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Anne Favard, David Bellicaud, Yannick Nochez, Christine Collin, Pierre Jean Pisella, Eric E. Gabison, Jean Christophe Pagès; Corneal Cellular And Gene Therapy: Transduction Of Mesenchymal Stem Cells With An Ecdysone Inductible Lentiviral Vector Expressing Luciferase Gene. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5149.
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Several studies, using limbal stem cells or mesenchymal stem cells (MCSs), have confirmed the usefulness of these cells to repair epithelial and stromalcorneal tissue. However the reparation process might be hampered by a strong inflammatory reaction inducing non-physiologic remodelling of healing tissue. The use of genetically modified cells with inducible gene to control the stromal reaction might offer an opportunity to improve new treatments in ophthalmology.
We aimed at transducing MSCs with an Ecdysone inducible lentivirus. To the end, we generated a conditional expression vector, expressing the luciferase under an ecdysone expression system. We have transduced MSCs and evaluated their ability to synthetize on « demand » the luciferase. Following validation of the process in cultured cells, the genetically modified cells will be evaluated in vivo, in rat.
Using an in vitro recombination system, we generated a lentiviral vector containing the luciferase as transgene, placed under an Ecdysone promoter (« E/Luc »). To obtain an effective control of the luciferase, we generated a second vector containing the RXR gene and VgEcR gene under a constitutive promoter (« V/R »). Viral recombinant vectors were produced through 293T cells. MSCs were first transduced by the « V/R » vectors followed by the "E/Luc" lentiviruses. At each step, we have tested the transduction by PCR. Finally, we have tested the responsiveness of the Ecdysone system in MSCs by adding Ponasterone and enzymatic activity of the Luc gene. Preliminary experiments using GFP-labeled MSC injected in normal rat corneal stroma allowed their detection ex-vivo after one month without any loss of transparency.
The expression of the luciferase, using the above conditional expression vectors, has been validated in vitro. We predict that it could be applied to follow in vivo MSCs after their corneal engraftment. Upon validation of the system, the use of inducible gene expression could be applied to pathological wound-healing in a Sprague Dawley rat alkali burn injury model.
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