Louis C. Smith

Optimization of Electroporation Parameters for the Intramuscular Delivery of Plasmids in Pigs

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.

Growth hormone releasing hormone plasmid supplementation, a potential treatment for cancer cachexia, does not increase tumor growth in nude mice

Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a muscle-specific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors (P<.02), a 48% increase in white blood cells (P<.025) and a 300% increase in monocyte count (P<.0001), as well as an increase in the frequency of activated CD3+ and CD4+ cells in the tumors, compared to tumors of control animals. No adverse effects were observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the GHRH–GH–IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia in humans.

Effects of plasmid-mediated growth hormone-releasing hormone supplementation on LL-2 adenocarcinoma in mice

This study was designed to measure the effects of plasmid growth hormone-releasing hormone (GHRH) supplementation on LL-2 (Lewis lung adenocarcinoma) tumor-bearing immunocompetent mice. Male and female mice (n = 20/group/experiment) received 2.5 x 10(6) LL-2 cells in the left flank. One day later, we injected the mice intramuscularly with 20 micro g of a myogenic plasmid, pSP-hGHRH or pSP-betagal, as a control. Mean serum IGF-I was significantly higher in treated animals versus controls (P < 0.05). Male and female mice constitutively expressing GHRH exhibited a decline in tumor growth rate relative to controls (20% for males, P < 0.03, and 11% for females, P < 0.13). Histopathological analysis revealed that treated animals were less likely to develop lung metastases than controls (11%) and had no alternate-organ metastases. The number of metastases/lung was reduced by 57% in female mice with GHRH treatment (P < 0.006). When tumor size exceeded 8% of body weight, GHRH-treated mice showed normal urea, creatinine, and kidney volume, while controls displayed signs of renal insufficiency. This study provides evidence that with plasmid-mediated GHRH supplementation in tumor-bearing mice, tumor growth rate is not increased but is actually attenuated.