Suicide gene approach using a dual-expression lentiviral vector to enhance the safety of ex vivo gene therapy for bone repair

摘要

'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, δtk (LV-δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 μgml-1 in MBMCs vs 90-95% cell death at 5 days by 0.1 μgml-1 of GCV in W-20-17 cells). Implantation of LV-δtk-T2A-BMP-2 transduced MBMCs healed a 2mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.