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

严重联合免疫缺陷病（SCID）是一种致命的先天性 儿童的疾病。 大约1/3的病例与 与 腺苷脱氨酶（ADA）的缺乏和一些剩余的 X连锁变异型病例有IL-2 γ受体突变 基因 这种疾病的严重性，单一的缺乏 肽蛋白，以及通过同种异体移植来纠正疾病的能力。 骨髓移植导致许多研究人员探索 使用基因转移技术作为治疗选择， 关于SCID 本文提出的工作的长期目标是利用基因 转让技术，有效地将技术转让给重建 造血干细胞和在这些细胞后代中的稳定表达 以提供终生治疗 这种疾病。 提出的基因转移方法的基础 在这项赠款包括实现，公布的数据，包括从 我们自己的实验室，证明目前的协议感染 重建大型动物物种的干细胞不是最佳的。 此外，大量的实验数据表明， 造血微环境是阳性和阴性来源 调节造血，这些信号可能是重要的 原始造血干细胞成功有效转导 细胞 在这个提议中要检验的假设是， 干细胞的长期重建可以在大规模 动物移植模型的逆转录病毒感染的存在下， 造血微环境的重要组成部分。 我们将 利用我们先前表征的简化逆转录病毒载体， 表达ADA cDNA和一组新的编码IL-2的载体 用于这些实验的γ受体cDNA。 我们将在体外同时利用 以及用于分析转导的人干细胞的体内测定 导入序列的效率和表达。 此外，我们将 继续使用灵长类动物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.