Vega, Rodolfo de la

When long bones fracture, healing occurs spontaneously in about 90% of cases, while the remaining 10% result in non-union, presenting significant clinical challenges. The standard treatment for non-unions is autologous bone grafting, but this approach has limitations, including high complication rates, donor site morbidity, and limited graft availability. An alternative strategy involves using growth factors to promote bone regeneration, with recombinant bone morphogenetic protein-2 (rBMP-2) being the most common. Although effective, rBMP-2 requires supra-physiological doses that cause inflammation, poor bone quality, and severe side effects. Our previous work suggests that genetic delivery of BMP-2 may offer more controlled healing. We aim to develop a more effective therapy for non-unions using adeno-associated viral vectors. By lyophilizing the vector onto a scaffold, we also aim to increase treatment potency, reduce leakage, and improve transduction using cryoprotectant buffers.AAV6-GFP-Luciferase was used to compare the activity of AAV6 stored at −80°C versus lyophilized in cryoprotectant buffer. A rat critical-sized defect model was employed, as described in [1]. For standard storage, AAV was added to the collagen sponge before implantation into a 5mm defect. For lyophilization, AAV was mixed with cryoprotectant, loaded onto the sponge, and lyophilized. Rats were imaged at 7- and 10-days using IVIS. To assess therapeutic relevance, the experiment was repeated with AAV6-BMP2-IL1Ra, implanted into 5mm femoral defects on collagen sponges, with healing monitored by X-ray every 4 weeks (continuing through 12-weeks post-operation).The reporter and therapeutic vectors showed improved transduction efficiency with lyophilization. The reporter vector exhibited higher transgene expression at days 7 and 10. This boost in expression either remained significant through 4 weeks or continued to show better signal at that time, correlating with improved healing seen in the X-rays (Fig. 2).Preliminary data suggest that lyophilization could reduce the therapeutic dose of AAV needed for treating osseous nonunion with a bicistronic vector. Lyophilization offers various benefits: enhanced transduction efficiency, improved stability of the gene product at logistically feasible conditions, and reduced off-target effects due to less AAV leakage from the implantation site. While in vivo experiments with AAV6-BMP2-IL1Ra are ongoing, these findings warrant further investigation into lyophilized AAV therapies for this application.For any figures or tables, please contact the authors directly.