568. Ex Vivo Mesenchymal Stem Cell Therapy with rAAV2 Encoding BMP-2 Enhances Bone Regeneration in Ovariectomized Mouse Model

Abstract

Mesenchymal stem cells (MSC) represent an ideal source for ex- vivo therapy for treatment of genetic, age-related, and segmental bone defects to increase bone mass. Recent studies have demonstrated that pluripotent MSC can home to the bone marrow and participate in new bone formation, raising the possibility that MSC-based therapies may be developed for effective intervention of bone loss. The pluripotent property of MSC necessitates selection of lineage- specific cells or restricted expression of therapeutic genes into committed progenitors with specific phenotypes for regulated gene therapy. An ideal approach to utilize them is by ex vivo gene therapy for osteopenia by modulating the MSC to function as a continuous source for providing progeny osteoblasts by retaining the capacity of self-renewal and direct the differentiated early progenitors towards osteoblast lineage, which would enhance new bone formation. Our earlier studies indicated efficient transduction of MSC by rAAV2. In the present study, we determined the efficacy of MSC transduced by rAAV-bone morphogenetic protein-2 (BMP-2) to repopulate and enhance bone density in a mouse model of osteoporosis. Low- passage MSC were transduced ex vivo with rAAV2 encoding BMP-2 under the transcriptional control of collagen type 1a promoter. Following rAAV transduction, the cells were maintained in culture for 10 days without induction of differentiation by maintaining the culture with FGF2. Approximately 107 MSC were then injected to ovariectomized female C57BL/6 mice intravenously. Prior to the injections, the cells were transfected with a plasmid encoding murine a4 integrin for bone-specific homing. Groups of mice also received rAAV2-GFP transduced MSC ectopically expressing |[alpha]|4 integrin or unmodified MSC. Dual Energy X-ray absorptiometry (DEXA) analysis was performed on all the animals prior to MSC injection and periodically after the treatments. Results indicated that the group of mice transplanted with MSC expressing BMP-2 showed statistically significant (p<0.035) increase in the bone mineral density (BMD) values compared to the rAAV2-GFP transduced MSC or unmodified MSC transplanted groups. Cohorts of mice from each group were sacrificed after 5 weeks and 10 weeks for bone histomorphometry, osteoclast/osteoblast ratio and the identification of engrafted MSC. Histomorphometric analysis using trichrome stain also confirmed the DEXA data where more osteoblasts and less osteoclasts were seen in the trabecular and growth plate area ofthe bone in BMP-2 treated group compared to controls. The long term effects of this ex vivo therapy on BMD will be discussed.