Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair

Abstract

‘Ex vivo’ gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90–95% cell death at 12 days with GCV at 1 μg ml−1 in MBMCs vs 90–95% cell death at 5 days by 0.1 μg ml−1 of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0–14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  1. Anderson DW, Burton DC, Jackson RS . Postoperative cervical myelopathy and cord compression associated with the use of recombinant bone morphogenetic protein-2 in posterior cervical decompression, instrumentation, and arthrodesis: a report of two cases. Spine 36: E682–E686.

  2. Carragee EJ, Hurwitz EL, Weiner BK . A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J 2011; 11: 471–491.

    Article  PubMed  Google Scholar 

  3. Eufinger H, Leppanen H . Iliac crest donor site morbidity following open and closed methods of bone harvest for alveolar cleft osteoplasty. J Craniomaxillofac Surg 2000; 28: 31–38.

    Article  CAS  PubMed  Google Scholar 

  4. Singh K, Phillips FM, Kuo E, Campbell M . A prospective, randomized, double-blind study of the efficacy of postoperative continuous local anesthetic infusion at the iliac crest bone graft site after posterior spinal arthrodesis: a minimum of 4-year follow-up. Spine 2007; 32: 2790–2796.

    Article  PubMed  Google Scholar 

  5. Park J, Ries J, Gelse K, Kloss F, von der Mark K, Wiltfang J et al. Bone regeneration in critical size defects by cell-mediated BMP-2 gene transfer: a comparison of adenoviral vectors and liposomes. Gene Therapy 2003; 10: 1089–1098.

    Article  CAS  PubMed  Google Scholar 

  6. Baltzer AW, Lattermann C, Whalen JD, Wooley P, Weiss K, Grimm M et al. Genetic enhancement of fracture repair: healing of an experimental segmental defect by adenoviral transfer of the BMP-2 gene. Gene Therapy 2000; 7: 734–739.

    Article  CAS  PubMed  Google Scholar 

  7. Alden TD, Varady P, Kallmes DF, Jane JA Jr, Helm GA . Bone morphogenetic protein gene therapy. Spine 2002; 27 (16 Suppl 1): S87–S93.

    Article  PubMed  Google Scholar 

  8. Lieberman JR, Daluiski A, Stevenson S, Wu L, McAllister P, Lee YP et al. The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. J Bone Joint Surg Am 1999; 81: 905–917.

    Article  CAS  PubMed  Google Scholar 

  9. Sugiyama O, An DS, Kung SP, Feeley BT, Gamradt S, Liu NQ et al. Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo. Mol Ther 2005; 11: 390–398.

    Article  CAS  PubMed  Google Scholar 

  10. Virk MS, Conduah A, Park SH, Liu N, Sugiyama O, Cuomo A et al. Influence of short-term adenoviral vector and prolonged lentiviral vector mediated bone morphogenetic protein-2 expression on the quality of bone repair in a rat femoral defect model. Bone 2008; 42: 921–931.

    Article  CAS  PubMed  Google Scholar 

  11. Virk MS, Sugiyama O, Park SH, Gambhir SS, Adams DJ, Drissi H et al. ‘Same day’ ex-vivo regional gene therapy: a novel strategy to enhance bone repair. Mol Ther 2011; 19: 960–968.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ahmed A . Antiviral Treatment of Cytomegalovirus Infection. Infect Disord Drug Targets 2011; 11: 475–503.

    Article  CAS  PubMed  Google Scholar 

  13. Henderly DE, Jampol LM . Diagnosis and treatment of cytomegalovirus retinitis. J Acquir Immune Defic Syndr 1991; 4 (Suppl 1): S6–10.

    PubMed  Google Scholar 

  14. Jacobson MA, O'Donnell JJ . Approaches to the treatment of cytomegalovirus retinitis: ganciclovir and foscarnet. J Acquir Immune Defic Syndr 1991; 4 (Suppl 1): S11–S15.

    PubMed  Google Scholar 

  15. Brown DG, Visse R, Sandhu G, Davies A, Rizkallah PJ, Melitz C et al. Crystal structures of the thymidine kinase from herpes simplex virus type-1 in complex with deoxythymidine and ganciclovir. Nat Struct Biol 1995; 2: 876–881.

    Article  CAS  PubMed  Google Scholar 

  16. Cihova M, Altanerova V, Altaner C . Stem Cell Based Cancer Gene Therapy. Mol Pharm 2011; 8: 1480–1487.

    Article  CAS  PubMed  Google Scholar 

  17. Li S, Gao Y, Pu K, Ma L, Song X, Liu Y . All-trans retinoic acid enhances bystander effect of suicide-gene therapy against medulloblastomas. Neurosci Lett 2011; 503: 115–119.

    Article  CAS  PubMed  Google Scholar 

  18. Liu T, Ye L, Chen X, Peng J, Zhang X, Yi H et al. Combination gene therapy using VEGF-shRNA and fusion suicide gene yCDglyTK inhibits gastric carcinoma growth. Exp Mol Pathol 2011; 91: 745–752.

    Article  CAS  PubMed  Google Scholar 

  19. Pulkkanen KJ, Yla-Herttuala S . Gene therapy for malignant glioma: current clinical status. Mol Ther 2005; 12: 585–598.

    Article  CAS  PubMed  Google Scholar 

  20. Spitzweg C, Morris JC . Gene therapy for thyroid cancer: current status and future prospects. Thyroid 2004; 14: 424–434.

    Article  CAS  PubMed  Google Scholar 

  21. Salomon B, Maury S, Loubiere L, Caruso M, Onclercq R, Klatzmann D . A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice. Mol Cell Biol 1995; 15: 5322–5328.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Pensak MJ, Lieberman JR . Gene therapy for bone regeneration. Curr Pharmaceut Design 2013; 19: 3466–3473.

    Article  CAS  Google Scholar 

  23. Moutsatsos IK, Turgeman G, Zhou S, Kurkalli BG, Pelled G, Tzur L et al. Exogenously regulated stem cell-mediated gene therapy for bone regeneration. Mol Ther 2001; 3: 449–461.

    Article  CAS  PubMed  Google Scholar 

  24. Lieberman JR, Le LQ, Wu L, Finerman GA, Berk A, Witte ON et al. Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents. J Orthopaedic Res 1998; 16: 330–339.

    Article  CAS  Google Scholar 

  25. Gafni Y, Pelled G, Zilberman Y, Turgeman G, Apparailly F, Yotvat H et al. Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system. Mol Ther 2004; 9: 587–595.

    Article  CAS  PubMed  Google Scholar 

  26. Baird SD, Turcotte M, Korneluk RG, Holcik M . Searching for IRES. RNA 2006; 12: 1755–1785.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Licursi M, Christian SL, Pongnopparat T, Hirasawa K . In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression. Gene Ther 2011; 18: 631–636.

    Article  CAS  PubMed  Google Scholar 

  28. Ngoi SM, Chien AC, Lee CG . Exploiting internal ribosome entry sites in gene therapy vector design. Curr Gene Ther 2004; 4: 15–31.

    Article  CAS  PubMed  Google Scholar 

  29. 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 

  30. Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 2011; 6: e18556.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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 

  32. 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 (Pt 11): 2727–2732.

    Article  CAS  PubMed  Google Scholar 

  33. Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D et al. Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J Gen Virol 2001; 82 (Pt 5): 1013–1025.

    Article  CAS  PubMed  Google Scholar 

  34. Luke G, Escuin H, De Felipe P, Ryan M . 2A to the fore - research, technology and applications. Biotechnol Genet Eng Rev 2010; 26: 223–260.

    Article  CAS  PubMed  Google Scholar 

  35. Altaner C . Prodrug cancer gene therapy. Cancer Lett 2008; 270: 191–201.

    Article  CAS  PubMed  Google Scholar 

  36. Ciceri F, Bonini C, Gallo-Stampino C, Bordignon C . Modulation of GvHD by suicide-gene transduced donor T lymphocytes: clinical applications in mismatched transplantation. Cytotherapy 2005; 7: 144–149.

    Article  CAS  PubMed  Google Scholar 

  37. Ciceri F, Bordignon C . Suicide-gene-Transduced donor T-cells for controlled graft-versus-host disease and graft-versus-tumor. Int J Hematol 2002; 76: 305–309.

    Article  CAS  PubMed  Google Scholar 

  38. Matuskova M, Hlubinova K, Pastorakova A, Hunakova L, Altanerova V, Altaner C et al. HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells. Cancer Lett 2009; 290: 58–67.

    Article  PubMed  Google Scholar 

  39. Mesnil M, Yamasaki H . Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication. Cancer Res 2000; 60: 3989–3999.

    CAS  PubMed  Google Scholar 

  40. Nicholas TW, Read SB, Burrows FJ, Kruse CA . Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects. Histol Histopathol 2003; 18: 495–507.

    CAS  PubMed  Google Scholar 

  41. 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 

  42. Kuriyama S, Masui K, Sakamoto T, Nakatani T, Kikukawa M, Tsujinoue H et al. Bystander effect caused by cytosine deaminase gene and 5-fluorocytosine in vitro is substantially mediated by generated 5-fluorouracil. Anticancer Res 1998; 18: 3399–3406.

    CAS  PubMed  Google Scholar 

  43. Boucher PD, Im MM, Freytag SO, Shewach DS . A novel mechanism of synergistic cytotoxicity with 5-fluorocytosine and ganciclovir in double suicide gene therapy. Cancer Res 2006; 66: 3230–3237.

    Article  CAS  PubMed  Google Scholar 

  44. Feeley BT, Conduah AH, Sugiyama O, Krenek L, Chen IS, Lieberman JR . In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation models. J Orthop Res 2006; 24: 1709–1721.

    Article  CAS  PubMed  Google Scholar 

  45. Hassan W, Sanford MA, Woo SL, Chen SH, Hall SJ . Prospects for herpes-simplex-virus thymidine-kinase and cytokine gene transduction as immunomodulatory gene therapy for prostate cancer. World J Urol 2000; 18: 130–135.

    Article  CAS  PubMed  Google Scholar 

  46. 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 

  47. Shalev M, Kadmon D, Teh BS, Butler EB, Aguilar-Cordova E, Thompson TC et al. Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer. J Urol 2000; 163: 1747–1750.

    Article  CAS  PubMed  Google Scholar 

  48. Visnjic D, Kalajzic I, Gronowicz G, Aguila HL, Clark SH, Lichtler AC et al. Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice. J Bone Miner Res 2001; 16: 2222–2231.

    Article  CAS  PubMed  Google Scholar 

  49. Kung SK, An DS, Chen IS . A murine leukemia virus (MuLV) long terminal repeat derived from rhesus macaques in the context of a lentivirus vector and MuLV gag sequence results in high-level gene expression in human T lymphocytes. J Virol 2000; 74: 3668–3681.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hsu WK, Sugiyama O, Park SH, Conduah A, Feeley BT, Liu NQ et al. Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats. Bone 2007; 40: 931–938.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by NIH grant 1R01AR057076-01A1 to JRL.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to J R Lieberman.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alaee, F., Sugiyama, O., Virk, M. et al. Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair. Gene Ther 21, 139–147 (2014). https://doi.org/10.1038/gt.2013.66

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2013.66

Keywords

This article is cited by

Search

Quick links