ORIGINAL ARTICLE Preclinical study of an ex vivo gene therapy protocol for hepatocarcinoma B Lortal 1 , F Gross 2 , JM Peron 3 , M Pe ´nary 1,4 , D Berg 1 , I Hennebelle 5 , G Favre 1,4 and B Couderc 1,4,5 1 INSERM U563, CPTP, Institut Claudius Regaud, Toulouse, France; 2 CIC Biothe´rapies, CHU Rangueil, Toulouse, France; 3 INSERM U858, Toulouse, France; 4 Universite ´ Toulouse III, Faculte ´ des Sciences Pharmaceutiques, Toulouse, France and 5 EA 3035, Institut Claudius Regaud, Toulouse, France 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. Cancer Gene Therapy (2009) 16, 329–337; doi:10.1038/cgt.2008.88; published online 7 November 2008 Keywords: IL-12; retrovirus vectors; ex vivo; fibroblasts Introduction Hepatocarcinoma (HC) is the most frequent liver cancer with 250 000–1 000 000 new cases each year and represents the fourth leading cause of cancer death worldwide. Its incidence is increasing every year because of (i) an increase in life expectancy of patients presenting cirrhosis, (ii) improved diagnosis or treatment of hepatic diseases, and (iii) a higher frequency of the hepatic pathologies connected to the hepatitis C virus. 1–4 The three curative treatments of HC are transplantation, resection and percutaneous destruction, 5 all of which improve the natural history of the HC and prolong the survival of patients with a unique tumor of less than 5cm or a maximum of three lesions measuring at most 3 cm. In all other cases, however, these treatments become palliative. Thus, for about 90% of patients, no effective treatment can be proposed, 6 and HC remains a world health problem. The heterodimeric cytokine interleukin 12 (IL-12) consists of two subunits, p40 and p35. 7 It is recognized as a master regulator of adaptive type 1 cell-mediated immunity, the critical pathway involved in protection against neoplasia and many viruses. 8 It enhances tumor destruction by activating cytotoxic T lymphocytes, prim- ing the differentiation of naive CD4 þ T cells to helper T type 1 cells that can subsequently help tumor-specific cytotoxic T lymphocytes, and stimulating natural killer cells. 7,9–11 IL-12 presents immunomodulating and anti- angiogenic functions, which have provided the rationale for exploiting this cytokine as an anticancer agent. In this way, IL-12 has been applied as a recombinant protein in cancer patients in various phase I and II trials and found to show some efficacy. 12–15 However, its use induced severe toxicity leading to the death of two patients in one phase II trial. 16,17 On the basis of the promising results obtained in a large series of preclinical IL-12 gene therapy studies, 8 clinical trials have been designed with the aim of achieving cytokine production at the tumor site, while at the same time maintaining low serum concentrations to reduce systemic toxicity. 18–21 Various strategies involving immune stimulation have shown occasional activity against HC leading to the execution of phase I immuno gene therapy involving Received 21 April 2008; revised 8 August 2008; accepted 24 September 2008; published online 7 November 2008 Correspondence: Professor B Couderc, EA3035, Institut Claudius Regaud, 20–24 rue du pont St Pierre, Toulouse 31052, France. E-mail: couderc.bettina@claudiusregaud.fr Cancer Gene Therapy (2009) 16, 329–337 r 2009 Nature Publishing Group All rights reserved 0929-1903/09 $32.00 www.nature.com/cgt