C. Qian

Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin-12 (IL-12)

The use of gene therapy to enhance antitumor immunity has emerged as a promising procedure to fight cancer. In this study we have tested the ability of an adenovirus carrying interleukin 12 (IL-12) gene (AdCMVIL-12) to eliminate tumoral lesions in 3 animal models of orthotopic hepatocellular carcinoma (HCC). Intratumoral injection of AdCMVIL-12 in animals with a single big tumor nodule implanted in the liver resulted in significant inhibition of tumor growth in a dose-dependent manner. Fifty percent of animals that received a dose of 5 x 10(9) plaque-forming units, showed complete regression of the tumor 2 weeks after treatment. In animals with 2 independent tumor nodules in the left liver lobe, injection in only one of them of 5 x 10(9) pfu AdCMVIL-12 induced, 15 days after therapy, complete regression of 50% of treated tumors and also of 50% of untreated lesions, with 60% long-term survival. Rats that were tumor free after therapy with AdCMVIL-12 showed protection against tumor rechallenge. A group of rats received the carcinogen diethylnitrosamine and developed multiple hepatic dysplasic nodules of 1 to 5 mm in diameter. These animals were treated by intrahepatic artery injection of either AdCMVIL-12 (5 x 10(9) pfu) or control vector. In this model AdCMVIL-12 induced complete tumor regression in 20% of treated rats and inhibited tumor growth in 60% of cases with an increase in rat survival. Activation of natural killer (NK) cells and inhibition of angiogenesis were found to be antitumor mechanisms set in motion by AdCMVIL-12. Our data indicate that experimental HCC can be efficiently treated by intratumoral or intravascular injection of adenovirus expressing IL-12.

Treatment of murine fulminant hepatitis with genetically engineered endothelial progenitor cells

BACKGROUND & AIMSnCell therapy has been used to attenuate liver injury. Here we evaluated whether genetic engineering of either bone marrow-derived mononuclear cells (MNC) or endothelial progenitor cells (EPC) many enhance their hepatoprotective properties.nnnMETHODSnMice with ConA-induced hepatitis or with lethal fulminant hepatitis resulting from administration of an adenovirus encoding CD40L (AdCD40L) received an intra-splenic injection of saline or 2 × 10(6) unmodified MNC or EPC or the same cells transduced ex vivo with an adenovirus expressing luciferase (MNCLUC and EPCLUC) or encoding the hepatoprotective cytokine cardiotrophin-1 (CT-1) (MNCCT-1 and EPCCT-1). We analyzed the extent of liver damage, the intensity of inflammatory reaction, and animal survival.nnnRESULTSnLuciferase immunohistochemistry showed that after injection into the spleen, the engineered cells migrated efficiently to the damaged liver. In mice with ConA hepatitis EPCCT-1, but not other forms of cell therapy, significantly decreased serum transaminases and induced more intense histological improvement than other treatments. This superior therapeutic effect was associated with upregulation of cytoprotective molecules including IGF-I and EGF, lower expression of proinflammatory cytokines, IL-1b and TNFα, and decreased granzyme B levels. In AdCD40L-induced lethal fulminant hepatitis, EPCCT-1 also exceeded other cell therapies in attenuating the expression of proinflammatory mediators and hepatic injury enabling 35.7% survival while mortality was 100% in the other treatment groups.nnnCONCLUSIONSnGenetic engineering of EPC to overexpress CT-1 enhances the hepatoprotective properties of EPC and constitutes a therapy that deserves consideration for acute liver failure.

Alpha-V-beta-3• integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment

We previously reported that systemic injection of recombinant adenovirus resulted in a rim of gene transduction around experimental liver tumor nodules. This zone of higher infection is dependent on the alpha(v)beta(3) integrin, acting as an adenovirus internalization receptor, which is overexpressed in tissues surrounding liver metastases. When a recombinant adenovirus encoding interleukin-12 (AdCMVIL-12) is given into a subcutaneous tumor nodule in mice also bearing concomitant liver tumors, a fraction of AdCMVIL-12 reaches the systemic circulation and infects liver tissue, especially at the malignant/healthy tissue interface. As a result of the expression at this location of the interleukin-12 transgenes, VCAM-1 is induced on vessel cells and mediates the recruitment of adoptively transferred anti-tumor cytolytic T-lymphocytes. These studies provide mechanistic explanations for the potent therapeutic synergy observed between interleukin-12 gene transfer and adoptive T-cell therapy.

Optimization of the ‘tet on’ system to regulate interleukin 12 expression in the liver for the treatment of hepatic tumors

Interleukin 12 (IL-12) is a potent antitumoral cytokine, but it can be toxic at high doses. Therapy of liver tumors might benefit from the use of vectors enabling tight control of IL-12 expression in hepatic tissue for long periods of time. To this aim, we have improved the Tet-on system by modifying the minimal region of the inducible promoter and adjusting the level of the trans-activator using liver-specific promoters with graded activities. The resulting vectors allowed hepato-specific gene regulation with lower basal activity and higher inducibility compared with the original system in the absence of repressor molecules. The basal and final protein levels depend on the strength of the promoter that directs the transcripcional activator as well as the relative orientation of the two genes in the same plasmid. We have selected the construct combining minimal leakage with higher level of induced gene expression to regulate IL-12 after DNA transfer to mouse liver. Administration of doxycycline (Dox) enhanced IL-12 expression in a dose-dependent manner, whereas it was undetectable in serum in the noninduced state. Gene activation could be repeated several times, and sustained levels of IL-12 were achieved by daily administration of Dox. The antitumor effect of IL-12 was evaluated in a mouse model of metastatic colon cancer to the liver. Complete eradication of liver metastasis and prolonged survival was observed in all mice receiving Dox for 10 days. These data demonstrate the potential of a naked DNA gene therapy strategy to achieve tight control of IL-12 within the liver for the treatment of cancer.

547. Improvement of Liver Gene Transfer by First and Third Generation Adenoviral Vectors

Recombinant adenoviruses are among the most extensively used vectors in gene therapy studies. To optimize adenovirus-mediated liver transduction we have studied the effect of the route of administration using first and third generation adenovirus vectors. We compared the local delivery of those vectors, using direct intrahepatic injection (IH), and the systemic administration via tail vein (IV). IH injection of first generation adenovirus resulted in a three-fold increase transgene expression when a high dose of virus was administered. More importantly when a relatively low dose of virus was used, IV administration resulted in no detectable protein expression while IH administration resulted in high levels of protein in serum. Significant differences in the kinetic and location of protein expression were observed depending on the route, IH injection resulted in a faster protein expression which localized to the site of injection, compared to widespread liver expression following IV administration. A 3 fold increase in the level of protein expression was also obtained after IH injection of a helper-dependent adenovirus expressing hIL-12 (HD-hIL12). Furthermore, IH injection using a HD-Ad carrying murine IL12 (HD-mIL12) correlated with the improvement of its antitumoral efficacy in a murine tumor model. Macrophage depletion experiments using clodronate loaded-liposomes showed that IH injection partially circumvent macrophage phagocytic activity. Interestingly, the administration of empty liposomes increases transgene expression without macrophage depletion regardless of the route of administration. In summary, our study describes two ways to improve liver transduction using adenovirus-based vectors: 1, direct liver injection of the adenovirus 2, administration of empty liposomes prior to virus injection.

808. Monitoring of Transgene Expression in Cancer Patients with Positron Emission Tomography (PET)

Background & Goal: Clinical development of gene therapy is hindered by the lack of tools that may help evaluating the intensity and duration of transgene expression. In animal models, PET imaging using a fluorine-18 labeled penciclovir analog (18FHBG) as a radiotracer can monitor in vivo the expression of thymidine kinase, a viral enzyme used for genetic prodrug activation therapy. We have explored this strategy in patients with liver cancer.