GENE REPLACEMENT THERAPY IN HEMATOPOIETIC STEM CELLS

该计划的总体目标是制定战略 通过基因治疗纠正骨髓来源细胞的遗传性疾病 造血干细胞的修饰。 本次活动的具体目标 提案的重点是使用重组逆转录病毒和腺病毒 用于将基因转移至原始基因的相关病毒 (AAV) 载体 造血干细胞。 针对的具体疾病有两种形式 严重联合免疫缺陷（X连锁和腺苷脱氨酶 缺乏症）、S 连锁慢性肉芽肿病 (CGD) 和 Fanconi C 组贫血。将为有效地制定方案 用逆转录病毒和 AAV 转导人类造血干细胞 向量。 不同人类造血来源的相对优点 将评估干细胞作为这些病毒载体的靶标。 的 纳入离体扩增以增加数量的可行性 干细胞靶点也将被研究。 最后，重组病毒 将鉴定出导致高水平、稳定和 转导基因在造血后代中的功能表达 干细胞。 实验方法将包括使用体外 培养系统、异种动物模型和 X-鼠模型 链接的 CGD。 这些目标的实施将由 4 项目和3个核心单位。 该提案来自一组 在造血方面具有不同但互补经验的研究人员 和干细胞生物学、逆转录病毒和 AAV 介导的基因转移、 分子遗传学、病毒学、骨髓移植等 新生儿学。 这些目标的实现将使 这项基础工作旨在制定有效的临床方案 遗传性血液疾病的病毒介导的基因转移疗法。 上一个 更广泛的层面上，这些研究应该提供对生物学的见解 造血干细胞的行为和操纵它们的能力 离体。

The overall goal of this program is to develop strategies for the correction of inherited diseases of bone marrow-derived cells by genetic modification of hematopoietic stem cells. The specific aims of this proposal are focused on the use of recombinant retroviral and adeno- associated virus (AAV) vectors for gene transfer into primitive hematopoietic stem cells. The specific diseases targeted are two forms of severe combined immunodeficiency (X-linked and adenosine deaminase deficiency), S-linked chronic granulomatous disease (CGD), and Fanconi anemia Group C. Protocols will be developed for the efficient transduction of human hematopoietic stem cells with retroviral and AAV vectors. The relative merit of different sources of human hematopoietic stem cells as targets for these viral vectors will be evaluated. The feasibility of incorporating ex vivo expansion to increase the number of stem cell targets will also be investigated. Finally, recombinant viral vectors will be identified that result in high level, stable, and functional expression of the transduced gene in progeny of hematopoietic stem cells. Experimental approaches will include the use of in vitro culture systems, xenographic animal models, and a murine model of X- linked CGD. The implementation of these aims will be shared among 4 projects and 3 core units. This proposal draws from a group of investigators with diverse but complementary experience in hematopoiesis and stem cell biology, retroviral- and AAV-mediated gene transfer, molecular genetics, virology, bone marrow transplantation, and neonatology. Achievement of these goals will permit the translation of this basic work to the development of clinical protocols for effective viral-mediated gene transfer therapy of genetic blood diseases. On a broader level, these studies should provide insight into the biologic behavior of hematopoietic stem cells and the ability to manipulate them ex vivo.