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Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

Gene Therapy volume 15, pages 1411–1423 (2008)Cite this article

Abstract

In human gene therapy applications, lentiviral vectors may have advantages over γ-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike γ-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20–30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

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References

  1. Thomas S, Hart DP, Xue SA, Cesco-Gaspere M, Stauss HJ . T-cell receptor gene therapy for cancer: the progress to date and future objectives. Expert Opin Biol Ther 2007; 7: 1207–1218.

    Article  CAS  PubMed  Google Scholar 

  2. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006; 314: 126–129.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cooper LJ, Topp MS, Pinzon C, Plavec I, Jensen MC, Riddell SR et al. Enhanced transgene expression in quiescent and activated human CD8+ T cells. Hum Gene Ther 2004; 15: 648–658.

    Article  CAS  PubMed  Google Scholar 

  4. Ikawa M, Tanaka N, Kao WW, Verma IM . Generation of transgenic mice using lentiviral vectors: a novel preclinical assessment of lentiviral vectors for gene therapy. Mol Ther 2003; 8: 666–673.

    Article  CAS  PubMed  Google Scholar 

  5. Pfeifer A, Ikawa M, Dayn Y, Verma IM . Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 2002; 99: 2140–2145.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol 2006; 24: 687–696.

    Article  CAS  PubMed  Google Scholar 

  7. Sauce D, Bodinier M, Garin M, Petracca B, Tonnelier N, Duperrier A et al. Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein–Barr virus potential through both culture-dependent and selection process-dependent mechanisms. Blood 2002; 99: 1165–1173.

    Article  CAS  PubMed  Google Scholar 

  8. Sauce D, Tonnelier N, Duperrier A, Petracca B, de Carvalho Bittencourt M, Saadi M et al. Influence of ex vivo expansion and retrovirus-mediated gene transfer on primary T lymphocyte phenotype and functions. J Hematother Stem Cell Res 2002; 11: 929–940.

    Article  CAS  PubMed  Google Scholar 

  9. Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest 2005; 115: 1616–1626.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Chang AH, Sadelain M . The genetic engineering of hematopoietic stem cells: the rise of lentiviral vectors, the conundrum of the LTR, and the promise of lineage-restricted vectors. Mol Ther 2007; 15: 445–456.

    Article  CAS  PubMed  Google Scholar 

  11. Wiznerowicz M, Trono D . Harnessing HIV for therapy, basic research and biotechnology. Trends Biotechnol 2005; 23: 42–47.

    Article  CAS  PubMed  Google Scholar 

  12. Krogsgaard M, Davis MM . How T cells ‘see’ antigen. Nat Immunol 2005; 6: 239–245.

    Article  CAS  PubMed  Google Scholar 

  13. Schumacher TN . T-cell-receptor gene therapy. Nat Rev Immunol 2002; 2: 512–519.

    Article  CAS  PubMed  Google Scholar 

  14. Sadelain M, Riviere I, Brentjens R . Targeting tumours with genetically enhanced T lymphocytes. Nat Rev Cancer 2003; 3: 35–45.

    Article  CAS  PubMed  Google Scholar 

  15. Zhao Y, Zheng Z, Robbins PF, Khong HT, Rosenberg SA, Morgan RA . Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines. J Immunol 2005; 174: 4415–4423.

    Article  CAS  PubMed  Google Scholar 

  16. Morgan RA, Dudley ME, Yu YY, Zheng Z, Robbins PF, Theoret MR et al. High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens. J Immunol 2003; 171: 3287–3295.

    Article  CAS  PubMed  Google Scholar 

  17. Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16: 457–472.

    Article  CAS  PubMed  Google Scholar 

  18. Cohen CJ, Zheng Z, Bray R, Zhao Y, Sherman LA, Rosenberg SA et al. Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR. J Immunol 2005; 175: 5799–5808.

    Article  CAS  PubMed  Google Scholar 

  19. Morgan RA, Couture L, Elroy-Stein O, Ragheb J, Moss B, Anderson WF . Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res 1992; 20: 1293–1299.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yu X, Zhan X, D’Costa J, Tanavde VM, Ye Z, Peng T et al. Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells. Mol Ther 2003; 7: 827–838.

    Article  CAS  PubMed  Google Scholar 

  21. Amendola M, Venneri MA, Biffi A, Vigna E, Naldini L . Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol 2005; 23: 108–116.

    Article  CAS  PubMed  Google Scholar 

  22. Osti D, Marras E, Ceriani I, Grassini G, Rubino T, Vigano D et al. Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes. J Virol Methods 2006; 136: 93–101.

    Article  CAS  PubMed  Google Scholar 

  23. Szymczak AL, Vignali DA . Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin Biol Ther 2005; 5: 627–638.

    Article  CAS  PubMed  Google Scholar 

  24. Quintarelli C, Vera JF, Savoldo B, Giordano Attianese GM, Pule M, Foster AE et al. Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes. Blood 2007; 110: 2793–2802.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 2004; 22: 589–594.

    Article  CAS  PubMed  Google Scholar 

  26. van de Ven WJ, Voorberg J, Fontijn R, Pannekoek H, van den Ouweland AM, van Duijnhoven HL et al. Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes. Mol Biol Rep 1990; 14: 265–275.

    Article  CAS  PubMed  Google Scholar 

  27. Holst J, Vignali KM, Burton AR, Vignali DA . Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice. Nat Methods 2006; 3: 191–197.

    Article  CAS  PubMed  Google Scholar 

  28. Krysan DJ, Rockwell NC, Fuller RS . Quantitative characterization of furin specificity. Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides. J Biol Chem 1999; 274: 23229–23234.

    Article  CAS  PubMed  Google Scholar 

  29. Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M et al. Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 2005; 23: 584–590.

    Article  CAS  PubMed  Google Scholar 

  30. Simmons A, Whitehead RP, Kolokoltsov AA, Davey RA . Use of recombinant lentivirus pseudotyped with vesicular stomatitis virus glycoprotein G for efficient generation of human anti-cancer chimeric T cells by transduction of human peripheral blood lymphocytes in vitro. Virol J 2006; 3: 8.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Sumiyama K, Saitou N, Ueda S . Adaptive evolution of the IgA hinge region in primates. Mol Biol Evol 2002; 19: 1093–1099.

    Article  CAS  PubMed  Google Scholar 

  32. Johnson LA, Heemskerk B, Powell Jr DJ, Cohen CJ, Morgan RA, Dudley ME et al. Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. J Immunol 2006; 177: 6548–6559.

    Article  CAS  PubMed  Google Scholar 

  33. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D . Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997; 15: 871–875.

    Article  CAS  PubMed  Google Scholar 

  34. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263–267.

    Article  CAS  PubMed  Google Scholar 

  35. Kohn DB . Lentiviral vectors ready for prime-time. Nat Biotechnol 2007; 25: 65–66.

    Article  CAS  PubMed  Google Scholar 

  36. Dardalhon V, Herpers B, Noraz N, Pflumio F, Guetard D, Leveau C et al. Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap. Gene Therapy 2001; 8: 190–198.

    Article  CAS  PubMed  Google Scholar 

  37. Barklis E, Mulligan RC, Jaenisch R . Chromosomal position or virus mutation permits retrovirus expression in embryonal carcinoma cells. Cell 1986; 47: 391–399.

    Article  CAS  PubMed  Google Scholar 

  38. Colicelli J, Goff SP . Isolation of a recombinant murine leukemia virus utilizing a new primer tRNA. J Virol 1986; 57: 37–45.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Schambach A, Galla M, Maetzig T, Loew R, Baum C . Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors. Mol Ther 2007; 15: 1167–1173.

    Article  CAS  PubMed  Google Scholar 

  40. Ellis J . Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum Gene Ther 2005; 16: 1241–1246.

    Article  CAS  PubMed  Google Scholar 

  41. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D . Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 2002; 295: 868–872.

    Article  CAS  PubMed  Google Scholar 

  42. Ma Y, Ramezani A, Lewis R, Hawley RG, Thomson JA . High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 2003; 21: 111–117.

    Article  CAS  PubMed  Google Scholar 

  43. Cavalieri S, Cazzaniga S, Geuna M, Magnani Z, Bordignon C, Naldini L et al. Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence. Blood 2003; 102: 497–505.

    Article  CAS  PubMed  Google Scholar 

  44. Deschamps M, Robinet E, Certoux JM, Mercier P, Sauce D, De Vos J et al. Transcriptome of retrovirally transduced CD8(+) lymphocytes: influence of cell activation, transgene integration, and selection process. Mol Immunol 2008; 45: 1112–1125.

    Article  CAS  PubMed  Google Scholar 

  45. Coito S, Sauce D, Duperrier A, Certoux JM, Bonyhadi M, Collette A et al. Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells. Stem Cells Dev 2004; 13: 71–81.

    Article  CAS  PubMed  Google Scholar 

  46. Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA et al. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J Exp Med 2003; 198: 569–580.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Abad JD, Wrzensinski C, Overwijk W, De Witte MA, Jorritsma A, Hsu C et al. T-cell receptor gene therapy of established tumors in a murine melanoma model. J Immunother 2008; 31: 1–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Cohen CJ, Li YF, El-Gamil M, Robbins PF, Rosenberg SA, Morgan RA . Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res 2007; 67: 3898–3903.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cohen CJ, Zhao Y, Zheng Z, Rosenberg SA, Morgan RA . Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. Cancer Res 2006; 66: 8878–8886.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Chinnasamy D, Milsom MD, Shaffer J, Neuenfeldt J, Shaaban AF, Margison GP et al. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI. Virol J 2006; 3: 14.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Holst J, Szymczak-Workman AL, Vignali KM, Burton AR, Workman CJ, Vignali DA . Generation of T-cell receptor retrogenic mice. Nat Protoc 2006; 1: 406–417.

    Article  CAS  PubMed  Google Scholar 

  52. de Felipe P, Ryan MD . Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences. Traffic (Copenhagen, Denmark) 2004; 5: 616–626.

    Article  CAS  Google Scholar 

  53. Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, Lu X et al. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci USA 2006; 103: 17372–17377.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ryan MD, King AM, Thomas GP . Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen Virol 1991; 72 (Part 11): 2727–2732.

    Article  CAS  PubMed  Google Scholar 

  55. Geraerts M, Willems S, Baekelandt V, Debyser Z, Gijsbers R . Comparison of lentiviral vector titration methods. BMC Biotechnol 2006; 6: 34.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Topalian SL, Solomon D, Rosenberg SA . Tumor-specific cytolysis by lymphocytes infiltrating human melanomas. J Immunol 1989; 142: 3714–3725.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank FACS laboratory and TIL laboratory in Surgery Branch for providing technical support and maintenance of tumor cells from patients. This work is supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.

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Authors and Affiliations

  1. Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    S Yang, C J Cohen, P D Peng, Y Zhao, L Cassard, Z Yu, Z Zheng, S Jones, N P Restifo, S A Rosenberg & R A Morgan

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Correspondence to R A Morgan.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Yang, S., Cohen, C., Peng, P. et al. Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Ther 15, 1411–1423 (2008). https://doi.org/10.1038/gt.2008.90

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