273. Long-Term Effects of Plasmid-Mediated Growth Hormone Releasing Hormone in Dogs

Abstract

Top of pageAbstract We have developed a capsid-modified oncolytic adenovirus (Ad) vector (Ad5/35.IR-E1A/TRAIL) that is highly efficient in the elimination of liver metastases derived from various human tumor cell lines in mouse models. However, testing for viral efficacy on established tumor cell lines and for safety in mouse models may not be predictive for the performance of these vectors in clinical trials. In contrast to cancer cell lines, the genotype and gene expression profiles of primary short term cultures more closely reflects that of actual tumors. We have therefore tested the oncolytic ability of our vector and a series of control vectors on primary ovarian and cervical cancer cultures. So far we have established two ovarian cancer cultures and three cultures of normal mesothelial cells from ascites. Ad.5/35.IR-E1A/TRAIL caused apoptosis in 100% and ~80% of ovarian cancer cells in cultures from two different patients, but did not cause cytolysis in mesothelial cells. The efficiency and specificity of oncolysis by Ad5/35.IR-E1a/TRAIL was compared to wild-type Ads. Wild-type Ad35 efficiently caused cytopathic effects in mesothelial cells and ovarian cancer cells (without the characteristic apoptotic features seen in the Ad.5/35.IR-E1A/TRAIL infected cells). Wild-type Ad5 did not efficiently lyse tumor cells, but did cause more CPE in mesothelial cells than wt Ad35, which probably reflects the differential expression of receptors for Ad5 and Ad35. We are in the process of establishing more primary cultures and the results will be reported. For safety studies, baboons were injected through the femoral vein with PBS (mock) or 1 × 1011 pfu/kg of Ad5/35.IR-E1a/TRAIL (in PBS) and blood samples were monitored for 30 days for biochemical parameters, blood cell counts, vector genome concentrations, and cytokine levels. Upon necropsy, tissue histology, presence of transgene expression, and vector genomes were analyzed in samples of brain, lymph nodes, bone marrow, heart, aorta, digestive tract, liver, pancreas, bladder, testis, muscle, spleen, lung, kidney, peripheral blood cells, and prostate. All laboratory blood test parameters were in normal ranges following injection of the oncolytic vectors. Among the cytokines, IL-6 was elevated at 6 hours after vector infusion, but reached pre-injection levels 24 hours post-infusion. We used real-time PCR for vector genomes to assess the kinetics of Ad clearance from the blood. When we compared the amount of vector DNA associated with blood cells and plasma with the total amount of injected Ad, we found that at 1 min post–infusion, ~2% of the input Ad dose is associated with blood cells, which could be due to binding to erythrocytes (Ad35 agglutinates monkey erythrocytes). Ad5/35 genomes in plasma and blood cells reached pre-injection levels by 24 hours p.i.. No histopathological changes were found in tissues analyzed 30 days post-infusion. Analysis of vector genomes and transgene expression in tissue samples is ongoing.