An Improved Plasmid DNA Expression Vector for Direct Injection into Skeletal Muscle

Abstract

In previous work, the direct injection of 50 μg of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. 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, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 μg 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 modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications. The direct injection of plasmid DNA into mouse skeletal muscle was used as an in vivo assay platform to evaluate vector design improvements. Preexisting plasmid DNA vectors were systematically altered and tested, resulting in the production of a new gene therapy vector that can express up to 1 μg of transgene product after a single injection into muscle. The new vector, termed VR1255, represents a significant improvement over previously generated plasmid DNA expression vectors.