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) 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来看，逆转录病毒的水平 HSC-IE多能祖细胞的转导 长期造血--目前尚不足以用于治疗 利益。Moloney小鼠转导效率低 基于白血病病毒(MoMuLV)的载体被认为是次要的 将前病毒整合到静止的HSC基因组中。一个 对转导的另一个假设限制是低表达 HSC上的病毒受体。绝大多数造血祖细胞 在短时间暴露期间，细胞因子是否有反应，并被有效地转导 对病毒没有作用，不利于长期多系造血。 标准的体外检测方法可以检测这些成熟的祖细胞。我们有 测定骨髓和脐带血CD34+CD38-免疫表型 血液虽然用于补充HSC，但在功能上定义了 细胞因子反应性方面的异质性群体[使用 延长的长期培养启动细胞(ELTC-IC)试验]，谱系 潜能(使用单细胞多潜能分析，即B淋巴系 和髓系电位)和被转导的能力。我们假设 通过研究稀有的、功能原始的和转导 异质性CD34+CD38-祖细胞池中的抗性HSC，我们可能 确定这种群体逃避转导的机制 并开发可以改善基因转移的方法。三 将评估增加HSC转导的方法：延长 病毒暴露、体外对HSC的操纵和操纵 病毒及其包膜的。这项提议的具体目的是 研究内容如下：(1)确定HSC的体外干细胞分化周期。 维持细胞功能(多潜能)，(2)确定 体外操作条件对体外培养效率的影响 逆转录病毒转导、病毒受体表达及其功能 (3)确定HSC的转导效率。 水疱性口炎病毒假型慢病毒载体的构建 信封。这些研究将揭示HSC的耐药机制。 逆转录病毒转导和提供新的手段来改善 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.