Enhancer and Promoter Chimeras in Plasmids Designed for Intramuscular Injection: A Comparative In Vivo and In Vitro Study

Abstract

Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.

Overview summary

Combinations of muscle-specific gene regulatory elements and the CMV enhancer/promoter were tested for their ability to increase plasmid-based gene expression after intramuscular plasmid injection and after transfection of muscle myoblast or nonmuscle cells in vitro. Expression levels from these chimeric plasmids ranged from insignificant to twice that of the parental plasmid. Most regulatory elements, while not significantly enhancing expression of plasmid injected in muscle, did exhibit significant modulation of expression in cultured cells. The results indicate that the goal of identifying high-expression plasmids for use in intramuscular injections is best achieved by in vivo testing.