Hematepoietic Stem Cell Transduction by AAV2 Vectors

描述：（研究者摘要）腺相关病毒2（AAV） 矢量作为更常用的 逆转录病毒和腺病毒载体。重组AAV载体已经被广泛应用。 显示出对某些细胞类型，如肌肉和大脑， 好.然而，关于AAV载体在治疗中的功效存在争议。 转导造血干细胞。为了解决这些争议， 我们进行了一项系统的研究，调查 AAV介导的造血干细胞转导。我们所获得 有证据表明，至少有三个障碍，必须克服之前， 可发生通过AAV载体高效转导。首先，靶细胞 必须表达受体和辅助受体才能成功感染。二是 靶细胞必须允许有效和快速的病毒运输到 原子核第三，靶细胞必须允许病毒的第二股DNA 合成.我们已经证明，除了细胞表面表达， 除了硫酸乙酰肝素蛋白聚糖（HSPG）作为受体外，AAV还需要一种 细胞辅助受体，成纤维细胞生长因子受体1（FGFR 1）， 成功感染。我们有证据表明受损的细胞内 AAV的运输可以显著影响其转导效率， 体外和体内。最后，我们发现一种细胞伴侣 蛋白质FKBP 52，其在酪氨酸和丝氨酸/苏氨酸处均被磷酸化 残基，与单链D-序列内的特异性相互作用 AAV反向末端重复序列，在病毒第二链中起关键作用 DNA合成因此，很明显， 在AAV生命周期中，需要更好地了解事件， 限制了造血干细胞的高效转导。这项建议 将测试以下假设：1。AAV有效进入初级 造血细胞需要HSPG和FGFR 1之间的复杂相互作用， 以及FGFR 1的其他下游靶标; 2.成功贩运AAV 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FKBP 52， 这种蛋白质的去磷酸化是 AAV介导的转导;和4. AAV前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， vivo.从这些研究中获得的知识将适用于进一步的研究。 AAV载体的开发及其在人类基因治疗中的最佳用途。

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.