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.
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