Control and augmentation of long-term plasmid transgene expression in vivo in murine muscle tissue and ex vivo in patient mesenchymal tissue

Abstract

Purpose: 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.

Figure 1

Plasmid Constructs. Schematic representations of firefly luciferase coding constructs used in this study. Promoters are outlined in grey.

Figure 2

Duration of plasmid-based CMV promoter activity in vivo in liver and muscle. pCMV-luc was delivered to liver and thigh muscle (n = 10) by electroporation and luminescence analysed in vivo over time using IVIS live whole body imaging. CMV activity in liver is initially high but reduced to background level by day 14. A gross reduction in expression over time was not apparent in muscle.

Figure 3

Adenoviral vector and electroporated plasmid-mediated transgene expression in vivo. Thighs of mice were in vivo administered pCMV-luc by electroporation or AdCMV-luc (n = 5). Luminescence was analysed in vivo over time using IVIS live whole body imaging. (a) Magnitude of electroporated plasmid-mediated luciferase expression compared with Ad-mediated expression (day 18 and day 1, resp.). (b) Plasmid versus Ad transgene expression over time. Although luminescence was higher with Ad delivery, it was subject to rapid silencing to background levels by day 21, unlike electroporated plasmid.

Figure 4

Long-term controllable transgene expression using doxycycline-inducible plasmid. pGTRTL was delivered to quadriceps muscle (n = 22) by electroporation and luminescence analysed in vivo over time using IVIS live whole body imaging. Doxycycline was administered from days 8–11 and days 105–118 inclusively (red bar). Efficient, reproducible, high-magnitude expression was noted on induction with doxycycline.

Figure 5

Murine ex vivo tissue survival in DMEM over time. Murine thigh muscle was harvested immediately after culling, immersed in DMEM, and stored at 37° centigrade. Cell survival was assessed at 0, 1, 4, and 24 hours with 38% viability at 24 hours when compared to 0 hours.

Figure 6

Human musculoskeletal tissue electrotransfected ex vivo with either pCMV-luc or pGTRTL. Expression was observed in all samples. Levels of expression for the “tet-on” plasmid were significantly increased in the presence of doxycycline.