Control and augmentation of long-term plasmid transgene expression in vivo in murine muscle tissue and ex vivo in patient mesenchymal tissue.
OBJECTIVE
In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. We aimed to assess the duration and magnitude of transene expression in vivo in mice and ex vivo in human tissues.


METHODS
Using bioluminescence imaging, plasmid and adenoviral vector-based transgene expression in murine quadriceps in vivo was examined. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue, and applied in ex vivo human tissue.


RESULTS
In vivo plasmid-based transgene expression did not silence in murine muscle, unlike in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery compared with plasmid, expression reduced over time. The inducible promoter cassette successfully regulated gene expression with maximum levels a factor of 11 greater than baseline. Expression was re-induced to a similar level on a temporal basis. Luciferase expression was readily detected ex vivo in human muscle and tendon.


CONCLUSIONS
Plasmid constructs resulted in long-term in vivo gene expression in skeletal muscle, in a controllable fashion utilising an inducible promoter in combination with oral agents. Successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.
Cork Cancer Research Centre, BioSciences Institute, University College Cork, Cork, Ireland. dimorrissey@hotmail.com
David
D
Morrissey
Jan P
JP
van Pijkeren
Simon
S
Rajendran
Sara A
SA
Collins
Garrett
G
Casey
Gerald C
GC
O'Sullivan
Mark
M
Tangney
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D018274:Electroporation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism; D049409:Luciferases, Firefly / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013997:Time Factors; D014022:Tissue Survival; D019076:Transgenes / Q000235:genetics*
D049409:Luciferases, Firefly