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Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo

Gene Therapy volume 11, pages 1165–1169 (2004)Cite this article

Abstract

Adeno-associated virus 2 (AAV) vectors are currently in use in Phase I/II clinical trials for gene therapy of cystic fibrosis and hemophilia B. Although 100% of murine hepatocytes can be targeted by AAV vectors, the transgene expression is limited to ∼5% of hepatocytes. Since the viral genome is a single-stranded DNA, and single strands of both polarities are encapsidated with equal frequency, it has been suggested that failure to undergo DNA strand-annealing accounts for the lack of efficient transgene expression. We and others, on the other hand, have proposed that failure to undergo viral second-strand DNA synthesis attributes to the observed low efficiency of transgene expression. We have previously documented that a cellular protein, designated FKBP52, when present in phosphorylated forms, inhibits the viral second-strand DNA synthesis, and consequently, limits transgene expression in nonhepatic cells, whereas unphosphorylated forms of FKBP52 have no effect. To further evaluate whether phosphorylated FKBP52 is also involved in regulating AAV-mediated transgene expression in murine hepatocytes, we generated transgenic mice overexpressing the cellular T-cell protein tyrosine phosphatase (TC-PTP) protein, known to catalyze dephosphorylation of FKBP52, as well as mice deficient in FKBP52. We demonstrate here that dephosphorylation of FKBP52 in TC-PTP transgenic (TC-PTP-TG) mice, and removal of FKBP52 in FKBP52-knockout (FKBP52-KO) mice results in efficient transduction of murine hepatocytes following tail-vein injection of recombinant AAV vectors. We also document efficient viral second-strand DNA synthesis in hepatocytes from both TC-PTP-TG and FKBP52-KO mice. Thus, our data strongly support the contention that the viral second-strand DNA synthesis, rather than DNA strand-annealing, is the rate-limiting step in the efficient transduction of hepatocytes, which should have implications in the optimal use of recombinant AAV vectors in human gene therapy.

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References

  1. Muzyczka N, Berns KI. In: Knipe D, Howley P, Griffin D, Lamb R, Martin M, Roizman B, Straus S (eds). Fields Virology, Vol. 2 Lippincott, Williams & Wilkins: Philadelphia, PA, 2001, pp 2327–2347.

    Google Scholar 

  2. Nakai H, Storm TA, Kay MA . Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo. J Virol 2000; 74: 9451–9463.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chen ZY et al. Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 2001; 3: 403–410.

    Article  CAS  PubMed  Google Scholar 

  4. Fisher KJ et al. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. J Virol 1996; 70: 520–532.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ferrari FK, Samulski T, Shenk T, Samulski RJ . Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol 1996; 70: 3227–3234.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Mah C et al. Adeno-associated virus 2-mediated gene transfer: role of epidermal growth factor receptor protein tyrosine kinase in transgene expression. J Virol 1998; 72: 9835–9841.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Qing KY et al. Adeno-associated virus type 2-mediated gene transfer: correlation of tyrosine phosphorylation of the cellular single-stranded D sequence-binding protein with transgene expression in human cells in vitro and murine tissues in vivo. J Virol 72; 1998: 1593–1599.

    Google Scholar 

  8. Qing KY et al. Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression. J Virol 2001; 75: 8968–8976.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Qing KY et al. Adeno-associated virus type 2-mediated gene transfer: role of cellular T-cell protein tyrosine phosphatase in transgene expression in established cell lines in vitro and transgenic mice in vivo. J Virol 2003; 77: 2741–2746.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Qing KY et al. Role of tyrosine phosphorylation of a cellular protein in adeno-associated virus 2-mediated transgene expression. Proc Natl Acad Sci USA 1997; 94: 10879–10884.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang X-S, Qing KY, Ponnazhagan S, Srivastava A . Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats. J Virol 1997; 71: 3077–3082.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ponnazhagan S et al. Adeno-associated virus 2-mediated gene transfer in vivo: organ-tropism and expression of transduced sequences in mice. Gene 1997; 190: 203–210.

    Article  CAS  PubMed  Google Scholar 

  13. Snyder RO et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet 1997; 16: 270–276.

    Article  CAS  PubMed  Google Scholar 

  14. Nakai H, Storm TA, Kay MA . Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors. Nat Biotechnol 2000; 18: 527–532.

    Article  CAS  PubMed  Google Scholar 

  15. Chen SF, Tazelaar J, Wilson JM . Selective repopulation of normal mouse liver by hepatocytes transduced in vivo with recombinant adeno-associated virus. Hum Gene Ther 2001; 12: 45–50.

    Article  CAS  PubMed  Google Scholar 

  16. Song S et al. Stable therapeutic serum levels of human α1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors. Gene Therapy 2001; 8: 1299–1306.

    Article  CAS  PubMed  Google Scholar 

  17. Miao CH et al. The kinetics of rAAV integration in the liver. Nat Genet 1998; 19: 13–15.

    Article  CAS  PubMed  Google Scholar 

  18. Miao CH et al. Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction. J Virol 2000; 74: 3793–3803.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nakai H et al. A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction. J Virol 2002; 76: 11343–11349.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Czar MJ et al. Evidence that the FK506-binding protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Mol Endocrinol 1995; 9: 1549–1560.

    CAS  PubMed  Google Scholar 

  21. Miyata Y et al. Phosphorylation of the immunosuppresant FK506-binding protein FKBP52 by casein kinase II: regulation of HSP90-binding activity of FKBP52. Proc Natl Acad Sci USA 1997; 94: 14500–14505.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lorenzen JA, Dadabay CY, Fischer EH . COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus. J Biol Chem 1995; 131: 631–643.

    CAS  Google Scholar 

  23. You-Ten KE et al. Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice. J Exp Med 1997; 186: 683–693.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Muise ES et al. Thermosensitive mutants of the MPTP and hPTP1B protein tyrosine phosphatases: isolation and structural analysis. Protein Sci 1996; 5: 604–613.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hansen J et al. Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts. J Virol 2000; 74: 992–997.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hansen J, Qing KY, Srivastava A . Infection of purified nuclei by adeno-associated virus. Mol Ther 2001; 4: 289–296.

    Article  CAS  PubMed  Google Scholar 

  27. McCarty DM, Monahan PE, Samulski RJ . Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy 2001; 8: 1248–1254.

    Article  CAS  PubMed  Google Scholar 

  28. Wang Z et al. Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Therapy 2003; 10: 2105–2111.

    Article  CAS  PubMed  Google Scholar 

  29. McCarty DM et al. Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Therapy 2003; 10: 2112–2118.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Michel Tremblay for generously providing the TC-PTP expression plasmids, and Dr Jacqueline A Hobbs for a critical review of this manuscript. This research was supported in part by Public Health Service Grant R01 HL-70259 (to WS), and R01 EB-002073 and R01 HL-65570 (to AS) from the National Institutes of Health. KAW-K was supported by an NIH training Grant T32 HL-07910 (to AS).

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Author notes

  1. K Qing

    Present address: Eli Lilly & Co., Indianapolis, IN, 46285, USA

  2. L Zhong, W Li and Z Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology & Immunology, Indiana University School of Medicine, Indianapolis, IN, USA

    L Zhong, L Chen, K Qing, K A Weigel-Kelley & A Srivastava

  2. Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA

    L Zhong, L Chen, K Qing, K A Weigel-Kelley & A Srivastava

  3. Walther Cancer Institute, Indiana University School of Medicine, Indianapolis, IN, USA

    L Zhong, L Chen, K Qing, K A Weigel-Kelley & A Srivastava

  4. Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN, USA

    W Li, Z Yang, Y Li, M C Yoder & W Shou

  5. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    W Li, Z Yang, Y Li, M C Yoder & W Shou

  6. Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA

    W Li, Z Yang, Y Li, M C Yoder & W Shou

  7. Division of Hematology/Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

    A Srivastava

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Zhong, L., Li, W., Yang, Z. et al. Improved transduction of primary murine hepatocytes by recombinant adeno-associated virus 2 vectors in vivo. Gene Ther 11, 1165–1169 (2004). https://doi.org/10.1038/sj.gt.3302283

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