Enhanced disc regeneration through CRISPR/Cas9-mediated SOX9 and TGFβ1 coexpression in tonsil-derived mesenchymal stromal cells

Intervertebral disc (IVD) degeneration, a primary cause of chronic low back pain, currently lacks treatments that target its underlying pathological mechanisms. Tonsil-derived mesenchymal stromal cells (ToMSCs) have shown promise for IVD regeneration; however, their therapeutic potential is limited by the harsh microenvironment of degenerated discs. This study investigated whether ToMSCs engineered to co‐overexpress SOX9 and TGFβ1 using a tetracycline‐off (Tet‐off) regulatory system could enhance extracellular matrix (ECM) restoration and reduce inflammation in degenerative IVDs. We used CRISPR/Cas9 technology to generate ToMSCs that express SOX9, TGFβ1, or both factors under Tet-off regulation. Gene expression was confirmed by Western blot and qRT-PCR analyses. In vitro studies assessed chondrogenic differentiation capacity, while in vivo assessments were performed using a rat tail needle puncture model of IVD degeneration. After administering the CRISPR-engineered ToMSCs, we monitored mechanical allodynia with the von Frey test over six weeks. Therapeutic outcomes were evaluated through T2‐weighted MRI and histological analysis. In vitro experiments showed that ToMSCs co-expressing SOX9 and TGFβ1 exhibited superior chondrogenic differentiation compared to cells expressing a single factor. In vivo studies demonstrated that dual-factor expressing ToMSCs significantly improved disc hydration (as confirmed by MRI), enhanced ECM synthesis—particularly aggrecan and type II collagen—and reduced inflammation compared to single-factor treatments. These improvements were accompanied by reduced mechanical allodynia, indicating functional recovery. Our study demonstrates that ToMSCs engineered to co-express SOX9 and TGFβ1 effectively promote IVD regeneration by enhancing ECM production and reducing inflammation. This dual-factor approach represents a promising therapeutic strategy for treating degenerative disc disease and warrants further investigation for clinical application.