8:42 An in vivo study of Sox9 gene therapy for intervertebral disc degeneration

Abstract

Purpose of study: The concentration of type II collagen, an essential constituent of the healthy nucleus pulposus, declines with intervertebral disc degeneration. Interventions that stimulate type II collagen production by disc cells may represent a novel therapy for the treatment of degenerative disc disease. Recent studies have shown that Sox9 is an essential transcription factor for type II collagen synthesis, and it has been termed a “master regulator” of chondrocyte phenotype. The goals of this study were to assess the effects of delivering a gene expressing Sox9 to cultured rabbit chondrocytes and in vivo in rabbit intervertebral discs.Methods used: Adenoviral vectors expressing Sox9 (AdSox9) and green fluorescent protein were constructed using the AdEasy system. Isolated New Zealand rabbit intervertebral disc cells were infected with the vectors. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical analysis were performed to document increased type II collagen expression. After an anterior transabdominal approach, the AdSox9 virus was directly injected into the lumbar discs of two live NZ rabbits. After 5 weeks, rabbits were sacrificed, and discs were evaluated with magnetic resonance imaging (MRI) and histological examination.of findings: The AdSox9 virus efficiently transduced rabbit disc cells (greater than 60%). Western blot analysis confirmed increased Sox9 production. Using a luciferase reporter gene driven by type II collagen promoter, we demonstrated that Sox9 induced Pro-alphaI(II) mRNA expression through interaction with its promoter. Sox9-mediated type II collagen production was detected using RT-PCR and immunohistochemical staining. In the rabbit model at 5 weeks postinjection, control discs showed degenerative changes consistent with annulotomy-induced degeneration. In the nucleus, cells became spindle-shaped fibrocytes and the extracellular matrix assumed a fibrous appearance. However, the disc injected with AdSox9 retained the chondroid appearance of normal nucleus pulposus and the cells maintained a chondrocytic phenotype. MRI demonstrated lesser grades of disc degeneration in the Sox9-treated discs.Relationship between findings and existing knowledge: Successful adenovirus-mediated transduction of Sox9 transcription factor with stimulation of type II collagen was demonstrated in rabbit disc cells. In an annulotomy model of disc degeneration, in vivo transduction with exogenous Sox9 decreased disc degeneration.Overall significance of findings: These findings suggest a potential role for Sox9 gene therapy in the treatment of degenerative disc disease.Disclosures: No disclosures.Conflict of interest: No conflicts.