Stem cell gene therapy for canine hemophilia

描述(申请人提供)：血友病可以通过体外逆转录病毒介导的将因子VIII或因子IX基因转移到自体造血干细胞中，然后将经过基因修正的细胞注入干细胞捐赠者中来治愈。到目前为止，使用Moloney小鼠白血病病毒(MoMLV)载体将其导入大型动物和人类的造血干细胞的效率太低，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，而MoMLV载体不能转导未分裂的靶细胞。相比之下，基于人类免疫缺陷病毒1型(HIV-1)的载体已被证明可以感染未分裂的细胞。因此，我们希望比较含有犬B区缺失因子VIII基因的HIV-1载体和相应的MoMLV载体转导正常犬干细胞的效率。然后，利用最有效的载体系统，我们希望评估通过部分骨髓切除增加转导的造血干细胞植入的低毒策略：a)环磷酰胺，b)白头翁，或c)100cGy全身照射(TBI)。我们希望将这些策略与200cGy全身照射进行比较，在过去的研究中，200cGy全身照射被证明在促进转导的骨髓细胞植入方面最有效。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反应。我们建议在正常的比格犬中，对转导了“外来”人第VIII因子基因的自体骨髓细胞的免疫反应进行表征，并在移植后通过免疫抑制治疗来预防或减少这种免疫反应。最有效的载体系统、增加转导的骨髓细胞植入的毒性最低的策略以及防止宿主对凝血因子VIII的免疫反应的最有效的方法将被用于研究血友病A犬是否可以通过逆转录病毒介导的犬凝血因子VIII基因转移到造血干细胞来实现犬血友病的长期纠正。概述的研究将解决当前基因转移努力中的重要问题，如果成功，将促进未来人类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.