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Morrissey, D.,van Pijkeren, J.P. ,Rajendran, S.,Collins, S.,Casey, G.,O’Sullivan, G.C. ,Tangney, M.
2012
Control and augmentation of long-term plasmid transgene expression in vivo in murine muscle tissue and ex vivo in patient mesenchymal tissue
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Purpose In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. Our aim was to assess the duration and magnitude of skeletal muscle non-viral plasmid gene expression and examine the applicability of the technique in human ex-vivo tissue. Methods Using luminescent imaging, in vivo murine muscle gene expression was examined and compared to that of adenoviral gene expression. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue. This model was subsequently used to assess the application of non-viral gene therapy in ex vivo human tissue. Results Murine muscle luciferase expression did not silence over the study period, while silencing was rapid in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery, compared to plasmid delivery, silencing occurred over time with the viral construct. 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. Gene expression was achieved in ex vivo human muscle and tendon. Conclusions Plasmid constructs result in long term in vivo gene expression in skeletal muscle, while expression can be simply controlled by utilising an inducible promoter in combination with oral agents. Finally, successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time. Purpose In vivo gene therapy directed at tissues of mesenchymal origin could potentially augment healing. Our aim was to assess the duration and magnitude of skeletal muscle non-viral plasmid gene expression and examine the applicability of the technique in human ex-vivo tissue. Methods Using luminescent imaging, in vivo murine muscle gene expression was examined and compared to that of adenoviral gene expression. Temporal control was assessed using a doxycycline-inducible system. An ex vivo model was developed and optimised using murine tissue. This model was subsequently used to assess the application of non-viral gene therapy in ex vivo human tissue. Results Murine muscle luciferase expression did not silence over the study period, while silencing was rapid in liver. Although maximum luciferase expression was higher in muscle with adenoviral delivery, compared to plasmid delivery, silencing occurred over time with the viral construct. 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. Gene expression was achieved in ex vivo human muscle and tendon. Conclusions Plasmid constructs result in long term in vivo gene expression in skeletal muscle, while expression can be simply controlled by utilising an inducible promoter in combination with oral agents. Finally, successful plasmid gene transfection in human ex vivo mesenchymal tissue was demonstrated for the first time.
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