Hematepoietic Stem Cell Transduction by AAV2 Vectors

AAV2 载体的造血干细胞转导

• 批准号: 6337984
• 负责人: Arun Srivastava
• 金额: $ 37.06万
• 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 2005-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6337984
• 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：（研究者摘要）腺相关病毒 2 (AAV) 载体作为更常用载体的替代品而受到关注。 基于逆转录病毒和腺病毒的载体。重组AAV载体已被 被证明可以非常有效地转导某些细胞类型，例如肌肉和大脑 出色地。然而，AAV 载体的功效存在争议。 转导造血干细胞。为了解决这些争议， 我们进行了系统的研究来调查以下基本步骤： AAV 介导的造血干细胞转导。我们已经获得了 有证据表明，在此之前至少必须克服三个障碍 AAV 载体可以实现高效转导。一、目标细胞 必须表达受体和辅助受体才能成功感染。其次， 靶细胞必须允许有效和快速的病毒运输到 核。第三，靶细胞必须允许病毒第二链 DNA 合成。我们已经记录了除了细胞表面表达之外 硫酸乙酰肝素蛋白多糖（HSPG）作为受体，AAV 还需要 细胞辅助受体，成纤维细胞生长因子受体 1 (FGFR1)，用于 感染成功。我们已经获得证据表明细胞内 AAV 的贩运可显着影响其转导效率 体外和体内。最后，我们发现了一个细胞伴侣 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 序列特异性相互作用 AAV反向末端重复序列在病毒第二链中起着至关重要的作用 DNA合成。因此，很明显，早期步骤的系统描述 为了更好地理解 AAV 生命周期中的事件， 限制造血干细胞的高效转导。这个提议 将测试以下假设： 1. AAV 有效进入初级阶段 造血细胞需要 HSPG 和 FGFR1 之间复杂的相互作用 以及 FGFR1 的其他下游靶标； 2. AAV 成功贩运 进入细胞核是由特定的细胞蛋白介导的； 3. 具体 细胞蛋白酪氨酸和/或丝氨酸/苏氨酸激酶磷酸化 FKBP52， 该蛋白质的去磷酸化是一个重要的决定因素 AAV 介导的转导； 4. AAV 原病毒基因组的整合确实 不影响原代造血干细胞的分化潜能 体内。从这些研究中获得的知识将适用于进一步 AAV 载体的开发及其在人类基因治疗中的最佳应用。