Potential use of Sox9 gene therapy for intervertebral degenerative disc disease.

STUDY DESIGN: A new recombinant adenoviral vector expressing Sox9, a chondrocyte-specific transcription factor, was tested in a chondroblastic cell line and primary human intervertebral disc cells in vitro. Direct infection of intervertebral disc cells then was assessed in a rabbit model. OBJECTIVES: To deliver a potentially therapeutic viral vector expressing Sox9 to degenerative human and rabbit intervertebral discs cells, and to assess the effect of Sox9 expression on Type 2 collagen production. SUMMARY OF THE BACKGROUND DATA: The concentration of competent Type 2 collagen, an essential constituent of the healthy nucleus pulposus, declines with intervertebral disc degeneration. Recent studies suggest that Sox9 upregulates Type 2 collagen production. Interventions that augment Type 2 collagen production by intervertebral disc cells may represent a novel therapeutic method for patients with degenerative disc disease. METHODS: Adenoviral delivery vectors expressing Sox9 and green fluorescent protein were constructed using the AdEasy system. The chondroblastic cell line, HTB-94, and cultured human degenerated intervertebral disc cells were infected with the vectors. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses were performed to document increased Type 2 collagen expression. The AdSox9 virus then was injected directly into the intervertebral discs of three rabbits. After 5 weeks, the injected discs were evaluated histologically. RESULTS: The AdSox9 virus efficiently transduced HTB-94 cells and degenerated human disc cells. Western blot analysis confirmed increased Sox9 production. Increased Type 2 collagen production was demonstrated in infected HTB-94 and human disc cells using both reverse transcriptase-polymerase chain reaction and immunohistochemical staining. In the rabbit model, cells infected with AdSox9 maintained a chondrocytic phenotype, and the architecture of the nucleus pulposus was preserved over a 5-week study period compared to control discs. CONCLUSIONS: A novel adenoviral vector efficiently increased Sox9 and Type 2 collagen synthesis in cultured chondroblastic cells and human degenerated disc cells. In a rabbit model, sustained Sox9 production preserved the histologic appearance of the nucleus pulposus cells in vivo. These findings suggest a potential role for Sox9 gene therapy in the treatment of human degenerative disc disease.

Induction of early transcription in one-cell mouse embryos by microinjection of the nonhistone chromosomal protein HMG-I.

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state.

In vivo gene transfer method in keratinocyte gene therapy: intradermal injection of DNA complexed with high mobility group-1 protein in rats.

In order to develop a more efficient method of introducing genes into keratinocytes in vivo, we intradermally injected DNA bound to high mobility group 1 protein, thereby taking advantages of the naked DNA and hemagglutinating virus of the Japan-liposome method reported previously. First we performed a gel mobility shift assay, which confirmed DNA binding to high mobility group 1. Then we injected beta-galactosidase expression vector complexed with high mobility group 1 into the rat skin and the activity of sample with the protein was 2-3 times higher than that without the protein as control. Semiquantification of transferred-DNA content using polymerase chain reaction and a time course of transgene expression in keratinocytes suggested that high mobility group 1 protein increased transfer of the DNA from the cytoplasm to the nucleus. Direct injection of the DNA-high-mobility-group-1 complex is a highly efficient method for introducing genes into keratinocytes in connection with gene therapy.