GENE REPLACEMENT THERAPY--HUMAN AND MOUSE MODELS

腺苷脱氨酶缺乏症--一种罕见的常染色体隐性遗传病 导致一种严重的联合免疫缺陷，提供了一种最佳的 建立疾病模型以开发疾病的基因治疗策略，这些疾病可能 通过将基因转移到造血组织中而得到改善。我们最近 成功获得人ADA在小鼠中的长期稳定表达 在造血细胞中进行逆转录病毒基因转移，鼓励 该技术可进一步完善并应用于肺癌的基因治疗。 遗传性人类疾病。人类造血干细胞感染将是 尝试使用长期转换成功的载体 在小鼠中表达(Delta N2ADA)，并利用新的载体 建造的。人骨髓中原始干细胞的浓缩 允许我们检查这些细胞对逆转录病毒的感染性 向量。为此，用抗CD34单抗进行阳性选择 抗体将与阴性选择结合使用抗CD33和 抗Ia抗体可丰富原始目标人群 造血祖细胞。原始干细胞的感染将是 通过长期骨髓培养和移植进行评估 免疫缺陷小鼠体内的人造血祖细胞。问题 关于安全问题和将技术扩展到 血液学重建所需的细胞将被解决。 鸟氨酸氨基转移酶(OTC)缺乏症，最常见和最严重 人类先天性尿失禁，是一种极好的发展和发展的模型疾病 旨在纠正肝脏先天错误的测试基因治疗策略 细胞新陈代谢。我们最近确定了非处方药的分子基础 稀毛(SPF)小鼠的缺乏症，为人类的小鼠模型 叛逃。逆转录病毒和腺相关病毒载体的构建将 并测试媒介感染和感染的能力 在体内外将OTC活性转导入SPF肝细胞。这个 小肠表达人OTC基因的SPF转基因小鼠的建立 在生化和表型上对非处方药缺乏点是正确的 一种新的靶组织，用于这种疾病的基因治疗。重组 编码OTC的逆转录病毒和腺相关病毒将用于 感染SPF小肠上皮细胞以确定基因转移 OTC在该组织中的表达是可能的，如果成功 该组织中OTC活性的转导将纠正先天错误 这些老鼠。

Adenosine deaminase (ADA) deficiency, a rare autosomal recessive condition causing a form of severe combined immunodeficiency, provides an optimal model disease to develop strategies for gene therapy of diseases which may be ameliorated by gene transfer into hematopoietic tissue. Our recent success in obtaining stable long term expression of human ADA in mice, via retroviral gene transfer in hematopoietic cells, offers encouragement that the techniques can be further improved and applied to gene therapy of inherited human diseases. Human hematopoietic stem cell infection will be attempted with the vector that was successful in transducing long term expression in mice (delta N2ADA) and with new vectors which will be constructed. Enrichment for primitive stem cells from hum bone marrow will allow us to examine the infectability of those cells with retroviral vectors. For this purpose, positive selection with anti-CD34 monoclonal antibodies will be combined with negative selection using anti-CD33 and anti-Ia antibodies to enrich the target population for primitive hematopoietic progenitor cells. Infection of primitive stem cells will be evaluated in long term bone marrow culture and through transplantation of human hematopoietic progenitors in immuno-deficient mice. Questions regarding issues of safety and expansion of the technology to the number of cells required for hematology reconstitution will be addressed. Ornithine transcarbamylase (OTC) deficiency, the most common and severe human defect in ureagenesis, is an excellent model disease to develop and test gene therapy strategies aimed at correcting inborn errors of liver cell metabolism. We have recently identified the molecular basis of OTC deficiency in the sparse fur (spf) mouse, the mouse model for the human defect. Construction of retroviral and adeno-associated viral vectors will be undertaken and the vectors tested for their ability to infect and transduce OTC activity into spf hepatocytes in vitro and in vivo. The creation of transgenic spf mice which express human OTC cDNA in small bowel and are biochemically and phenotypically correct for OTC deficiency points to a new target tissue for gene therapy of this disease. Recombinant retrovirus and adeno-associated virus which encode for OTC will be used to infect spf small intestinal epithelial cells to determine if gene transfer and expression of OTC in this tissue is possible and if successful transduction of OTC activity in this tissue will correct the inborn error of these mice.