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

描述(申请人提供)：这项建议的目标是开发一种有效的基因治疗策略，通过将编码因子VIII(FVIII)的慢病毒载体直接导入骨髓细胞来治疗血友病A(HEMA)。目前HEMA患者的治疗包括反复输注FVIII蛋白，这既昂贵又不方便。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的表型纠正，而不会出现抗FVIII抗体的并发症，是非常理想的治疗方法。骨髓中的造血干细胞(HSCs)是遗传病基因和细胞治疗的理想靶点，因为它们可以自我更新并分化为成熟的血细胞。骨髓内注射慢病毒载体已被证明可以有效地转导小鼠的骨髓细胞，而不需要进行预适应。这种方法避免了体外HSC基因转移遇到的困难，如干细胞特性的维持，细胞转移后植入潜力的丧失，以及潜在的细胞因子刺激。此外，使用这种方法不需要对干细胞进行体外操作和对受试者进行预适应。为了减少抗FVIII抗体形成的可能性，我们将把编码免疫原性较低的B区FVIII变异体的人FVIII基因整合到慢病毒载体中。两个慢病毒载体由两个不同的启动子驱动，一个普遍存在的人类延长因子-1？(EF1？)启动子(E-LV)和人巨核细胞特异性糖蛋白1b？(GP1b？)构建了启动子(G-LV)。在血友病A(HEMA)小鼠骨髓内注射这两种慢病毒载体后，将评估和比较FVIII基因的表达、HEMA表型的纠正和抗FVIII免疫应答的产生。我们将研究G-LV组小鼠血小板中FVIII基因的低水平表达或E-LV加或不加免疫调节的E-LV组小鼠血浆中高水平循环FVIII基因的表达是否对纠正小鼠长期的HEMA表型有效。我们将测试假设：1)在非条件小鼠的骨髓内注射慢病毒载体后，重要的骨髓细胞，包括原始的HSCs，将被转导并成熟为表达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表达，而由血小板特异性启动子驱动的慢病毒载体将仅在巨核细胞和血小板中引导FVIII基因的表达。3)血友病A的长期表型纠正将通过骨髓内注射慢病毒载体来实现，慢病毒载体由无处不在的或血小板特异的启动子驱动，并有或没有免疫调节。 公共卫生相关性：我们这个项目的目标是开发一种有效的基因治疗策略来治疗血友病A。造血干细胞基因治疗。骨髓中的造血干细胞(HSCs)是遗传病基因和细胞治疗的理想靶点。我们将开发一种新的策略，将慢病毒或泡沫病毒载体直接转移到骨髓细胞。这一策略可以避免体外HSC基因转移遇到的许多困难。使用这种方法不需要对干细胞进行体外操作和对受试者进行预适应。在体内，HSC基因治疗可能提供长期的治疗益处，要么通过血小板局部传递FVIII，要么在有或没有免疫调节的情况下通过血浆无处不在地传递FVIII。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Our goal of this project is to develop an effective gene therapy strategy to treat hemophilia A. Hematopoietic stem cell gene therapy. Hematopoietic stem cells (HSCs) in the bone marrow are an ideal target for gene- and cell-based therapy of genetic diseases. We will develop a new novel strategy to directly transfer lentiviral or foamy viral vectors into bone marrow cells. This strategy can avoid many difficulties encountered by ex vivo HSC gene transfer. No in vitro manipulation of stem cells and pre-conditioning of the subject will be needed using this approach. In vivo HSC gene therapy may provide long-term therapeutic benefit using either local delivery of FVIII via platelets or ubiquitous delivery of FVIII in plasma with or without immunomodulation.