766. Bone Regeneration Induced by Combining In Vivo Electroporation of an Osteogenic Gene and Human Mesenchymal Stem Cells

Abstract

Top of pageAbstract A major obstacle to be addressed in gene therapy remains finding a safe and effective gene delivery method. Recent studies indicate that in vivo electroporation is a safe, simple, and effective method. We hypothesized that combining in vivo electroporation of an osteogenic gene with implanted human mesenchymal stem cells (MSCs) would induce bone regeneration. First, in order to establish an in vivo electroporation approach, 12 ug of luciferase plasmid were injected into the thigh muscles of C3H/Hen mice and transcutaneous electric pulses were applied using an ECM 2001 Electro Cell Manipulator (BTX). A bioluminescence imaging system was used to monitor noninvasively and quantitatively luciferase activity in vivo. In the second phase, human (h)MSCs were obtained from bone marrow. To demonstrate that hMSCs implanted in vivo can be efficiently transfected using electroporation, we implanted hMSCs engineered to overexpress the GFP gene and embedded in fibrin gel into the thigh muscles of NOD/SCID mice. Three days later we injected a plasmid encoding for red fluorescent protein (pDsRed) into the implantation site and in vivo electroporation was performed. On Day 10 frozen sections of thigh muscle were prepared. In the third phase, to demonstrate bone regeneration, 2|[times]|10^6 hMSCs embedded in fibrin gel were implanted into a 2.5-mm defect created in radii of NOD/SCID mice. Three days later the defect site was injected with 12 ug of bone morphogenetic protein|[ndash]|9 plasmid and the electroporation protocol was repeated. The injury site was monitored for bone formation by using quantitative micro|[ndash]|CT analysis. We observed successful gene transfer to the skeletal muscle (Fig. 1A). Quantification of the bioluminescent signal demonstrated high levels of luciferase expression up to Day 24 (Fig. 1B). Using a fluorescence microscope we identified coexpression of the GFP and DsRed reporter genes in the implanted hMSCs (Fig. 2A). Moreover, the bone formation had led to defect closure in the radii (Fig. 2B). Our results indicate for the first time that bone in a nonhealing defect can be regenerated by the combination of in vivo electroporation of a therapeutic gene and human MSCs. Our method may pave the way for regeneration of other tissues such as cartilage and tendon.