Stem cell gene therapy for canine hemophilia

描述（由申请方提供）：血友病可通过离体逆转录病毒介导的因子VIII或因子IX基因转移至自体造血干细胞中，然后将遗传校正的细胞输注至干细胞供体中来治愈。迄今为止，使用莫洛尼鼠白血病病毒（MoMLV）载体转导到大型动物和人的造血干细胞中的效率太低，无法达到治疗效果。原因之一是造血干细胞被认为主要是静止的，并且MoMLV载体不能使非分裂的靶细胞增殖。相反，基于人类免疫缺陷病毒1型（HIV-1）的载体已显示感染非分裂细胞。因此，我们希望将含有犬B结构域缺失的因子VIII基因的HIV-1载体与相应的MoMLV载体对正常犬的造血干细胞的效率进行比较。使用最有效的载体系统，然后我们希望评估具有低毒性的策略，以通过用以下进行部分骨髓消融来增加转导的造血干细胞的植入：a）环磷酰胺，B）白消安，或c）100 cGy全身照射（TBI）。我们希望将这些策略与200 cGy的全身照射进行比较，在我们过去的研究中，200 cGy的全身照射在增强转导的骨髓细胞的植入方面是最有效的。基因治疗的另一个障碍是可能诱导宿主对转导细胞和转基因编码蛋白的免疫应答。我们建议在正常的比格犬的自体骨髓细胞转导的“外国”的人因子VIII基因的免疫反应的特点，并通过移植后的免疫抑制治疗，以防止或减少这种免疫反应。最有效的载体系统，毒性最小的策略，以增加移植的转导骨髓细胞和最有效的方法，以防止对因子VIII的宿主免疫反应，然后将被用来调查在血友病A犬是否可以实现长期纠正犬血友病的逆转录病毒介导的转移犬因子VIII的cDNA到造血干细胞。概述的研究将解决目前基因转移工作的重要问题，如果成功，将促进未来血友病和其他疾病的基因治疗。

DESCRIPTION (provided by applicant): Hemophilia can potentially be cured by ex vivo retrovirus-mediated transfer of the factor VIII or factor IX gene into autologous hematopoietic stem cells followed by infusion of the genetically corrected cells into the stem cell donor. Transduction efficiency into hematopoietic stem cells of large animals and humans using Moloney murine leukemia virus (MoMLV) vectors has been too low so far to achieve a therapeutic effect. One of the reasons is that hematopoietic stem cells are considered to be predominantly quiescent and MoMLV vectors are not able to transduce non-dividing target cells. In contrast, human immunodeficiency virus type 1 (HIV-1) based vectors have been shown to infect nondividing cells. We, therefore, wish to compare the efficiency of HIV-1 vectors containing the canine B-domain deleted factor VIII gene with corresponding MoMLV vectors to transduce stem cells of normal dogs. Using the most efficient vector system, we then wish to evaluate strategies with low toxicity to increase engraftment of transduced hematopoietic stem cells by partial marrow ablation with: a) cyclophosphamide, b) busulfan, or c) 100 cGy total body irradiation (TBI). We wish to compare these strategies with 200 cGy total body irradiation, shown to be most effective in enhancing engraftment of transduced marrow cells in our past studies. Another hurdle towards gene therapy is the possible induction of host immune responses to transduced cells and transgene encoded proteins. We propose to characterize in normal beagles the immune response to autologous marrow cells transduced with the "foreign" human factor VIII gene and to prevent or decrease this immune response by immunosuppressive treatment after transplantation. The most efficient vector system, the least toxic strategy to increase engraftment of transduced marrow cells and the most effective method to prevent a host immune response against factor VIII will then be used to investigate in hemophilia A dogs whether long-term correction of canine hemophilia can be achieved by retrovirus-mediated transfer of the canine factor VIII cDNA into hematopoietic stem cells. The outlined studies will address important problems of current gene transfer efforts and, if successful, will facilitate future gene therapy of hemophilia and other diseases in man.