Parvovirus Vectors for Human Gene Therapy

描述（由申请人提供）：人血红蛋白病，如 地中海贫血和镰状细胞病是可能的疾病 适合基因治疗。腺相关病毒2（AAV 2），一种非致病性的 人细小病毒作为一种潜在的有用载体已经引起了人们的注意。虽然 在理解《公约》的初步步骤方面取得了重大进展， AAV 2感染途径中，参与的细胞蛋白质的确切作用， 这些方法尚未阐明。例如，细胞表面乙酰肝素 硫酸蛋白聚糖（HSPG）已被证明是AAV 2的主要受体 结合，但病毒进入细胞的机制并不完全 明白此外，其他血清型，如AAV 4和AAV 5，还没有被发现。 评估造血干细胞转导。同样，虽然A 第二种人细小病毒，命名为细小病毒B19，已知具有 对红系中的人造血细胞具有显著的向性， 作为一种载体，在病毒宿主中的精确步骤 细胞间的相互作用尚未完全了解。我们还记录了 除了HSPG作为受体外，AAV 2还需要一个辅助受体，即成纤维细胞的生长 因子受体1（FGFR 1），用于成功感染。我们还记录了 据报道，红细胞P抗原细胞表面表达是一种 细小病毒B19受体，是必要的，但不是足够的成功 细小病毒B19感染。使用重组AAV 2-、AAV 4-、AAV 5-和 细小病毒B19-珠蛋白载体，我们将测试以下假设：1。 原代人造血干/祖细胞的有效转导可 由AAV 2、AAV 4和/或AAV 5载体介导，细小病毒的有效进入 B19在原代人造血细胞中由假定的细胞因子介导。 共受体，和3.细小病毒载体将被证明是安全和有效的， 在体内动物模型中治疗性纠正血红蛋白病。的 将实现以下四个具体目标：1.阐明基础 原代造血干细胞差异转导机制 AAV 2-、AAV 4-和AAV 6-从骨髓和脐带血中分离细胞， AAV 5-珠蛋白载体。2. AAV介导的红系限制性表达 人珠蛋白基因在体外人造血细胞中的表达， 在体内纯合β-地中海贫血小鼠中的校正。3.识别和 对有效的细胞共受体的表征 用细小病毒转导原代人造血祖细胞 B19-珠蛋白载体。4. AAV和细小病毒B19介导的转导和 人球蛋白基因在造血系统中的长期、调节表达 非人灵长类动物体内的祖细胞。从这些中获得的知识 研究将适用于AAV和细小病毒B19的进一步开发 载体及其在β-地中海贫血基因治疗中的最佳用途， 镰状细胞病

DESCRIPTION (provided by applicant): Human hemoglobinopathies, such as beta-thalassemia and sickle-cell disease, are among the likely diseases amenable to gene therapy. The adeno-associated virus 2 (AAV2), a non-pathogenic human parvovirus, has gained attention as a potentially useful vector. Although significant progress has been made in understanding the initial steps in the AAV2 infection pathway, the precise role of the cellular proteins involved in these processes has not been elucidated. For example, cell surface heparan sulfate proteoglycan (HSPG) has been shown to be the primary receptor for AAV2 binding, but the mechanism of viral entry into the cell is not completely understood. Also, other serotypes, such as AAV4 and AAV5, have not been evaluated for hematopoietic stem cell transduction. Similarly, although a second human parvovirus, designated parvovirus B19, known to possess a remarkable tropism for human hematopoietic cells in the erythroid lineage, has been developed as a vector, the precise steps in the virus host cell-interaction are not fully understood. We have documented that in addition to HSPG as a receptor, AAV2 also requires a co-receptor, fibroblast growth factor receptor 1 (FGFR1), for successful infection. We have also documented that cell surface expression of erythrocyte P antigen, reported to be a receptor for parvovirus B19, is necessary but not sufficient for a successful infection by parvovirus B19. Using recombinant AAV2-, AAV4-, AAV5-, and parvovirus B19-globin vectors, we will test the following hypotheses: 1. Efficient transduction of primary human hematopoietic stem/progenitor cells can be mediated by AAV2, AAV4 and/or AAV5 vectors, 2. Efficient entry of parvovirus B19 in primary human hematopoietic cells is mediated by a putative cellular co-receptor, and 3. Parvovirus vectors will prove to be safe and effective for therapeutic correction of hemoglobinopathies in animal models in vivo. The following four Specific Aims will be pursued: 1. Elucidation of underlying mechanisms of differential transduction of primary human hematopoietic stem cells from bone marrow and umbilical cord blood by AAV2-, AAV4-, and AAV5-globin vectors. 2. AAV-mediated erythroid lineage-restricted expression of human globin genes in human hematopoietic cells in vitro, and therapeutic correction in homozygous beta-thalassemic mice in vivo. 3. Identification and characterization of the putative cellular co-receptor for efficient transduction of primary human hematopoietic progenitor cells by parvovirus B19-globin vectors. 4. AAV- and parvovirus B19-mediated transduction and long-term, regulated expression of human globin genes in hematopoietic progenitor cells in non-human primates in vivo. The knowledge gained from these studies will be applicable in further development of AAV and parvovirus B19 vectors and their optimal use in gene therapy of beta-thalassemia and sickle-cell disease.