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Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo

Gene Therapy volume 15, pages 1287–1293 (2008)Cite this article

Abstract

Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to ∼5% of murine hepatocytes. Viral second-strand DNA synthesis continues to be a rate-limiting step for efficient transduction by the single-stranded AAV (ssAAV) vectors. This is due, in part, to the presence of a cellular chaperone (FK506-binding) protein, FKBP52, phosphorylated forms of which interact with the D-sequence in the inverted terminal repeats of AAV2 genome and inhibit the viral second-strand DNA synthesis. Our previous studies have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T-cell protein tyrosine phosphatase (TC-PTP), and at serine/threonine residues by protein phosphatase 5 (PP5) enhances viral second-strand DNA synthesis and consequently, the transgene expression. We have also reported that coinfection with a self-complementary AAV (scAAV)-TC-PTP vector results in up to sixfold increase in the transduction efficiency of conventional ssAAV2 vectors in primary murine hepatocytes in vivo. We reasoned that coinfection with scAAV-TC-PTP and scAAV-PP5 vectors may lead to a further increase in the transduction efficiency of ssAAV2 vectors. We demonstrate here that this strategy does indeed lead to ∼16-fold increase in the transduction efficiency of conventional ssAAV vectors in primary murine hepatocytes in vivo following tail-vein injections. Neither scAAV2-TC-PTP nor scAAV2-PP5 vectors alone or together had any adverse effect on the hepatocytes. Thus, this coinfection strategy may be useful for achieving expression from recombinant ssAAV2 vectors containing larger genes, such as coagulation factor VIII, which exceed the packaging capacity of scAAV vectors, for the potential gene therapy of hemophilia A.

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Acknowledgements

We thank Dr Michel Tremblay, Dr David Chen and Dr Benjamin Chen for their kind gifts of TC-PTP and PP5 expression plasmids, respectively, and Dr Xiao Xiao for generously providing the pdsCBAp-EGFP vector. This research was supported in part by a grant no. 8187368876 from the Roche Foundation for Anemia Research (to LZ), and 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). GRJ was supported in part by an ‘Overseas Associate Fellowship-2006’ from the Department of Biotechnology, Government of India. BK gratefully acknowledges the support from the University of Florida Science for Life Program supported by the Howard Hughes Medical Institute.

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  1. G R Jayandharan and L Zhong: These authors contributed equally to this work.

Authors and Affiliations

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

    G R Jayandharan, L Zhong, B Li, B Kachniarz & A Srivastava

  2. Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA

    G R Jayandharan, L Zhong & A Srivastava

  3. Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL, USA

    L Zhong & A Srivastava

  4. Genetics Institute, University of Florida College of Medicine, Gainesville, FL, USA

    L Zhong & A Srivastava

  5. Columbia University, New York, NY, USA

    B Kachniarz

  6. Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA

    A Srivastava

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  1. G R Jayandharan
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Correspondence to A Srivastava.

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Jayandharan, G., Zhong, L., Li, B. et al. Strategies for improving the transduction efficiency of single-stranded adeno-associated virus vectors in vitro and in vivo. Gene Ther 15, 1287–1293 (2008). https://doi.org/10.1038/gt.2008.89

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