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

以腺相关病毒2(AAV)为基础的载体系统已经吸引了 相当多的关注作为更常用的 逆转录病毒载体在基因治疗中的潜在应用 因为AAV对人类来说是一种非致病性病毒，而野生型(Wt) AAV基因组已被证明整合到人类染色体DNA中 一种特定于站点的方式。然而，一些与 野生型AAV与正常人相互作用的基础分子生物学 一般二倍体细胞，尤其是重组AAV的二倍体细胞， 很大程度上仍未被勘探。我们已经启动了系统研究，以 寻求这些问题的答案，并获得了初步证据 Wt AAV与正常人二倍体成纤维细胞以某种方式相互作用 这与人类非整倍体细胞的情况截然不同。假说 在这个子项目中要测试的是wt AAV集成到 正常人体造血细胞定位于不同的部位 来自人类非整倍体细胞的特征，以及重组 缺少病毒编码序列(REP和/或CAP)的AAV基因组整合 在与wt不同部位的二倍体造血细胞中 AAV基因组。将致力于实现以下具体目标： 1.人野生型甲型流感病毒基因组整合模式比较 非整倍体和二倍体造血细胞，包括纯化群体 从正常人骨髓和脐带血中分离原始祖细胞 脐带血：Southern印迹和聚合酶链式反应 分析，wt AAV基因组的整合模式将是 比较了人非整倍体和二倍体造血细胞。 2.评价AAV编码蛋白在位点特异性中的作用 重组腺病毒与重组AAV基因组的整合：重组病毒 包含病毒代表或帽基因序列的载体将 被用来评估AAV编码的蛋白质是否在该部位发挥作用- 具体整合。 3.AAV整合位点的分子克隆及鉴定 在二倍体细胞中，并与来自非整倍体细胞的比较：In 除了直接克隆到噬菌体lambda和质粒载体上， 将采用基于聚合酶链式反应的克隆策略获得DNA序列 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能 也将被确定。 4.腺相关病毒介导的体内转导的评价及应用前景 转导基因在小鼠模型中的长期表达 系统：小鼠的造血干细胞和祖细胞将被 受体的体外转导和体内重建 小鼠重组腺病毒与重组腺相关病毒的整合模式 基于AAV的载体系统的基因组以及安全性和有效性 将会被检查。 这些研究将为基础分子生物学提供新的见解。 AAV与正常二倍体细胞的相互作用，并评估在 以AAV为基础的载体系统的体内有效性和安全性 在人类基因治疗中的潜在应用。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.