Hematepoietic Stem Cell Transduction by AAV2 Vectors

AAV2 载体的造血干细胞转导

• 批准号: 6638693
• 负责人: Arun Srivastava
• 金额: $ 37.25万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-15 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638693
• 关键词: DNA replication; NOD mouse; SCID mouse; adeno associated virus group; biological signal transduction; fibroblast growth factor; gene expression; gene therapy; genetic transduction; genome; growth factor receptors; hematopoietic stem cells; human tissue; intracellular transport; molecular chaperones; phosphorylation; protein protein interaction; protein structure function; protein tyrosine kinase; proteoglycan; receptor expression; transfection /expression vector; virus genetics

DESCRIPTION: (Investigator's abstract) The adeno-associated virus 2 (AAV) vectors have gained attention as an alternative to the more commonly used retrovirus- and adenovirus-based vectors. Recombinant AAV vectors have been shown to transduce certain cell types, such as muscle and brain, exceedingly well. However, controversies exist with regard to efficacy of AAV vectors in transducing hematopoietic stem cells. In order to resolve these controversies, we have undertaken a systematic study to investigate the fundamental steps in AAV-mediated transduction of hematopoietic stem cells. We have obtained evidence that there are at least three obstacles that must be overcome before high-efficiency transduction by AAV vectors can occur. First, the target cell must express a receptor and a co-receptor for successful infection. Second, the target cell must allow for efficient and rapid viral trafficking to the nucleus. And third, the target cell must allow for viral second-strand DNA synthesis. We have documented that in addition to the cell surface expression of heparan sulfate proteoglycan (HSPG) as a receptor, AAV also requires a cellular co-receptor, fibroblast growth factor receptor 1 (FGFR1), for successful infection. We have obtained evidence that impaired intracellular trafficking of AAV can significantly affect its transduction efficiency both in vitro and in vivo. And finally, we have identified that a cellular chaperone protein, FKBP52, which is phosphorylated at both tyrosine and serine/threonine residues, interacts specifically with the single-stranded D-sequence within the AAV inverted terminal repeats, and plays a crucial role in viral second-strand DNA synthesis. Thus, it is clear that a systematic delineation of early steps in the AAV life cycle is required to gain a better understanding of events that limit high-efficiency transduction of hematopoietic stem cells. This proposal will test the following hypotheses: 1. Efficient entry of AAV into primary hematopoietic cells requires a complex interaction between HSPG and FGFR1 as well as other downstream targets of FGFR1; 2. Successful trafficking of AAV into the nucleus is mediated by specific cellular proteins; 3. Specific cellular protein tyrosine and/or serine/threonine kinases phosphorylate FKBP52, and dephosphorylation of this protein is an important determinant of AAV-mediated transduction; and 4. Integration of the AAV proviral genome does not affect the differentiation potential of primary hematopoietic stem cells in vivo. The knowledge gained from these studies will be applicable in further development of AAV vectors and their optimal use in human gene therapy.

描述：(研究人员摘要)腺相关病毒2(AAV)载体作为更常用的 逆转录病毒和腺病毒载体。重组AAV载体已成功构建 对某些类型的细胞，如肌肉和脑，有极强的转导作用 井。然而，关于AAV载体的有效性仍然存在争议。 转导造血干细胞。为了解决这些争议， 我们已经进行了一项系统的研究，以调查 AAV介导的造血干细胞转导。我们已经获得了 有证据表明，至少有三个障碍必须在 可以通过AAV载体进行高效的转导。首先，目标单元格 必须表达一个受体和一个共同受体才能成功感染。第二， 目标细胞必须允许有效和快速地将病毒运送到 原子核。第三，目标细胞必须允许病毒第二链DNA 综合。我们已经记录了除了细胞表面的表达 作为受体的硫酸肝素蛋白多糖(HSPG)，AAV还需要 成纤维细胞生长因子受体1(FGFR1) 成功感染。我们已经获得了细胞内受损的证据 AAV的贩运可显著影响其转导效率 体外和体内。最后，我们发现了一种细胞伴侣 蛋白质FKBP52，酪氨酸和丝氨酸/苏氨酸均被磷酸化 残基，与单链D序列特异性地相互作用于 AAV反向末端重复，在病毒第二链中起关键作用 DNA合成。因此，很明显，对早期步骤的系统描述 在AAV生命周期中需要更好地了解 限制造血干细胞的高效转导。这项建议 将检验以下假设：1.甲型肝炎病毒有效进入小学 造血细胞需要HSPG和FGFR1之间复杂的相互作用 以及FGFR1的其他下游靶点；2.成功贩运AAV 进入细胞核是由特定的细胞蛋白介导的；3.特定的 细胞蛋白酪氨酸和/或丝氨酸/苏氨酸激酶磷酸化FKBP52， 而该蛋白的去磷酸化是决定 AAV介导的转导；以及4.AAV前病毒基因组的整合 不影响原代造血干细胞在体内的分化潜能 活着。从这些研究中获得的知识将在今后的研究中应用 腺相关病毒载体的发展及其在人类基因治疗中的优化应用。