PARVOVIRUS VECTORS FOR HUMAN GENE THERAPY

描述：腺相关病毒2型(AAV)，一种非致病性人类 细小病毒，已被认为是一个潜在的有用的人类基因载体 心理治疗。然而，与AAV-HOST相关的一些基本问题 细胞间的相互作用在很大程度上仍未被探索。其中包括身份 甲型肝炎病毒的细胞受体及其与病毒相互作用的性质 正常人类二倍体细胞，最有可能成为天然AAV的靶点 感染，也是利用该载体进行基因治疗的潜在靶点 系统。同样，尽管另一种人类起源的细小病毒，被指定为 细小病毒B19对人类有显著的嗜性 红系B19载体中的造血祖细胞 能够特异性地运送红系细胞的基因还没有 发展起来的。斯里瓦斯塔瓦博士和他的同事们提议确定 AAV的细胞受体，并评估病毒宿主的相互作用 人脐血造血干/祖细胞的原代培养 脐带血。细小病毒B19嗜红细胞性有待开发 开发基于B19的新型载体以实现人的靶向传递 珠蛋白基因。这项建议中要检验的假设是：1. AAV进入人体细胞是特异性的，是由受体介导的。2.该条 红系细胞特异性递送和转导基因A的高水平表达 造血细胞中的β-珠蛋白基因在体外和体外都是可行的 活着。四个具体目标是：1.相互作用的表征 含造血干/祖细胞的重组AAV载体的构建 人脐血的分离鉴定及细胞的鉴定 AAV受体。2.AAV介导的转导与红系的评价 人正常β-珠蛋白基因的细胞特异性高效表达 脐血中的造血祖细胞。3.细小病毒的发展 基于B19的载体和B19介导的红细胞靶向递送 正常人β-珠蛋白基因在造血祖细胞中的表达 脐带血中的细胞。4.AAV和B19介导的研究 体外转导及其在体内长期表达的可能性 转导的人β-珠蛋白基因在β-地中海贫血小鼠和 非人灵长类动物模型。 这些研究将为人类免疫缺陷病毒的基础分子生物学提供新的见解。 细小病毒-二倍体细胞相互作用是SAFE发展的前奏 和有效载体，并有助于评估体内疗效和 它们在人类基因治疗中潜在应用之前的安全性 一般的血红蛋白病，镰状细胞病和β-地中海贫血 尤其是。

DESCRIPTION: The adeno-associated virus 2 (AAV), a non-pathogenic human parvovirus, has been suggested as a potentially useful vector for human gene therapy. However, a number of fundamental questions related to AAV-host cell interactions remain largely unexplored. These include the identity of the cellular receptor for AAV, and the nature of interaction of AAV with normal human diploid cells, the most likely target for a natural AAV infection, and also a potential target for gene therapy with this vector system. Similarly, although a second parvovirus of human origin, designated parvovirus B19, has been shown to possess a remarkable tropism for human hematopoietic progenitor cell in the erythroid lineage, B19-based vectors capable of erythroid cell-specific delivery of genes have not been developed. Dr. Srivastava and his colleagues propose to identify the cellular receptor for AAV, and evaluate the virus host interaction using primary human hematopoietic stem and progenitor cells in human umbilical cord blood. The erythroid cell-tropism of parvovirus B19 will be exploited to develop novel B19-based vectors to achieve targeted delivery of human globin genes. The hypotheses to be tested in this proposal are: 1. That entry of AAV in human cells is specific and receptor-mediated. 2. That erythroid cell-specific delivery and high level expression of a transduced beta-globin gene in hematopoietic cells is feasible in vitro as well as in vivo. The four specific aims are: 1. Characterization of interaction of recombinant AAV vectors with hematopoietic stem and progenitor cells in human umbilical cord blood, and identification of the putative cellular receptor for AAV. 2. Evaluation of AAV-mediated transduction and erythroid cell-specific, high level expression of a normal human beta-globin gene in hematopoietic progenitor cells in cord blood. 3. Development of parvovirus B19-based vector and B19-mediated erythroid cell-targeted delivery and expression of a normal human beta-globin gene in hematopoietic progenitor cells in cord blood. 4. Investigation of AAV- and B19- mediated transduction ex vivo, and the potential for long-term in vivo expression of the transduced human beta-globin gene in beta-thalassemic murine and non-human primate models. These studies will provide new insights into the basic molecular biology of parvovirus-diploid cell interactions as a prelude to the development of safe and effective vectors, and help in evaluating the in vivo efficacy and safety prior to their potential use in gene therapy for human hemoglobinopathies in general, and sickle cell disease and beta-thalassemia in particular.