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:

Preclinical study of an ex vivo gene therapy protocol for hepatocarcinoma

Cancer Gene Therapy volume 16, pages 329–337 (2009)Cite this article

Abstract

Preclinical studies in several animal models as well as clinical trials have shown a reduction in tumor growth following immunotherapy with interleukin-12 (IL-12). This cytokine is appropriate to test in therapeutic clinical trials to treat hepatocarcinoma (HC), a pathology often associated with hepatitis B or C-induced cirrhosis. The local delivery into the liver would be achieved through ex vivo gene transfer using retroviral (rv) vectors in autologous fibroblast carriers. In support of this clinical trial, a rv vector has been constructed to express coordinately both chains p35 and p40 of human IL-12. Here, we have tested good manufacturing practices (GMP) clinical lots of viral vectors derived from the transfected packaging cell line, PG13rvIL-12. We have also devised methods to facilitate the isolation of fibroblasts from freshly harvested skin specimens, enhance their outgrowth in large-scale cultures and assay IL-12 production following transduction, without any selection and irradiation. Twenty-four human skin specimens were processed to obtain fibroblast suspensions that were typically maintained for up to 8 or 12 passages. The mean ±s.d. overall time for obtaining the required number of transduced cells for the highest IL-12 need was 40 days. The procedure, in accordance with the French medical agency for gene therapy clinical trials, is now ready to begin a clinical trial.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Cottone M, Turri M, Caltagirone M, Parisi P, Orlando A, Fiorentino G et al. Screening for hepatocellular carcinoma in patients with Child's A cirrhosis: an 8-year prospective study by ultrasound and alphafetoprotein. J Hepatol 1994; 21: 1029–1034.

    Article  CAS  PubMed  Google Scholar 

  2. Kato Y, Nakata K, Omagari K, Furukawa R, Kusumoto Y, Mori I et al. Risk of hepatocellular carcinoma in patients with cirrhosis in Japan. Analysis of infectious hepatitis viruses. Cancer 1994; 74: 2234–2238.

    Article  CAS  PubMed  Google Scholar 

  3. Ikeda K, Saitoh S, Koida I, Arase Y, Tsubota A, Chayama K et al. A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis. Hepatology 1993; 18: 47–53.

    Article  CAS  PubMed  Google Scholar 

  4. Ikeda K, Saitoh S, Tsubota A, Arase Y, Chayama K, Kumada H et al. Risk factors for tumor recurrence and prognosis after curative resection of hepatocellular carcinoma. Cancer 1993; 71: 19–25.

    Article  CAS  PubMed  Google Scholar 

  5. Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001; 35: 421–430.

    Article  CAS  PubMed  Google Scholar 

  6. Trinchet JC, Beaugrand M . Treatment of hepatocellular carcinoma in patients with cirrhosis. J Hepatol 1997; 27: 756–765.

    Article  CAS  PubMed  Google Scholar 

  7. Trinchieri G . Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 1994; 84: 4008–4027.

    CAS  PubMed  Google Scholar 

  8. Del Vecchio M, Bajetta E, Canova S, Lotze MT, Wesa A, Parmiani G et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res 2007; 13: 4677–4685.

    Article  CAS  PubMed  Google Scholar 

  9. Kobayashi M, Fitz L, Ryan M, Hewick RM, Clark SC, Chan S et al. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989; 170: 827–845.

    Article  CAS  PubMed  Google Scholar 

  10. Folkman J . Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995; 1: 27–31.

    Article  CAS  PubMed  Google Scholar 

  11. Kanegane C, Sgadari C, Kanegane H, Teruya-Feldstein J, Yao L, Gupta G et al. Contribution of the CXC chemokines IP-10 and Mig to the antitumor effects of IL-12. J Leukoc Biol 1998; 64: 384–392.

    Article  CAS  PubMed  Google Scholar 

  12. Atkins MB, Robertson MJ, Gordon M, Lotze MT, DeCoste M, DuBois JS et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997; 3: 409–417.

    CAS  PubMed  Google Scholar 

  13. Bajetta E, Del Vecchio M, Mortarini R, Nadeau R, Rakhit A, Rimassa L et al. Pilot study of subcutaneous recombinant human interleukin 12 in metastatic melanoma. Clin Cancer Res 1998; 4: 75–85.

    CAS  PubMed  Google Scholar 

  14. Robertson MJ, Cameron C, Atkins MB, Gordon MS, Lotze MT, Sherman ML et al. Immunological effects of interleukin 12 administered by bolus intravenous injection to patients with cancer. Clin Cancer Res 1999; 5: 9–16.

    CAS  PubMed  Google Scholar 

  15. Heinzerling L, Burg G, Dummer R, Maier T, Oberholzer PA, Schultz J et al. Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy. Hum Gene Ther 2005; 16: 35–48.

    Article  CAS  PubMed  Google Scholar 

  16. Cohen J . IL-12 deaths: explanation and a puzzle. Science 1995; 270: 908.

    Article  CAS  PubMed  Google Scholar 

  17. Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 1997; 90: 2541–2548.

    CAS  PubMed  Google Scholar 

  18. Nanni P, Forni G, Lollini PL . Cytokine gene therapy: hopes and pitfalls. Ann Oncol 1999; 10: 261–266.

    Article  CAS  PubMed  Google Scholar 

  19. Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I et al. Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors. J Clin Oncol 2004; 22: 1389–1397.

    Article  CAS  PubMed  Google Scholar 

  20. Sangro B, Melero I, Qian C, Prieto J . Gene therapy of cancer based on interleukin 12. Curr Gene Ther 2005; 5: 573–581.

    Article  CAS  PubMed  Google Scholar 

  21. Kang WK, Park C, Yoon HL, Kim WS, Yoon SS, Lee MH et al. Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study. Hum Gene Ther 2001; 12: 671–684.

    Article  CAS  PubMed  Google Scholar 

  22. Mazzolini G, Alfaro C, Sangro B, Feijoo E, Ruiz J, Benito A et al. Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas. J Clin Oncol 2005; 23: 999–1010.

    Article  CAS  PubMed  Google Scholar 

  23. Peron JM, Couderc B, Rochaix P, Douin-Echinard V, Asnacios A, Souque A et al. Treatment of murine hepatocellular carcinoma using genetically modified cells to express interleukin-12. J Gastroenterol Hepatol 2004; 19: 388–396.

    Article  CAS  PubMed  Google Scholar 

  24. Peron JM, Bureau C, Gourdy P, Lulka H, Souque A, Calippe B et al. Treatment of experimental murine pancreatic peritoneal carcinomatosis with fibroblasts genetically modified to express IL12: a role for peritoneal innate immunity. Gut 2007; 56: 107–114.

    Article  CAS  PubMed  Google Scholar 

  25. Kimura E, Han JJ, Li S, Fall B, Ra J, Haraguchi M et al. Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy. Hum Mol Genet 2008; 17: 2507–2517.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cavorsi J, Vicari F, Wirthlin DJ, Ennis W, Kirsner R, O’Connell SM et al. Best-practice algorithms for the use of a bilayered living cell therapy (Apligraf) in the treatment of lower-extremity ulcers. Wound Repair Regen 2006; 14: 102–109.

    Article  PubMed  Google Scholar 

  27. Migliaccio AR, Quarto R, Piacibello W . Cell therapy: filling the gap between basic science and clinical trials October 15–17, 2001, Rome, Italy. Stem Cells 2003; 21: 348–356.

    Article  PubMed  Google Scholar 

  28. Tuszynski MH, Thal L, Pay M, Salmon DP, U HS, Bakay R et al. A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 2005; 11: 551–555.

    Article  CAS  PubMed  Google Scholar 

  29. Indraccolo S, Moserle L, Tisato V, Gola E, Minuzzo S, Roni V et al. Gene therapy of ovarian cancer with IFN-alpha-producing fibroblasts: comparison of constitutive and inducible vectors. Gene Ther 2006; 13: 953–965.

    Article  CAS  PubMed  Google Scholar 

  30. Elder EM, Lotze MT, Whiteside TL . Successful culture and selection of cytokine gene-modified human dermal fibroblasts for the biologic therapy of patients with cancer. Hum Gene Ther 1996; 7: 479–487.

    Article  CAS  PubMed  Google Scholar 

  31. Lotze MT, Hellerstedt B, Stolinski L, Tueting T, Wilson C, Kinzler D et al. The role of interleukin-2, interleukin-12, and dendritic cells in cancer therapy. Cancer J Sci Am 1997; 3 (Suppl 1): S109–S114.

    PubMed  Google Scholar 

  32. Towers GJ, Stockholm D, Labrousse-Najburg V, Carlier F, Danos O, Pages JC . One step screening of retroviral producer clones by real time quantitative PCR. J Gene Med 1999; 1: 352–359.

    Article  CAS  PubMed  Google Scholar 

  33. Zitvogel L, Tahara H, Cai Q, Storkus WJ, Muller G, Wolf SF et al. Construction and characterization of retroviral vectors expressing biologically active human interleukin-12. Hum Gene Ther 1994; 5: 1493–1506.

    Article  CAS  PubMed  Google Scholar 

  34. Danos O, Mulligan RC . Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA 1988; 85: 6460–6464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Miller AD, Garcia JV, von Suhr N, Lynch CM, Wilson C, Eiden MV . Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol 1991; 65: 2220–2224.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Gepstein L, Feld Y, Yankelson L . Somatic gene and cell therapy strategies for the treatment of cardiac arrhythmias. Am J Physiol Heart Circ Physiol 2004; 286: H815–H822.

    Article  CAS  PubMed  Google Scholar 

  37. Gache Y, Baldeschi C, Del Rio M, Gagnoux-Palacios L, Larcher F, Lacour JP et al. Construction of skin equivalents for gene therapy of recessive dystrophic epidermolysis bullosa. Hum Gene Ther 2004; 15: 921–933.

    Article  CAS  PubMed  Google Scholar 

  38. Featherstone C . Epidermolysis bullosa: from fundamental molecular biology to clinical therapies. J Invest Dermatol 2007; 127: 256–259.

    Article  CAS  PubMed  Google Scholar 

  39. Yamanaka R, Homma J, Yajima N, Tsuchiya N, Sano M, Kobayashi T et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical phase I/II trial. Clin Cancer Res 2005; 11: 4160–4167.

    Article  CAS  PubMed  Google Scholar 

  40. Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346: 1185–1193.

    Article  CAS  PubMed  Google Scholar 

  41. Gansbacher B, Zier K, Cronin K, Hantzopoulos PA, Bouchard B, Houghton A et al. Retroviral gene transfer induced constitutive expression of interleukin-2 or interferon-gamma in irradiated human melanoma cells. Blood 1992; 80: 2817–2825.

    CAS  PubMed  Google Scholar 

  42. Brach MA, Hass R, Sherman ML, Gunji H, Weichselbaum R, Kufe D . Ionizing radiation induces expression and binding activity of the nuclear factor kappa B. J Clin Invest 1991; 88: 691–695.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rosenthal FM, Fruh R, Henschler R, Veelken H, Kulmburg P, Mackensen A et al. Cytokine therapy with gene-transfected cells: single injection of irradiated granulocyte-macrophage colony-stimulating factor-transduced cells accelerates hematopoietic recovery after cytotoxic chemotherapy in mice. Blood 1994; 84: 2960–2965.

    CAS  PubMed  Google Scholar 

  44. Moret-Tatay I, Diaz J, Marco FM, Crespo A, Alino SF . Complete tumor prevention by engineered tumor cell vaccines employing nonviral vectors. Cancer Gene Ther 2003; 10: 887–897.

    Article  CAS  PubMed  Google Scholar 

  45. Santin AD, Ioli GR, Hiserodt JC, Manetta A, Pecorelli S, DiSaia PJ et al. Development and characterization of an interleukin-2-transduced human ovarian carcinoma tumor vaccine not expressing major histocompatibility complex molecules. Am J Obstet Gynecol 1996; 174: 633–640.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to Genethon, (Patricia Noguiey's team) which constructed the pMFG-hIL-12 plasmid and synthesized the clinical-grade retroviral vectors. We thank Dr J. Bonnet who performed the γ-irradiation of the cells. We are also grateful to Emily Witty (AngloScribe) for the careful reading and correction of this paper. We also thank Pr JP Vinel and Pr L Buscail for their critical input. This study was supported by La ligue contre le cancer, The Region Midi Pyrenees, the GRICR (Groupe de Recherche Institut Claudius Regaud) and hospitals of Toulouse III (PHRC; France).

Grant support: La ligue contre le cancer, the Region Midi Pyrenees, the GRICR (Groupe de Recherche Institut Claudius Regaud) and hospitals of Toulouse III (PHRC; France).

Author information

Authors and Affiliations

  1. INSERM U563, CPTP, Institut Claudius Regaud, Toulouse, France

    B Lortal, M Pénary, D Berg, G Favre & B Couderc

  2. CIC Biothérapies, CHU Rangueil, Toulouse, France

    F Gross

  3. INSERM U858, Toulouse, France

    J M Peron

  4. Université Toulouse III, Faculté des Sciences Pharmaceutiques, Toulouse, France

    M Pénary, G Favre & B Couderc

  5. EA 3035, Institut Claudius Regaud, Toulouse, France

    I Hennebelle & B Couderc

Authors
  1. B Lortal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F Gross
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J M Peron
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Pénary
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D Berg
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. I Hennebelle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G Favre
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. B Couderc
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B Couderc.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lortal, B., Gross, F., Peron, J. et al. Preclinical study of an ex vivo gene therapy protocol for hepatocarcinoma. Cancer Gene Ther 16, 329–337 (2009). https://doi.org/10.1038/cgt.2008.88

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cgt.2008.88

Keywords

This article is cited by

Search

Advanced search

Quick links