Increased level and duration of expression in muscle by co-expression of a transactivator using plasmid systems.
Skeletal muscle is an attractive target for gene therapies to treat either local or systemic disorders, as well as for genetic vaccination. An ideal expression system for skeletal muscle would be characterized by high level, extended duration of expression and muscle specificity. Viral promoters, such as the cytomegalovirus (CMV) promoter, produce high levels of transgene expression, which last for only a few days at high levels. Moreover, many promoters lack muscle tissue specificity. A muscle-specific skeletal alpha-actin promoter (SkA) has shown tissue specificity but lower peak activity than that of the CMV promoter in vivo. It has been reported in vitro that serum response factor (SRF) can stimulate the transcriptional activity of some muscle-specific promoters. In this study, we show that co- expression of SRF in vivo is able to up-regulate SkA promoter-driven expression about 10-fold and CMV/SkA chimeric promoter activity by five-fold in both mouse gastrocnemius and tibialis muscle. In addition, co-expression of transactivator with the CMV/SkA chimeric promoter in muscle has produced significantly enhanced duration of expression compared with that shown by the CMV promoter-driven expression system. A dominant negative mutant of SRF, SRFpm, abrogated the enhancement to SkA promoter activity, confirming the specificity of the response. Since all the known muscle-specific promoters contain SRF binding sites, this strategy for enhanced expression may apply to other muscle-specific promoters in vivo.
Li
S
S
Otolaryngology-Head and Neck Surgery, UAMS, Little Rock, AR, USA.
MacLaughlin
F C
FC
Fewell
J G
JG
Li
Y
Y
Mehta
V
V
French
M F
MF
Nordstrom
J L
JL
Coleman
M
M
Belagali
N S
NS
Schwartz
R J
RJ
Smith
L C
LC
D000199:Actins / Q000235:genetics*; D000704:Analysis of Variance; D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007273:Injections, Intramuscular; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D026362:Serum Response Factor; D013997:Time Factors; D015534:Trans-Activators; D014157:Transcription Factors
D000199:Actins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D026362:Serum Response Factor; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases