Expression of Heterodimeric Bone Morphogenetic Protein by Combination Gene Therapy Increases In Vitro Osteogenic Potency and Enhances Spine Fusion

Introduction: While recombinant bone morphogenetic proteins (BMPs) are homodimers, consisting of two identical monomeric proteins, BMP heterodimers resulting from the co-expression of two different BMP genes may be more potent.Aim: To test the osteogenic potency of BMP2/7 heterodimer using combination BMP2 and BMP7 gene therapy in vitro and in vivo in a rat spine fusion model.

Methods: A549 bronchial epithelial cells were treated with adenovirus (Ad) vectors encoding BMP2 (AdBMP2), or BMP7 (AdBMP7) or a combination of the two. BMP2/7 heterodimer was detected by immunoprecipitation with anti-BMP7 antibody followed by enzyme-linked immunosorbant assay (ELISA) for BMP2. To test in vitro osteogenic activity, C2C12 myoblastic cells were stimulated with supernatants of A549 cells containing BMP2, BMP7 or BMP2/7 proteins for 1 week, and osteoblastic differentiation assessed. To test in vivo bone formation, rats underwent single level posterior spine fusion (lumbar levels 4 and 5) with implantation of freeze-dried allograft bone and hemostatic cellulose sponge soaked with AdBMP2 (n=8, 1.5x10^10 particle units [pu]) or AdBMP7 (n=10, same dose), or a combination of the two (n=9, 0.75x10^10 pu each vector). Fusion rates were assessed at 8 week post-surgery by x-rays and manual palpation in a blinded fashion. For both in vitro and in vivo studies, AdNull (with no transgene) and saline were used as negative controls.

Results: Only A549 cells treated with both AdBMP2 and AdBMP7 expressed BMP2/7 heterodimer. In C2C12 cells, BMP2/7 heterodimer induced a 200-fold higher alkaline phosphatase activity and 10-fold higher osteocalcin expression than either homodimer (p<0 .05, all comparisons). Moreover, spines treated with AdBMP2 and AdBMP7 had a bilateral fusion rate of 89% as assessed by either parameter. This was 9-fold greater than treatment alone (10% fusion) 7-fold (12.5% fusion, p<0.05 No fusions were observed in the negative control spines.

Conclusion and Discussion: Our findings suggest that BMP2/7 heterodimers generated by combination AdBMP2 and AdBMP7 gene therapy have more osteogenic potency in vitro and in vivo, than either BMP2 or BMP7 homodimer. This combination gene therapy strategy may lead to the development of clinically efficient BMP therapies for spine fusion.