RAAV TRANSDUCTION IN HUMAN HEMATOPOIETIC STEM CELL

基因治疗的最大愿望之一是最终开发出 这项技术将提供一种可行方法， 缺陷和对抗传染病。 我们致力于研究 缺陷型人细小病毒腺相关病毒的分子生物学 病毒（AAV），希望开发一种安全有效的病毒载体， 人类基因疗法 AAV是一种依赖性细小病毒，其需要共- 感染另一种病毒（腺病毒或某些成员） 疱疹病毒组），以便进行生产性感染， 培养细胞 在不存在辅助病毒的共感染的情况下， 型（wt）AAV基因组通过其末端整合到宿主染色体中 以特定地点的方式，并以潜伏状态驻留在那里，直到 细胞被辅助病毒感染。 对AAV作为真核生物的兴趣 围绕着这种病毒的生物学。 除了 由于这种独特的生命周期，AAV具有广泛的感染性宿主范围 （人、小鼠、猴等），它在人类中无处不在， 完全非致病性整合病毒。 我们的研究开创了 重组AAV（rAAV）作为基因递送系统用于 造血细胞 我们启动了对人类珠蛋白基因 以及范可尼贫血C基因（FAC）。以前的努力利用 珠蛋白基因座控制区（LCR）序列遇到了困难 使用其他载体系统。 我们已经证明了高水平的，组织 人珠蛋白基因在人红系细胞中的特异性表达， 其中检测表达和珠蛋白。 虽然珠蛋白 在rAAV感染后检测到表达，转导在rAAV感染后低。 体外 在对该系统的继续分析中，我们证明了表达 和使用AAV校正来自范科尼患者的淋巴母细胞 携带范可尼贫血基因（FAC）的载体。 利用这种 患者CD 34富集骨髓细胞中的载体显示基因 在集落形成测定中的转导和选择性生长优势。 这些研究扩展到非人灵长类动物的骨髓细胞， 初步结果表明，基因标记了3个月。 这最后一 来自体外系统的观察和数据表明， 在造血祖细胞中rAVV的有效转导中， 缺乏稳定的融合。 建议的总体目标 这项工作是研究骨髓干细胞的整合， 表征的野生型AAv位点特异性整合系统和新动物 我们开发的模型。 长期目标是更好地理解 这些分子步骤在原代骨髓干细胞与最终 开发具有有效转导的特异性病毒载体的目标 有针对性的整合能力。

One of the great aspirations of gene therapy is to eventually develop technology which will provide a feasible approach to correct genetic defects and combat infectious diseases. We are engaged in studying the molecular biology of the defective human parvovirus adeno-associated virus (AAV) in hopes of developing a safe and efficient viral vector for human gene therapy. AAV is a dependent parvovirus which requires co- infection with another virus (either adenovirus or certain members of the herpes virus group) in order to undergo a productive infection in cultured cells. In the absence of co-infection with helper virus, wild type (wt) AAV genome integrates via its ends into the host chromosome in a site-specific manner and resides there in a latent state until the cell is infected with helper virus. The interest in AAV as a eukaryotic vector has centered around the biology of this virus. In addition to this unique life-cycle, AAV has a broad host range for infectivity (human, mouse, monkey, etc.), it is ubiquitous in humans, and is completely nonpathogenic integrating virus. Our research pioneered the use of recombinant AAV (rAAV) as a gene delivery system for hemopoietic cells. We initiated studies testing both human globin genes as well as the Fanconi anemia C gene (FAC). Previous efforts utilizing the globin locus control region (LCR) sequence has met with difficulty using other vector systems. We have demonstrated high level, tissue specific expression of human globin genes in human erythroid cells, where expression and globin protein was detected. Although globin expression was detected after rAAV infection, transduction was low in vitro. In continued analysis of this system, we demonstrated expression and correction of lymphoblast cells from a Fanconi patient using an AAV vector carrying the Fanconi anemia gene (FAC). Utilization of this vector in patient CD34 enriched bone marrow cells demonstrated gene transduction and selective growth advantage in colony forming assay. These studies were extended to non-human primates bone marrow cells with preliminary results suggesting gene marking out to 3 months. This last observation and data from in vitro systems suggests a rate limiting step in efficient transduction of rAVV in hematopoietic progenitor cells is the lack of stable integration. The overall objective of the proposed work is to study integration in bone marrow stem cells using the well characterized wt AAv site-specific integration system and a new animal model we have developed. The long range goal is to better understand these molecular step in primary bone marrow stem cells with the ultimate goal of developing specific viral vectors with efficient transducing capability as a result of targeted integration.