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

描述（由申请人提供）：本申请的目标是通过将编码因子VIII （FVIII）基因的慢病毒载体直接转移到骨髓细胞中，开发一种有效的治疗A型血友病（HemA）的基因治疗策略。目前对HemA患者的治疗涉及反复输注FVIII蛋白，这既昂贵又不方便。此外，约25%的治疗患者出现抗fviii免疫反应。基因治疗治疗，可以实现长期的表型纠正，而没有抗fviii抗体形成的并发症是非常需要的。骨髓中的造血干细胞（hsc）是基于基因和细胞治疗遗传病的理想靶点，因为它们具有自我更新能力，可以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骨髓细胞而无需预处理。这种方法避免了体外HSC基因转移所遇到的困难，如干细胞特性的维持、细胞转移后移植物潜能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处理。为了降低抗FVIII抗体形成的可能性，我们将编码免疫原性较低的b结构域FVIII变体的人FVIII cDNA整合到慢病毒载体中。两种慢病毒载体由两种不同的启动子驱动，一种普遍存在的人类延伸因子- 1？（EF1?）启动子（E-LV）和人巨核细胞特异性糖蛋白1b？（GP1b?）启动子（G-LV）已构建。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基因表达、HemA表型纠正和抗FVIII免疫反应的产生。我们将研究G-LV治疗小鼠血小板中低水平FVIII基因表达或E-LV治疗小鼠血浆中高水平FVIII基因表达是否能有效纠正小鼠长期的HemA表型。我们将验证以下假设：1)在无条件小鼠骨髓内注射慢病毒载体后，包括原始造血干细胞在内的大量骨髓细胞将被转导并成熟为表达fviii的血细胞；2)由无所不在的启动子驱动的慢病毒载体将在各种成熟血细胞中指导FVIII或GFP基因的表达，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指导FVIII基因的表达。3)通过骨髓内注射慢病毒载体（由泛在启动子或血小板特异性启动子驱动，有或没有免疫调节），可以实现血友病A的长期表型纠正。

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.