要的。 为了减少产生抗FVIII抗体的可能性，我们将加入一个人FVIII的cDNA 将免疫原性较低的B区FVIII变异体编码到慢病毒载体中。驱动的两个慢病毒载体 两个不同的启动子，一个是普遍存在的人类延长因子-1(EF1)启动子(E-LV)，另一个是人类 构建了巨核细胞特异性糖蛋白1b(GP1b)启动子(G-LV)。FVIII基因 将评估HEMA的表达、HEMA表型的纠正和抗FVIII免疫反应的产生 并在骨髓内注射这两种慢病毒载体治疗血友病A(HEMA)后进行比较 老鼠。我们将研究G-LV或GLV处理的小鼠血小板中FVIII基因的低水平表达 经免疫调节或不经免疫调节的E-LV治疗后，小鼠血浆循环FVIII水平升高 能有效纠正小鼠长期的HEMA表型。 我们将检验以下假设：1)骨髓内注射慢病毒载体后， 没有条件的小鼠，重要的骨髓细胞，包括原始的HSCs将被转化和成熟 进入表达FVIII的血细胞；2)由普遍存在的启动子驱动的慢病毒载体将引导FVIII或GFP 基因在各种成熟血细胞中的表达，而慢病毒载体由血小板特异性驱动 启动子将只在巨核细胞和血小板中引导FVIII基因的表达。3)长期表型 血友病A的纠正将通过骨髓内注射慢病毒载体来实现，由 无处不在的或具有或不具有免疫调节作用的血小板特异性启动子。

PROJECT SUMMARY The goal of this proposal is to develop an effective gene therapy strategy to treat hemophilia A (HemA) by directly transferring lentiviral vectors encoding factor VIII (FVIII) gene into bone marrow cells. Current treatment of HemA patients involves repeated infusions of FVIII proteins which is both costly and inconvenient. In addition, approximately 25% of treated patients develop anti-FVIII immune responses. Gene therapy treatment that can achieve long-term phenotypic correction without the complication of anti-FVIII antibody formation is highly desired. Hematopoietic stem cells (HSCs) in the bone marrow are an ideal target for gene- and cell- based therapy of genetic diseases, because they are self-renewal and can differentiate into mature blood cells. Intra-bone marrow injection of lentiviral vectors has been shown to effectively transduce bone marrow cells in mice without pre-conditioning. This approach avoids the difficulties encountered by ex vivo HSC gene transfer such as maintenance of stem cell properties, the loss of engraftment potential after cell transfer, and potential cytokine stimulation. Furthermore, no in vitro manipulation of stem cells and pre-conditioning of the subject will be needed using this approach. In order to reduce the potential of anti-FVIII antibody formation, we will incorporate a human FVIII cDNA encoding a less immunogenic B-domain FVIII variant into the lentiviral vectors. Two lentiviral vectors driven by two different promoters, a ubiquitous human elongation factor - 1¿ (EF1 ¿) promoter (E-LV) and a human megakaryocytic-specific glycoprotein 1b¿ (GP1b¿) promoter (G-LV) have been constructed. FVIII gene expression, correction of HemA phenotype and generation of anti-FVIII immune responses will be evaluated and compared following intra-bone marrow injection of these two lentiviral vectors in hemophilia A (HemA) mice. We will investigate whether low levels of FVIII gene expression in platelets in mice treated with G-LV or high levels of circulatory FVIII in plasma in mice treated with E-LV with or without immunomodulation will be effective to correct HemA phenotype for long-term in mice. We will test the hypotheses that: 1) Following intra-bone marrow injection of lentiviral vectors in unconditioned mice, significant bone marrow cells including primitive HSCs will be transduced and matured into FVIII-expressing blood cells; 2) lentiviral vectors driven by a ubiquitous promoter will direct FVIII or GFP gene expression in a variety of matured blood cells, whereas lentiviral vectors driven by a platelet-specific promoter will direct FVIII gene expression only in megakaryocytes and platelets. 3) Long-term phenotypic correction of hemophilia A will be achieved using intra-bone marrow injection of lentiviral vectors driven by either ubiquitous or platelet-specific promoters with or without immunomodulation.