Ovine Pilot Study Utilizing PMIS-miR-200a DNA Plasmid Gene Therapy for Regenerative Cranioplasty

Background: Reliable autologous regeneration of cranial bone remains an important unmet objective in craniomaxillofacial reconstruction. MicroRNA (miR)-based gene therapy, including inhibition of specific miRs, may provide a regenerative strategy. Methods: Two young adult male sheep were used to evaluate cranial bone regeneration using a plasmid-based microRNA inhibitor system ( PMIS ) targeting miR-200a . Variables included collagen matrix versus decellularized bone scaffolds and naked plasmid DNA versus PEG-peptide nanoparticle delivery. Results: Gross examination at 6 weeks demonstrated osseous regeneration in both 15 mm circular full-thickness calvarial defects treated with PMIS-miR-200a delivered on either a collagen sponge or a decellularized bone scaffold. Microcomputed tomography (µCT) and histologic analyses demonstrated new bone formation in both defects. In the second sheep, the 15 mm defect treated with PMIS-miR-200a delivered by PEG-peptide nanoparticles on a collagen sponge exhibited greater new bone fill than the empty-vector control defect. A larger 30 × 15 mm defect treated with PMIS-miR-200a delivered by the same nanoparticle system on a collagen sponge demonstrated peripheral new bone formation at 12 weeks. Conclusion: These pilot findings provide preliminary evidence that PMIS-miR-200a gene therapy can promote cranial bone regeneration in an ovine model. Adequately powered studies are required to optimize dose, scaffold, delivery system, and follow-up interval and to establish safety, efficacy, and mechanistic target engagement.