GENE-THERAPY BY INTRAMUSCULAR INJECTION OF PLASMID DNA - STUDIES ON FIREFLY LUCIFERASE GENE-EXPRESSION IN MICE

Direct injection of nonviral, covalently closed circular plasmid DNA i nto muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in lucifera se enzyme expression was noted among constructs with different regulat ory elements, among different batches of the same DNA construct, and a mong similar transfection experiments performed at different times. Th is variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitat ive experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct conta ining the human cytomegalovirus immediate-early gene promoter plus int ron A (a construct termed ''p-CMVint-lux'') showed the highest express ion among several constructs tested. Dose-response and time course ana lyses of p-CMVint-lux DNA injections showed that maximal luciferase ex pression was achieved with 25 mug of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for t heir influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endot oxin, cationic peptide, muscle stimulants or relaxants, vasoconstricto rs, metal chelators, or lysosomal lytic reagents had no significant ef fect on expression. However, linearization of the DNA, injection of th e DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.