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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065570-02
Application #
6537874
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Qasba, Pankaj
Project Start
2001-06-15
Project End
2005-05-31
Budget Start
2002-06-01
Budget End
2003-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$372,500
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Song, Liujiang; Kauss, M Ariel; Kopin, Etana et al. (2013) Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo. Cytotherapy 15:986-98
Silver, Jared N; Flotte, Terence R (2008) Towards a rAAV-based gene therapy for ADA-SCID: from ADA deficiency to current and future treatment strategies. Pharmacogenomics 9:947-68
Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao et al. (2008) Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression. Virology 381:194-202
Zhong, Li; Li, Baozheng; Mah, Cathryn S et al. (2008) Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci U S A 105:7827-32
Jayandharan, G R; Zhong, L; Li, B et al. (2008) Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther 15:1287-93
Zhong, Li; Zhou, Xiaohuai; Li, Yanjun et al. (2008) Single-polarity recombinant adeno-associated virus 2 vector-mediated transgene expression in vitro and in vivo: mechanism of transduction. Mol Ther 16:290-5
Zhong, Li; Zhao, Weihong; Wu, Jianqing et al. (2007) A dual role of EGFR protein tyrosine kinase signaling in ubiquitination of AAV2 capsids and viral second-strand DNA synthesis. Mol Ther 15:1323-30
Wu, Jianqing; Zhao, Weihong; Zhong, Li et al. (2007) Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep proteins in vector purity. Hum Gene Ther 18:171-82
Zhao, W; Wu, J; Zhong, L et al. (2007) Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency. Gene Ther 14:545-50
Zhong, Li; Li, Weiming; Li, Yanjun et al. (2006) Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5. Hum Gene Ther 17:321-33

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