|Draghia-Akli, Ruxandra - ADVISYS, INC.|
|Hill, Leigh-Anne - ADVISYS, INC.|
|Malone, Brandon - ADVISYS, INC.|
Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 25, 2002
Publication Date: January 2, 2003
Citation: Draghia-Akli, R., Ellis, K.M., Hill, L.A., Malone, P.B., Fiorotto, M.L. 2003. High-efficiency growth hormone-releasing hormone plasmid vector administration into skeletal muscle mediated by electroporation in pigs. Journal of The Federation of American Societies for Experimental Biology. 17(3):526-528. Interpretive Summary: We have shown that when DNA is injected into a muscle, it is taken up by the muscle fibers where it will express the protein that the gene encodes. The studies described demonstrated that the amount of DNA required to produce a biologically significant result can be reduced by improving the efficiency with which it is taken up by the fibers in pigs. Efficiency was improved when the DNA injection was followed by electroporation, in which a very minute electrical current is pulsed between needles injected into the muscle immediately following the DNA injection. Efficiency was also influenced by the age of the animal and the muscle injected. We further demonstrated that if the gene for growth hormone releasing hormone (GHRH) was injected using these optimized parameters, after 100 days as little as 0.1 mg of DNA resulted in pigs that were 9% heavier and leaner than controls and there were no associated adverse side-effects. This study represents a remarkable improvement in the use of a gene transfer approach that can be used to improve animal health and growth with great potential for therapeutic uses in humans.
Technical Abstract: We report here a very efficient method for the "in vivo" transfer of therapeutic plasmid DNA into porcine muscle fibers by using electric pulses of low field intensity. We evaluated delivery of 0.1-3 mg of plasmid vectors that encode reporter secreted-embryonic alkaline phosphatase (SEAP) or therapeutic growth hormone releasing hormone (GHRH). Reporter gene studies showed that internal needle electrodes give a 25-fold increase in expression levels compared with caliper electrodes in skeletal muscle in swine. Dose and time courses were performed. Pigs injected with 0.1 mg plasmid had significantly greater weight gain than controls over 53 days (22.4 +/- 0.8 kg vs. 19.7 +/- 0.03 kg, respectively; P<0.01). The group treated with GHRH-expressing plasmid at 14 days of age demonstrated greater weight gain than controls at every time point (25.8 +/- 1.5 kg vs. 19.7 +/- 0.03 kg; P<0.01). Body composition studies by dual X-ray absorbitometry showed a 22% decrease in fat deposition (P<0.05) and a 10% increase in bone mineral density (P<0.004). Our studies demonstrate that by optimizing the electroporation method, favorable physiological changes, such as enhanced weight gain and improved body composition, can be obtained at extremely low plasmid doses in a larger mammal.