GENE THERAPY--SEVERE COMBINED IMMUNODEFICIENCY DISEASE--HEMATOPOIETIC STEM CELLS

重症联合免疫缺陷病是一种致命的先天性疾病。 儿童疾病。大约1/3的病例与 腺苷脱氨酶(ADA)缺乏症及其部分残留 X连锁变异例有IL-2γ受体突变 吉恩。这种疾病的严重性，单纯性的不足 多肽蛋白和同种异体基因矫正疾病的能力 骨髓移植使许多研究人员探索 使用基因转移技术作为一种治疗选择 是SCID的。这项工作的长期目标是利用基因 将技术有效地转移到重构中 造血干细胞及其在后代中的稳定表达 对SCID中存在缺陷的基因序列进行终生治愈 这种疾病。提出了基因转移方法的基础 包括认识到已发布的数据，包括来自 我们自己的实验室，证明了目前的感染方案 重建大型动物物种的干细胞并不是最理想的。 此外，大量的实验数据表明， 造血微环境是正负两方面的来源 造血的调节器和这些信号可能对 成功高效地转导原始造血干细胞 细胞。在这项提议中要检验的假设是转导 长期重建干细胞的过程可以大量完成 存在逆转录病毒感染的动物移植模型 造血微环境的关键成分。我们会 利用我们之前表征的简化逆转录病毒载体 表达ADA基因和一组新的编码IL-2的载体 这些实验所需的伽马受体基因。我们将在体外利用这两种技术 以及人体干细胞体内试验以分析转导 导入序列的效率和表达。此外，我们还将 继续使用灵长类移植来评估基因技术 大动物自体移植模型。

Severe combined immunodeficiency disease (SCID) is a fatal congenital disease of children. Approximately 1/3 of cases are associated with the deficiency of adenosine deaminase (ADA) and some of the remaining cases with the X-linked variant have mutations of the IL-2 gamma receptor gene. The severe nature of this illness, the deficiency of single peptide proteins, and the ability to correct the disease by allogeneic bone marrow transplantation have led many investigators to explore the use of gene transfer technology as a therapeutic option for the treatment of SCID. The long term goal of the work proposed here is to utilize gene transfer technology to effect efficient transfer into reconstituting hematopoietic stem cells and stable expression in progeny of these cells to genetic sequences defective in SCID in order to provide life-long cure of this disease. The basis of the approaches to gene transfer proposed in this grant include the realization that published data, including from our own laboratory, demonstrate that current protocols for infection of reconstituting stem cells of large animals species are not optimal. Furthermore, a wealth of experimental data suggests that the hematopoietic microenvironment is a source of both positive and negative regulators of hematopoiesis and these signals may be important for successful and efficient transduction of primitive hematopoietic stem cells. The hypothesis to be tested in this proposal is that transduction of long term reconstituting stem cells can be accomplished in large animal transplant models by retroviral infection in the presence of critical components of the hematopoietic microenvironment. We will utilize our previously characterized simplified retroviral vector which expresses the ADA cDNA and a new set of vectors which encode the IL-2 gamma receptor cDNA for these experiments. We will utilize both in vitro and in vivo assays for human stem cells to analyze transduction efficiency and expression of introduced sequences. In addition, we will continue to use primate transplants to evaluate gene technology in a large animal autologous transplant model.