In vivo delivery of MMP3-shRNA and Sox9 lentivirus cocktail enhances matrix synthesis to prevent lumbar disc degeneration

Intervertebral disc degeneration (IDD) is characterized by increased proteolytic degradation of the extracellular matrix (ECM), leading to a loss of collagen II and proteoglycan in the nucleus pulposus (NP). Although MMP3 has been reported to play a central role in disc degeneration, it is still unknown whether gene therapy targeting MMP3 can inhibit IDD.To investigate whether lentivirus-mediated MMP3 knockdown is capable of attenuating IDD. More importantly, we also explored whether combined gene therapy that simultaneously antagonizes MMP3 and overexpresses Sox9 can synergistically inhibit IDD and induce augmented matrix reconstitution in the degenerative NP.We performed direct injection of lentiviral vectors LV-MMP3-shRNA and/or LV-Sox9 into rabbit lumbar discs. The animals were scanned using magnetic resonance imaging (MRI) at 8, 12 and 24 weeks after the operation. We also evaluated the gene expression and synthesis of NP matrix components, including collagen II, aggrecan and proteoglycan.The MRI scans showed remarkable needle-puncture-induced progressive IDD in animals injected with PBS or 10^7 viral particles (VP) of the control virus. In contrast, injection of 10^7 VP of LV-MMP3-shRNA or LV-Sox9 substantially inhibited IDD. MMP3 knockdown or Sox9 overexpression stimulated collagen II and aggrecan expression, as well as proteoglycan synthesis. Notably, the injection of a cocktail of LV-MMP3-shRNA and LV-Sox9 (5 × 10^6 VP each) greatly delayed the development of IDD and induced the highest levels of collagen II and proteoglycan production, indicating a synergistic effect in ECM induction.Our results suggest that gene therapy targeting MMP3 is an efficient way to delay IDD. Combined gene therapy possesses a stronger capacity to induce matrix components in degenerative NP tissue than single-gene delivery.