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Tumor-specific suicide gene therapy for hepatocellular carcinoma by transcriptionally targeted retroviral replicating vectors

Gene Therapy volume 22, pages 155–162 (2015)Cite this article

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Abstract

Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a major concern. To design a tumor-specific retroviral replicating vector (RRV), we replaced the U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the hepatitis B virus enhancer II (EII) and human α-fetoprotein (AFP) core promoter to produce ACE-GFP-EIIAFP, a hepatocellular carcinoma (HCC)-targeting RRV. Similar to ACE-GFP, ACE-GFP-EIIAFP exhibited robust green fluorescent protein (GFP) expression in HCC cells and, most importantly, it exhibited HCC-specific replication and did not replicate in non-HCC tumor cells or normal liver cells. We sequenced the promoter region of ACE-GFP-EIIAFP collected from serial infection cycles to examine the genomic stability of the vector during its replicative spread, and found that the vector could retain the hybrid promoter in the genome for at least six infection cycles. In vitro studies revealed that ACE-CD-EIIAFP and ACE-PNP-EIIAFP, which express the yeast cytosine deaminase and Escherichia coli purine nucleoside phosphorylase, respectively, exert a highly potent cytotoxic effect on HCC cells in the presence of their respective prodrugs. In vivo, ACE-CD-EIIAFP-mediated suicide gene therapy efficiently suppressed HCC tumor growth and no detectable RRV signal was observed in extratumoral tissues. These results suggest that the tumor-specific, suicide-gene-encoding RRV may fulfill the promise of retroviral gene therapy for cancer.

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References

  1. Ram Z, Culver KW, Walbridge S, Frank JA, Blaese RM, Oldfield EH . Toxicity studies of retroviral-mediated gene transfer for the treatment of brain tumors. J Neurosurg 1993; 79: 400–407.

    Article  CAS  PubMed  Google Scholar 

  2. Hein A, Czub S, Xiao LX, Schwender S, Dorries R, Czub M . Effects of adoptive immune transfers on murine leukemia virus-infection of rats. Virology 1995; 211: 408–417.

    Article  CAS  PubMed  Google Scholar 

  3. Traister RS, Lynch WP . Reexamination of amphotropic murine leukemia virus neurovirulence: neural stem cell-mediated microglial infection fails to induce acute neurodegeneration. Virology 2002; 293: 262–272.

    Article  CAS  PubMed  Google Scholar 

  4. Culver KW, Ram Z, Wallbridge S, Ishii H, Oldfield EH, Blaese RM . In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992; 256: 1550–1552.

    Article  CAS  PubMed  Google Scholar 

  5. Miller DG, Adam MA, Miller AD . Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990; 10: 4239–4242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Varmus HE, Padgett T, Heasley S, Simon G, Bishop JM . Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell 1977; 11: 307–319.

    Article  CAS  PubMed  Google Scholar 

  7. Thomas CE, Ehrhardt A, Kay MA . Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 2003; 4: 346–358.

    Article  CAS  PubMed  Google Scholar 

  8. Rainov NG . A phase III clinical evaluation of herpes simplex virus type 1 thymidine kinase and ganciclovir gene therapy as an adjuvant to surgical resection and radiation in adults with previously untreated glioblastoma multiforme. Hum Gene Ther 2000; 11: 2389–2401.

    Article  CAS  PubMed  Google Scholar 

  9. Harsh GR, Deisboeck TS, Louis DN, Hilton J, Colvin M, Silver JS et al. Thymidine kinase activation of ganciclovir in recurrent malignant gliomas: a gene-marking and neuropathological study. J Neurosurg 2000; 92: 804–811.

    Article  CAS  PubMed  Google Scholar 

  10. Kruse CA, Roper MD, Kleinschmidt-DeMasters BK, Banuelos SJ, Smiley WR, Robbins JM et al. Purified herpes simplex thymidine kinase Retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors. Cancer Gene Ther 1997; 4: 118–128.

    CAS  PubMed  Google Scholar 

  11. Puumalainen AM, Vapalahti M, Agrawal RS, Kossila M, Laukkanen J, Lehtolainen P et al. Beta-galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses. Hum Gene Ther 1998; 9: 1769–1774.

    Article  CAS  PubMed  Google Scholar 

  12. Ram Z, Culver KW, Oshiro EM, Viola JJ, DeVroom HL, Otto E et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med 1997; 3: 1354–1361.

    Article  CAS  PubMed  Google Scholar 

  13. Smiley WR, Laubert B, Howard BD, Ibanez C, Fong TC, Summers WS et al. Establishment of parameters for optimal transduction efficiency and antitumor effects with purified high-titer HSV-TK retroviral vector in established solid tumors. Hum Gene Ther 1997; 8: 965–977.

    Article  CAS  PubMed  Google Scholar 

  14. Logg CR, Logg A, Tai CK, Cannon PM, Kasahara N . Genomic stability of murine leukemia viruses containing insertions at the Env-3' untranslated region boundary. J Virol 2001; 75: 6989–6998.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tai CK, Logg CR, Park JM, Anderson WF, Press MF, Kasahara N . Antibody-mediated targeting of replication-competent retroviral vectors. Hum Gene Ther 2003; 14: 789–802.

    Article  CAS  PubMed  Google Scholar 

  16. Tai CK, Wang W, Lai YH, Logg CR, Parker WB, Li YF et al. Enhanced efficiency of prodrug activation therapy by tumor-selective replicating retrovirus vectors armed with the Escherichia coli purine nucleoside phosphorylase gene. Cancer Gene Ther 2010; 17: 614–623.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lu YC, Luo YP, Wang YW, Tai CK . Highly efficient gene transfer to solid tumors in vivo by tumor-selective replicating retrovirus vectors. Int J Mol Med 2010; 25: 769–775.

    CAS  PubMed  Google Scholar 

  18. Logg CR, Tai CK, Logg A, Anderson WF, Kasahara N . A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors. Hum Gene Ther 2001; 12: 921–932.

    Article  CAS  PubMed  Google Scholar 

  19. Wang WJ, Tai CK, Kasahara N, Chen TC . Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors. Hum Gene Ther 2003; 14: 117–127.

    Article  CAS  PubMed  Google Scholar 

  20. Tai CK, Wang WJ, Chen TC, Kasahara N . Single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma. Mol Ther 2005; 12: 842–851.

    Article  CAS  PubMed  Google Scholar 

  21. Diaz RM, Eisen T, Hart IR, Vile RG . Exchange of viral promoter/enhancer elements with heterologous regulatory sequences generates targeted hybrid long terminal repeat vectors for gene therapy of melanoma. J Virol 1998; 72: 789–795.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ferrari G, Salvatori G, Rossi C, Cossu G, Mavilio F . A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers. Hum Gene Ther 1995; 6: 733–742.

    Article  CAS  PubMed  Google Scholar 

  23. Moore KA, Scarpa M, Kooyer S, Utter A, Caskey CT, Belmont JW . Evaluation of lymphoid-specific enhancer addition or substitution in a basic retrovirus vector. Hum Gene Ther 1991; 2: 307–315.

    Article  CAS  PubMed  Google Scholar 

  24. Logg CR, Logg A, Matusik RJ, Bochner BH, Kasahara N . Tissue-specific transcriptional targeting of a replication-competent retroviral vector. J Virol 2002; 76: 12783–12791.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ido A, Nakata K, Kato Y, Nakao K, Murata K, Fujita M et al. Gene therapy for hepatoma cells using a retrovirus vector carrying herpes simplex virus thymidine kinase gene under the control of human alpha-fetoprotein gene promoter. Cancer Res 1995; 55: 3105–3109.

    CAS  PubMed  Google Scholar 

  26. Mawatari F, Tsuruta S, Ido A, Ueki T, Nakao K, Kato Y et al. Retrovirus-mediated gene therapy for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by human alpha-fetoprotein enhancer directly linked to its promoter. Cancer Gene Ther 1998; 5: 301–306.

    CAS  PubMed  Google Scholar 

  27. Nakabayashi H, Koyama Y, Suzuki H, Li HM, Sakai M, Miura Y et al. Functional mapping of tissue-specific elements of the human alpha-fetoprotein gene enhancer. Biochem Biophys Res Commun 2004; 318: 773–785.

    Article  CAS  PubMed  Google Scholar 

  28. Kramer MG, Barajas M, Razquin N, Berraondo P, Rodrigo M, Wu C et al. In vitro and in vivo comparative study of chimeric liver-specific promoters. Mol Ther 2003; 7: 375–385.

    Article  CAS  PubMed  Google Scholar 

  29. Kievit E, Bershad E, Ng E, Sethna P, Dev I, Lawrence TS et al. Superiority of yeast over bacterial cytosine deaminase for enzyme/prodrug gene therapy in colon cancer xenografts. Cancer Res 1999; 59: 1417–1421.

    CAS  PubMed  Google Scholar 

  30. Hong JS, Waud WR, Levasseur DN, Townes TM, Wen H, McPherson SA et al. Excellent in vivo bystander activity of fludarabine phosphate against human glioma xenografts that express the escherichia coli purine nucleoside phosphorylase gene. Cancer Res 2004; 64: 6610–6615.

    Article  CAS  PubMed  Google Scholar 

  31. Sorscher EJ, Peng S, Bebok Z, Allan PW, Bennett Jr LL, Parker WB . Tumor cell bystander killing in colonic carcinoma utilizing the Escherichia coli DeoD gene to generate toxic purines. Gene Therapy 1994; 1: 233–238.

    CAS  PubMed  Google Scholar 

  32. Hiraoka K, Kimura T, Logg CR, Tai CK, Haga K, Lawson GW et al. Therapeutic efficacy of replication-competent retrovirus vector-mediated suicide gene therapy in a multifocal colorectal cancer metastasis model. Cancer Res 2007; 67: 5345–5353.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tai CK, Kasahara N . Replication-competent retrovirus vectors for cancer gene therapy. Front Biosci 2008; 13: 3083–3095.

    Article  CAS  PubMed  Google Scholar 

  34. Duerner LJ, Schwantes A, Schneider IC, Cichutek K, Buchholz CJ . Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue. Gene Therapy 2008; 15: 1500–1510.

    Article  CAS  PubMed  Google Scholar 

  35. Lin AH, Timberlake N, Logg CR, Liu Y, Kamijima S, Diago O et al. MicroRNA 142-3p attenuates spread of replicating retroviral vector in hematopoietic lineage-derived cells while maintaining an antiviral immune response. Hum Gene Ther 2014; 25: 759–771.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Metzl C, Mischek D, Salmons B, Gunzburg WH, Renner M, Portsmouth D . Tissue- and tumor-specific targeting of murine leukemia virus-based replication-competent retroviral vectors. J Virol 2006; 80: 7070–7078.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nakamura H, Mullen JT, Chandrasekhar S, Pawlik TM, Yoon SS, Tanabe KK . Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosine deaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil. Cancer Res 2001; 61: 5447–5452.

    CAS  PubMed  Google Scholar 

  38. Parker WB, Allan PW, Shaddix SC, Rose LM, Speegle HF, Gillespie GY et al. Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells. Biochem Pharmacol 1998; 55: 1673–1681.

    Article  CAS  PubMed  Google Scholar 

  39. Huber BE, Austin EA, Richards CA, Davis ST, Good SS . Metabolism of 5-fluorocytosine to 5-fluorouracil in human colorectal tumor cells transduced with the cytosine deaminase gene: significant antitumor effects when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA 1994; 91: 8302–8306.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lu YC, Chen YJ, Yu YR, Lai YH, Cheng JC, Li YF et al. Replicating retroviral vectors for oncolytic virotherapy of experimental hepatocellular carcinoma. Oncol Rep 2012; 28: 21–26.

    CAS  PubMed  Google Scholar 

  41. Hersh MR, Kuhn JG, Phillips JL, Clark G, Ludden TM, Von Hoff DD . Pharmacokinetic study of fludarabine phosphate (NSC 312887). Cancer Chemother Pharmacol 1986; 17: 277–280.

    Article  CAS  PubMed  Google Scholar 

  42. Malspeis L, Grever MR, Staubus AE, Young D . Pharmacokinetics of 2-F-ara-A (9-beta-D-arabinofuranosyl-2-fluoroadenine) in cancer patients during the phase I clinical investigation of fludarabine phosphate. Semin Oncol 1990; 17 (5 Suppl 8): 18–32.

    CAS  PubMed  Google Scholar 

  43. Danhauser L, Plunkett W, Keating M, Cabanillas F . 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate pharmacokinetics in plasma and tumor cells of patients with relapsed leukemia and lymphoma. Cancer Chemother Pharmacol 1986; 18: 145–152.

    Article  CAS  PubMed  Google Scholar 

  44. Danhauser L, Plunkett W, Liliemark J, Gandhi V, Iacoboni S, Keating M . Comparison between the plasma and intracellular pharmacology of 1-beta-D-arabinofuranosylcytosine and 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate in patients with relapsed leukemia. Leukemia 1987; 1: 638–643.

    CAS  PubMed  Google Scholar 

  45. DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP . Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 1987; 7: 379–387.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Dr Noriyuki Kasahara for many helpful suggestions and discussion. We thank Chi Tsung Li for assistance with ELISA to detect AFP. This work was supported by a grant from the National Science Council of Taiwan (NSC99-2320-B-194-004-MY3).

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Authors and Affiliations

  1. Department of Life Science and Institutes of Molecular Biology and Biomedical Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan

    Y-H Lai, C-C Lin, S-H Chen & C-K Tai

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Correspondence to C-K Tai.

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Lai, YH., Lin, CC., Chen, SH. et al. Tumor-specific suicide gene therapy for hepatocellular carcinoma by transcriptionally targeted retroviral replicating vectors. Gene Ther 22, 155–162 (2015). https://doi.org/10.1038/gt.2014.98

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