ADENO ASSOCIATED VIRUS MEDIATED TRANSDUCTION--HEMATOPOIETIC STEM/PROGENITOR CELLS

基于腺相关病毒2（AAV）的载体系统吸引了 作为更常用的替代方案， 逆转录病毒载体在基因治疗中的潜在用途主要是 因为AAV对人类是非致病性病毒，而野生型（wt） 已经显示AAV基因组在重组中整合到人染色体DNA中。 一种特定的方式。 然而，一些问题涉及到 野生型AAV与正常人相互作用的基础分子生物学 通常是二倍体细胞，特别是重组AAV的二倍体细胞， 大部分尚未开发。 我们已展开有系统的研究， 寻求这些问题的答案，并获得了初步证据， 野生型AAV与正常人二倍体成纤维细胞相互作用的方式 这与人类非整倍体细胞的情况不同。 的假设 在这个子项目中要测试的是wt AAV整合到 正常人类造血细胞在不同位点特异性地定位 与人类非整倍体细胞中的特征不同， 缺乏病毒编码序列（rep和/或cap）的AAV基因组整合 在二倍体造血细胞中与野生型不同的位点 AAV基因组。 将追求以下具体目标： 1. 野生型AAV基因组在人类中整合模式的比较 非整倍体和二倍体造血细胞，包括纯化群体 来自正常人骨髓和脐带的原始祖细胞 脐带血：采用Southern杂交和聚合酶链反应（PCR） 分析中，wt AAV基因组的整合模式将是 在人类非整倍体和二倍体造血细胞中进行比较。 2. 评估AAV编码的蛋白质在位点特异性重组中的作用。 野生型和重组AAV基因组的整合：重组病毒 含有病毒Rep或Cap基因序列的载体将 用于评估AAV编码的蛋白质是否在该位点发挥作用- 具体整合。 3. AAV整合位点的分子克隆和表征 在二倍体细胞中，并与非整倍体细胞比较：在 除了直接克隆入λ噬菌体和质粒载体之外， 将采用基于PCR的克隆策略获得DNA序列 其含有来自人类造血细胞的AAV整合位点。 这些序列的一级结构和转录潜力 也将被确定。 4. AAV介导的体内转导的评估，以及其潜在的 用于小鼠模型中转导基因的长期表达 系统：小鼠造血干细胞和祖细胞将被 体外转导，并在受体的体内重建后 小鼠，wt和重组AAV的整合模式 基因组以及基于AAV的载体系统的安全性和有效性 将被审查。 这些研究将为基础分子生物学提供新的见解 的AAV-正常二倍体细胞的相互作用，并评估在 基于AAV的载体系统的体内功效和安全性， 在人类基因治疗中的潜在用途。 这些研究也与我们的 对细小病毒和人类疾病的长期兴趣。

The adeno-associated virus 2(AAV)-based vector system has attracted considerable attention as an alternative to the more commonly used retroviral vectors for its potential use in gene therapy primarily because AAV is a non-pathogenic virus for humans, and the wild-type (wt) AAV genome has been shown to integrate into the human chromosomal DNA in a site-specific manner. However, a number of questions related to the basic molecular biology of the wt AAV interactions with normal human diploid cells in general, and that of the recombinant AAV in particular, remain largely unexplored. We have initiated systematic studies to pursue answers to these questions, and obtained preliminary evidence that the wt AAV interacts with normal human diploid fibroblasts in a manner that is distinct from that with human aneuploid cells. The hypotheses to be tested in this subproject are that the wt AAV integrates into normal human hematopoietic cells site-specifically at a site distinct from that characterized in human aneuploid cells, and that recombinant AAV genomes lacking the viral coding sequences (rep and/or cap) integrate in diploid hematopoietic cells at sites different from that for the wt AAV genome. The following Specific Aims will be pursued: 1. Comparison of patterns of integration of the wt AAV genome in human aneuploid and diploid hematopoietic cells, including purified populations of primitive progenitor cells from normal human bone marrow and umbilical cord blood: Using Southern blot and polymerase-chain reaction (PCR) analyses, the patterns of integration of the wt AAV genome will be compared in human aneuploid and diploid hematopoietic cells. 2. Evaluation of the role of AAV-encoded proteins in the site-specific integration of the wt and the recombinant AAV genomes: Recombinant viral vectors containing either the viral rep or the cap gene sequences will be used to evaluate whether AAV-encoded proteins play a role in the site- specific integration. 3. Molecular cloning and characterization of the AAV-integration sites in diploid cells, and comparison with that from aneuploid cells: In addition to direct cloning into bacteriophage lambda and plasmid vectors, PCR-based cloning strategies will be employed to obtain DNA sequences that contain the AAV-integration sites from human hematopoietic cells. The primary structure and transcriptional potential of these sequences will also be determined. 4. Evaluation of AAV-mediated transduction in vivo, and the potential for long-term expression of the transduced genes in a murine model system: Murine hematopoietic stem and progenitor cells will be transduced ex vivo, and following in vivo reconstitution of recipient mice, the patterns of integration of the wt and the recombinant AAV genomes as well as safety and efficacy of the AAV-based vector system will be examined. These studies will provide new insights into the basic molecular biology of the AAV-normal diploid cell interactions, and also evaluate the in vivo efficacy and safety of the AAV-based vector system prior to its potential use in human gene therapy. These studies also relate to our long-term interests in parvoviruses and human disease.