TRANSDUCTION OF HUMAN HEMATOPOIETIC STEM CELLS

人类造血干细胞的转导

• 批准号: 6381255
• 负责人: Gay M Crooks
• 金额: $ 25.23万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6381255
• 关键词: Lentivirus; Vesiculovirus; genetic transduction; hematopoietic stem cells; human tissue; receptor expression; transfection /expression vector; virus envelope; virus receptors

The goal of this proposal is to determine the mechanisms by which genes can be efficiently transferred into hematopoietic stem cells (HSC) without concurrent loss of stem cell function. Based on the results from human clinical gene therapy trials, the level of retroviral transduction of HSC- ie pluripotent progenitors able to contribute to long term hematopoiesis- is currently inadequate for therapeutic benefit. The low efficiency of transduction using Moloney Murine Leukemia Virus (MoMuLV) based vectors is assumed to be secondary to non- integration of provirus into the genomes of quiescent HSC. An additional postulated limitation to transduction is low expression of viral receptors on HSC. The great majority of hematopoietic progenitors are cytokine responsive, efficiently transduced during short exposure to virus and do not contribute to long term multilineage hematopoiesis. Standard in vitro assays measure these mature progenitors. We have determined that the CD34+CD38-immunophenotype of bone marrow and cord blood, although used to enrich for HSC, defines a functionally heterogeneous population in terms of cytokine responsiveness [using the Extended Long Term Culture-Initiating Cell (ELTC-IC) assay], lineage potential (using a single cell assay of pluripotentiality ie B lymphoid and myeloid potential) and the ability to be transduced. We hypothesize that by studying the rare, functionally primitive and transduction resistant HSC within the heterogeneous CD34+CD38-progenitor pool, we may determine the mechanisms by which this population escape transduction and develop the means by which gene transfer can be improved. Three approaches to increase HSC transduction will be evaluated: prolonging exposure to virus, manipulation of the HSC in vitro, and manipulation of the virus and its envelope. The Specific Aims of this proposal are as follows: (1) To determine the period in vitro during which HSC stem cell function (pluripotentiality) is maintained, (2) To determine the effects of manipulating invitro conditions on the efficiency of retroviral transduction, expression of viral receptors and the function of HSC, and (3) To determine the efficiency of transduction of HSC using a lentiviral vector pseudotyped with a Vesicular Stomatitis Virus (VSV) envelope. These studies will reveal the mechanisms for resistance of HSC to retroviral transduction and provide novel means for improvement of transduction efficiency.

该提案的目的是确定基因的机制 可以有效地转移到造血干细胞中（HSC） 没有同时丧失干细胞功能。 基于结果 从人类临床基因治疗试验中，逆转录病毒水平 HSC-IE多能祖细胞的转导能够做出贡献 长期造血 - 目前的治疗不足 益处。 使用Moloney Murine的转导效率低 假定基于白血病病毒（MOMULV）载体继发于非 - 将病毒的整合到静态HSC的基因组中。 一个 转导的其他假设限制是低表达的 HSC上的病毒受体。 绝大多数造血祖细胞 细胞因子反应迅速，在短期暴露期间有效转导 为病毒，不导致长期多素造血。 标准体外测定测量这些成熟的祖细胞。 我们有 确定骨髓和绳索的CD34+CD38-免疫表型 血液虽然用来丰富HSC，但在功能上定义了 在细胞因子反应性方面，异质种群[使用 长期培养发射细胞（ELTC-IC）测定]，谱系 电位（使用单电位性的单细胞测定IE B淋巴样 和髓样的潜力）和转导的能力。 我们假设 通过研究罕见的功能原始和转导 在异质CD34+CD38-Progogenit池中的抗性HSC，我们可能 确定该人群逃脱转导的机制 并开发可以改善基因转移的手段。 三 将评估增加HSC转导方法的方法：延长 暴露于病毒，在体外操纵HSC和操纵 病毒及其信封。 该提议的具体目的是 如下：（1）确定HSC茎的体外周期 维持细胞功能（多电位性），（2）确定 操纵Invitro条件对效率的影响 逆转录转导，病毒受体的表达和功能 HSC和（3）使用HSC的转导效率 用囊泡口腔炎病毒（VSV）伪造载体拟型载体。 信封。这些研究将揭示HSC抗性的机制 逆转录病毒转导并提供了改进的新颖手段 转导效率。