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Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency

Gene Therapy volume 14, pages 545–550 (2007)Cite this article

Abstract

We have documented that a cellular chaperone protein, FKBP52, when phosphorylated at tyrosine and/or serine/threonine (Ser/Thr) residues, interacts with the D-sequence in the inverted terminal repeats of the adeno-associated virus 2 (AAV) genome, inhibits the viral second-strand DNA synthesis, and leads to inefficient transgene expression from recombinant AAV vectors in certain cell types. We have also demonstrated that FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and that deliberate overexpression of TC-PTP leads to more efficient viral second-strand DNA synthesis, and increased transgene expression. However, the identity of the putative Ser/Thr protein phosphatase that dephosphorylates FKBP52 at Ser/Thr residues has remained elusive. Using known inhibitors of Ser/Thr phosphatases, we have now identified protein phosphatase 5 (PP5) to be a candidate enzyme. Deliberate overexpression of PP5 in 293 cells, which does not influence cellular growth, leads to ∼5-fold increase in the transduction efficiency of conventional single-stranded AAV vectors, but no significant enhancement in the transduction efficiency of self-complementary AAV vectors, suggesting that PP5 plays a role in AAV second-strand DNA synthesis. Electrophoretic mobility-shift assays show that in cells overexpressing PP5, the extent of the complex formation between FKBP52 and the AAV D-sequence is significantly reduced. These studies suggest that PP5-mediated dephosphorylation of FKBP52 at Ser/Thr residues augments viral second-strand DNA synthesis and enhances AAV transduction efficiency, which has implications in the optimal use of these vectors in human gene therapy.

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Acknowledgements

We thank Drs David J Chen and Benjamin PC Chen for generously providing the human PP5 cDNA, and Dr Keyun Qing for his expert technical advice and general counsel. We also thank Dr Jacqueline A Hobbs for a critical review of this paper. This work was supported in part by United States Public Health Service Grants R01 EB-002073, R01 HL-65570, R01 HL-76901, and P01 DK-058327 (Project 1) from the National Institutes of Health (to AS).

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Authors and Affiliations

  1. Division of Cellular & Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA

    W Zhao, J Wu, L Zhong & A Srivastava

  2. Departments of Nephrology & Geriatrics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, P.R. China

    W Zhao & J Wu

  3. Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA

    A Srivastava

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Correspondence to A Srivastava.

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Zhao, W., Wu, J., Zhong, L. et al. Adeno-associated virus 2-mediated gene transfer: role of a cellular serine/threonine protein phosphatase in augmenting transduction efficiency. Gene Ther 14, 545–550 (2007). https://doi.org/10.1038/sj.gt.3302886

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