TRANSDUCTION OF HUMAN HEMATOPOIETIC STEM CELLS

这项提议的目标是确定基因 可以有效地转移到造血干细胞（HSC）中， 而不会同时丧失干细胞功能。 根据结果 从人类临床基因治疗试验中， HSC-1 e多能祖细胞的转导能够有助于 长期造血-目前不足以治疗 效益 使用莫洛尼鼠的转导效率低 假设基于白血病病毒（MoMuLV）的载体继发于非白血病病毒。 前病毒整合到静止期HSC的基因组中。 一个 对转导的另外的假定限制是低表达的 HSC上的病毒受体 绝大多数造血祖细胞 是细胞因子反应性的，在短时间暴露期间有效转导 对病毒没有作用，对长期的多系造血没有作用。 标准的体外测定测量这些成熟的祖细胞。 我们有 确定了骨髓和脐带中的CD 34 + CD 38-免疫表型， 血液，虽然用于富集HSC，但在功能上定义了 异质性人群的细胞因子反应性[使用 扩展长期培养起始细胞（ELTC-IC）试验]，谱系 潜能（使用多潜能性（即B淋巴）的单细胞测定 和骨髓潜能）和被转导的能力。 我们假设 通过研究罕见的，功能原始的 在异质性CD 34 + CD 38-祖细胞库中，我们可以 确定该群体逃避转导的机制 并开发出改善基因转移的方法。 三 将评估增加HSC转导的方法：延长 暴露于病毒、体外操作HSC和操作HSC 病毒和它的包膜 这项建议的具体目标是 （1）确定HSC干细胞在体外培养的时间， 维持细胞功能（多能性），（2）为了确定 操纵体外条件的影响， 逆转录病毒的转导、病毒受体的表达及其功能 和（3）为了确定HSC的转导效率， 用水泡性口炎病毒（VSV）假型化的慢病毒载体 信封.这些研究将有助于揭示肝星状细胞耐药的机制 逆转录病毒转导，并提供新的手段， 转导效率

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.