Adenoviral-mediated skeletal muscle transcriptional targeting using chimeric tissue-specific promoters.
BACKGROUND
Adenoviral vectors are promising tools to achieve skeletal muscle gene transfer for the treatment of peripheral ischemia. However, the use of ubiquitous viral promoters represents a major safety issue that could limit their use. Cellular regulatory sequences that allow strong and tissue-specific expression could circumvent this problem.


METHODS
Adenoviral vectors encoding the firefly luciferase under the control of the human skeletal a-actin promoter, alone or combined with the b-enolase or creatine kinase enhancer, were studied in vitro in murine C2C12 cells and in vivo in C57BL/6 mice. The expression of the reporter gene was measured in cell lysates and animal tissue homogenates. Adenoviral distribution was evaluated by PCR on DNA extracted from liver, spleen, heart and lungs.


RESULTS
Skeletal a-actin promoter-based expression cassettes follow the regulation of the endogenous skeletal a-actin gene in vitro as luciferase expression strongly increases with myoblast differentiation into myotubes. The addition of the cellular b enolase or the creatine kinase enhancer improves the specificity of the skeletal a-actin promoter in vitro as well as in vivo. When adenoviral vectors are locally injected into skeletal muscles, the chimeric promoters drive a relatively strong gene expression, ranging from 16 to 28% of the Rous sarcoma virus promoter-related expression.


CONCLUSIONS
Chimeric regulatory sequences based on the skeletal a-actin promoter are highly specific and allow transgene expression in vivo at high levels. These results indicate that expression cassettes designed for the treatment of peripheral ischemia by gene therapy can efficiently target gene expression to skeletal muscle.
Frauli
Mélanie
M
Cardiovascular Gene Therapy Laboratory, TRANSGENE S.A., Strasbourg, France.
Ribault
Sébastien
S
Neuville
Pascal
P
Augé
Fabrice
F
Calenda
Valérie
V
D000199:Actins / Q000235:genetics; D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D015870:Gene Expression; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018482:Muscle, Skeletal / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D000199:Actins; D011993:Recombinant Fusion Proteins