IDURONIDASE GENE TRANSFER INTO HEMATOPOIETIC CELLS

代谢储存疾病，如粘多糖病，可以通过体外移植缺陷基因到自体造血干细胞中，然后输注基因校正的细胞来治愈。然而，在大型动物和人类造血干细胞中的转导效率太低，无法达到治疗效果。在本提案中，我们希望评估低毒性策略，以增加移植物造血干细胞移植物，这些移植物是由Moloney小鼠白血病病毒（MoMLV）载体转导的骨髓细胞在没有条件或输注的情况下，在羟基脲、甲氨蝶呤或布硫凡部分骨髓消融后至少与200 cGy TBI一样有效，在我们过去的研究中显示更有效。基因治疗的另一个障碍是可能诱导对转导细胞和转基因编码蛋白的免疫反应。我们建议表征正常比格犬对外源人IDU基因转导的自体骨髓细胞的免疫反应，以将这些转导细胞的长期持久性与宿主的免疫反应联系起来。我们希望通过移植后的免疫抑制治疗来预防或减少这种反应。最有效的方法将在IDU缺乏的狗身上进行试验，这些狗将接受用“外源”犬IDU基因转导的自体骨髓细胞，然后进行免疫抑制治疗。造血干细胞被认为主要是静止的。通过MoMLV载体将基因转移到造血干细胞中效率不高，部分原因是它们无法转导非分裂细胞。由于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非分裂靶细胞，我们希望比较含有犬IDU基因的HIV-1载体与相应的MoMLV载体转导正常和IDU缺陷犬干细胞的效率。概述的研究将解决当前的基因转移问题，如果成功，将促进未来对赫勒综合征和其他疾病的基因治疗。

Metabolic storage disease like mucopolysaccharidosis can potentially be cured by ex vivo transfer of the deficient gene into autologous hematopoietic stem cells followed by infusion of the genetically corrected cells. Transduction efficiency into hematopoietic stem cells of large animals and humans though has been too low to achieve a therapeutic effect. In this proposal, we wish to evaluate strategies with low toxicity to increase engraftment of hematopoietic stem cells transduced with Moloney murine leukemia virus (MoMLV) vectors transduced marrow cells in the absence of conditioning or infusions after partial marrow ablation with either hydroxyurea, methotrexate or busulfan are at least as effective as 200 cGy TBI, shown to be more effective in our past studies. Another hurdle towards gene therapy is the possible induction of immune responses to transduced cells and the transgene encoded proteins. We propose to characterize the immune response in normal beagles to autologous marrow cells transduced with the "foreign" human IDU gene to correlate the long-term persistence of these transduced cells with the host's immune response. We wish to develop methods to prevent or decrease this response by immunosuppressive treatment after transplantation. The most effective method will be tested in IDU-deficient dogs which will receive autologous marrow cells transduced with the "foreign" canine IDU gene followed by immunosuppressive treatment. Hematopoietic stem cells are considered to be predominantly quiescent. Gene transfer into hematopoietic stem cells by MoMLV vectors is not efficient in part due to their inability to transduce non-dividing cells. As human immunodeficiency virus type 1 (HIV-1) based vectors have been shown to infect non-dividing target cells, we wish to compare the efficiency of HIV-1 vectors containing the canine IDU gene with corresponding MoMLV vectors to transduce stem cells of normal and IDU deficient dogs. The outlined studies will address current problems of gene transfer and, if successful, will facilitate future gene therapy of Hurler syndrome and other diseases.