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 into 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 luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed “p-CMVint-lux”) showed the highest expression among several constructs tested. Dose–response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 μg of DNA at 7–14 days post-injection. Selected manipulations of the transfection process were examined for their 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 endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the 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. The simplest method of human gene therapy would be to administer purified DNA directly to tissue using methods similar to those used for other drug molecules. Manthorpe et al. have examined in detail the transfection of mouse muscle tissue after intramuscular injection of microgram amounts of purified reporter DNA. They report that the type of DNA regulatory elements as well the dosage and the time after injection are critical in achieving maximal expression, and that many variations in the injection procedure do not significantly alter gene expression levels.