Gene Therapy for Bone Regeneration using Non-viral BMP Gene Expression Vector and in vivo Electroporation

Abstract

It is well known that bone morphogenetic protein (BMP) induces ectopic bone formation when the recombinant protein or BMP gene is transferred into the skeletal muscle. We developed a novel method for BMP gene transfer, which is combination with nonviral BMP gene expression vector and in vivo electroporation. Then, we applied this method to transfer BMP-2 gene into the skeletal muscles of rats and induced the ectopic bone formation in the target sites. We injected BMP gene expression vector, pCAGGS-BMP-2, in the skeletal muscles of rats and immediately electroporated under the conditions of 100 voltage, 50 msec., and 8 pulses. We found the ectopic bone formation in the skeletal muscles 21 days after BMP gene transfer. In the BMP family, BMP-2/4 or BMP-2/7 heterodimer has stronger potential for bone induction compared with BMP-2, BMP-4 or BMP-7 homodimer. Then, we constructed BMP-2/7 heterodimer produced vector: pCAGGS-BMP-2/7. It has no IRES site, therefore each of BMP-2 and BMP-7 gene expression is equal. When we injected pCAGGS-BMP-2/7 plasmid vector in the skeletal muscles and immediately performed in vivo electroporation, the ectopic bone formation was induced quickly on 10 days after gene transfer. For clinical application, we need more safe procedure on in vivo electroporation under the condition of lower voltage than 100 voltage. If we set the condition: 50 voltage and 8 pulses, the efficiency of gene transfer also reduced. But, when we induced pulse number, it recovered. We evaluated proper voltage and pulse number as the same gene transfer efficiency of 100 voltage. We tried to apply this gene transfer system for alveolar bone regeneration under the condition less 50 voltage. We often use bone prosthetic material and autogenous bone graft for alveolar bone defect caused by periodontal disease or trauma. But, these therapies sometimes have some risk for patients such as infection or fractures. Our developed gene therapy system for bone regeneration will be with more safety and with less burden on the patient.