STEM CELL ADHESION IN GROWTH AND TRANSDUCTION

干细胞生长和转导中的粘附

• 批准号: 6879595
• 负责人: David Collin Williams
• 金额: $ 35.69万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-29 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879595
• 关键词: Retroviridae; biotechnology; cell adhesion; cell cycle; chimeric proteins; cytokine receptors; fibronectins; gene therapy; genetic transduction; hematopoietic stem cells; hematopoietic tissue transplantation; messenger RNA; microarray technology; stem cell transplantation; tissue /cell culture; transfection /expression vector

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.

严重联合免疫缺陷病（SCID）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）和一些剩余的酶缺乏 X连锁变异病例的 IL-2 γ 受体发生突变 基因。 这种疾病的严重性，缺乏单一的 肽蛋白，以及通过同种异体纠正疾病的能力 骨髓移植促使许多研究者探索 使用基因转移技术作为治疗选择 SCID 的。 这里提出的工作的长期目标是利用基因 转移技术以实现高效转移至重组 造血干细胞及其在这些细胞后代中的稳定表达 针对 SCID 缺陷的基因序列以提供终身治愈 这种疾病。 提出的基因转移方法的基础 在这笔赠款中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当前的感染方案 重建大型动物物种的干细胞并不是最佳的。 此外，大量的实验数据表明 造血微环境是积极和消极的来源 造血调节因子，这些信号可能对于 成功高效的原始造血干转导 细胞。 本提案要检验的假设是转导 长期重建干细胞可以在大范围内完成 在存在逆转录病毒感染的情况下建立动物移植模型 造血微环境的关键组成部分。 我们将 利用我们之前表征的简化逆转录病毒载体 表达 ADA cDNA 和一组编码 IL-2 的新载体 用于这些实验的 γ 受体 cDNA。 我们将在体外利用两者 以及人体干细胞的体内测定以分析转导 引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 大型动物自体移植模型。