AN IMPROVED PLASMID DNA EXPRESSION VECTOR FOR DIRECT-INJECTION INTO SKELETAL-MUSCLE

In previous work, the direct injection of 50 mu g of a plasmid DNA vec tor encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days post injection, In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences, Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay, In general, modific ations to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect, However, modifications to the polyadenylation and transcriptional termination sequences, plas mid backbone elements, and the luciferase gene itself each increased l uciferase expression levels, The best-expressing vector, designated VR 1255, contained a combination of these incrementally beneficial change s, A single intramuscular injection of 50 mu g of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modificati ons to the luciferase gene) and 154-fold higher than a commercially av ailable luciferase expression vector, Thus, VR1255 represents an impro ved plasmid DNA vector that may be useful for gene therapy application s.