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Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_III | Pages 325 - 326
1 Nov 2002
Le Maitre CL Rajpura A Watkins A Watkins W Staley W Ross R Knight M Freemont AJ Hoyland. JA
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Background: Current treatments for Low back pain (LBP) are often empirical and few directed at the underlying disorder, altered discal cell metabolism, which precipitates the problem. The use of gene therapy to manipulate discal metabolism to treat LBP is an interesting possibility. The Intervertebral disc (IVD) is a therapeutic target in LBP, and one approach to gene therapy would be to isolate IVD chondrocytes (IVDC) and transfer genes Ex Vivo into these cells. Subsequent reinjection of these genetically altered cells into the lumbar IVD, would permit the expression of the trans-gene in vivo, generating the therapeutic protein within the IVD.

Methods: To test the viability of this approach, we isolated human IVDC from patients undergoing surgery, grew them Ex vivo and transfected them with the marker gene LacZ, using an adenovirus vector and the CMV promoter. Expression of the gene was then measured using X-gal staining for the gene product ~-galactosidase.

Results: IVDC infected with adenovirus/CMV-LacZ showed maximal LacZ expression 2 days post infection, with almost 50% of cells displaying X-gal positivity within monolayer cultures and 100% infection within alginate culture, gene expression was maintained up to 4 weeks and control cultures showed no LacZ expression.

Conclusion: This study shows that human IVDC can be transfected with a foreign gene using the adenovirus vector. The gene transduction of a therapeutic gene into IVDC, could provide long lasting effect. In addition the use of inducible promoters could allow for the autoregulation of gene expression.


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_II | Pages 142 - 142
1 Jul 2002
Le Maitre C Rajpura A Staley W Byers R Knight M Ross R Freemont A Hoyland J
Full Access

Background: Low back pain (LBP) is a major cause of disability. However, current treatments are often empirical and few are directed at the underlying disorder, altered discal cell metabolism, which precipitates the problem. The use of gene therapy to manipulate discal metabolism to treat LBP is an interesting possibility. The Intervertebral disc (IVD) is a therapeutic target in LBP, and one approach to gene therapy would be to isolate IVD chondrocytes (IVDC) and transfer genes ex vivo into these cells. Subsequent reinjection of these genetically altered cells into the lumbar IVD, would permit the expression of the transgene in vivo, generating the therapeutic protein within the IVD.

Methods: To test the viability of this approach, we isolated human IVDC from patients undergoing surgery, grew them ex vivo and transfected them with the marker gene LacZ, using an adenovirus vector and the CMV promoter. Expression of the gene was then measured using X-gal staining for the gene product _-galactosidase. Post infection, some cells were treated with forskolin for 24 hours to assess whether expression of the transgene could be manipulated.

Results: IVDC infected with adenovirus/CMV-LacZ showed maximal LacZ expression 2 days post infection, with almost 50% of cells displaying X-gal positivity. Cells maintained a low level of expression for the remaining 12 days of the study. Control cultures showed no LacZ expression. Cells treated with forskolin after infection with adenovirus/CMV-LacZ exhibited 4 times the level of _-galactosidase activity seen in unstimulated cultures.

Conclusion: This study shows that human IVDC can be transfected with a foreign gene using the adenovirus vector. The gene transduction of a therapeutic gene into IVDC could provide a long lasting effect. In addition, the use of inducible promoters could allow for the autoregulation of gene expression.