Gene Therapy for New Bone Formation Using Adeno-Associated Viral Bone Morphogenetic Protein-2 Vectors

Introduction: Delivery of bone morphogenetic proteins (BMP) via a gene therapy approach for in vivo bone regeneration has been reported. The most commonly used delivery vector is an adenovirus because of their high transduction rates, high BMP production titres, and that both replicating and non-replicating cells can become infected. However their major disadvantage is their high immunogenicity in the host. This has been shown to significantly limit the duration of BMP production in immunocompetent mice, and will likely limit their applicability to humans. Adeno-associated virus (AAV) is a replication-defective virus without any association with immunogenicity and human disease. This study was conducted to investigate whether orthotopic new bone formation could be induced by in vivo gene therapy using AAV based bone morphogenetic protein-2 (BMP2) vectors.

Methodology and Results: To test the feasibility of this approach, we constructed an AAV vector carrying human BMP2 gene. Mouse myoblast cells (C2C12) transduced with this vector could produce and secrete biologically active BMP2 protein and induce osteogenic activity, which was confirmed by ELISA and alkaline phosphatase activity assay. For in vivo study, AAV-BMP2 vectors were directly injected into the hind limb muscle of immuno-competent Sprague-Dawley rats. Significant new bone under X-ray films could be detected as early as three weeks post-injection. The ossification tissue was further examined by histological and immunohistochemical analysis. The results not only suggested that new bone was formed through endochondral mechanism, but also demonstrated that AAV could mediate long-term transduction for BMP2 expression.

Conclusion: This study is, to our knowledge, the first to establish the feasibility of AAV-based BMP2 gene therapy for endochondral ossification in immuno-competent animals, and provides a safe solution for application of such techniques in human patients.