Multi-Target Gene Therapy for Osteoarthritis: Dual-Axis Modeling and In Silico Validation

Objective Osteoarthritis (OA) remains therapeutically intractable, with no approved disease-modifying OA drug. This study proposes and tests an in silico Dual-Axis Model in which inflammatory signaling and mechano-structural degradation form a coupled system requiring simultaneous intervention. Design: We integrated structural analysis of therapeutic proteins (IL-1Ra, SOX9, IGF-1), published structural data for ADAMTS-5/GLPG1972 (PDB: 6YJM), Monte Carlo network perturbation modeling (n = 1,000 iterations), Reynolds-algorithm shRNA design with BLAST off-target screening, and human-canine sequence homology assessment. An ECM Recovery Score quantified simulated matrix restoration across control, IL-1Ra-only, SOX9-only, and multi-target intervention scenarios. Results Multi-target intervention achieved a higher simulated ECM Recovery Score (76.2 ± 8.3) than control (43.6 ± 12.1), IL-1Ra-only (52.3 ± 10.8), or SOX9-only (58.7 ± 9.4) conditions (p < 0.001 for model-output comparisons; Cohen's d = 3.16 vs control). Baseline-corrected additivity predicted 67.4, whereas the observed multi-target score was 76.2 (synergy index 1.13). Structural analysis of GLPG1972 binding generated a testable "Exosite Bypass" hypothesis in which catalytic-site blockade may not prevent substrate pre-positioning through exosite-mediated aggrecan binding. In silico screened shRNA candidates for ADAMTS-5 and MMP-13 achieved Reynolds scores of 8–9/9 with minimal predicted off-target risk. Human-canine homology analysis showed high conservation of functional domains (mean 95%). Conclusions The findings provide hypothesis-generating in silico evidence that simultaneous anti-inflammatory, pro-anabolic, and anti-catabolic targeting may overcome limitations of single-axis OA interventions. The proposed AAV system should be treated as a conceptual design requiring payload-size optimization, experimental knockdown validation, and in vivo safety/efficacy testing.