Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer: Implications for the clinical management of intervertebral disc disorders

Seong Hwan Moon, Lars G. Gilbertson, Kotaro Nishida, Mark Knaub, Thomas Muzzonigro, Paul D. Robbins, Christopher H Evans, James D. Kang

Research output: Contribution to journalArticle

93 Citations (Scopus)

Abstract

Study Design. Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. Objectives. To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. Summary of Background Data. Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. Methods. Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-β-galactosidase (X-Gal) staining and luciferase assay. Results. Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. Conclusions. In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genesefficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells (using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.

Original languageEnglish (US)
Pages (from-to)2573-2579
Number of pages7
JournalSpine
Volume25
Issue number20
DOIs
StatePublished - Oct 15 2000
Externally publishedYes

Fingerprint

Intervertebral Disc
Adenoviridae
Luciferases
Genes
Intervertebral Disc Degeneration
Genetic Therapy
Cultured Cells
Galactosidases
Transfer Factor
Gene Transfer Techniques
Lac Operon
Aptitude
Proteoglycans
Transgenes
Intercellular Signaling Peptides and Proteins
Cell Culture Techniques
Staining and Labeling
Rabbits

Keywords

  • Adenovirus
  • Degenerative disc disease
  • Human cells
  • Intervertebral disc
  • Intradiscal gene therapy

ASJC Scopus subject areas

  • Physiology
  • Clinical Neurology
  • Orthopedics and Sports Medicine

Cite this

Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer : Implications for the clinical management of intervertebral disc disorders. / Moon, Seong Hwan; Gilbertson, Lars G.; Nishida, Kotaro; Knaub, Mark; Muzzonigro, Thomas; Robbins, Paul D.; Evans, Christopher H; Kang, James D.

In: Spine, Vol. 25, No. 20, 15.10.2000, p. 2573-2579.

Research output: Contribution to journalArticle

Moon, Seong Hwan ; Gilbertson, Lars G. ; Nishida, Kotaro ; Knaub, Mark ; Muzzonigro, Thomas ; Robbins, Paul D. ; Evans, Christopher H ; Kang, James D. / Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer : Implications for the clinical management of intervertebral disc disorders. In: Spine. 2000 ; Vol. 25, No. 20. pp. 2573-2579.
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abstract = "Study Design. Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. Objectives. To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. Summary of Background Data. Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. Methods. Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-β-galactosidase (X-Gal) staining and luciferase assay. Results. Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100{\%} of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. Conclusions. In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genesefficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells (using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.",
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AU - Nishida, Kotaro

AU - Knaub, Mark

AU - Muzzonigro, Thomas

AU - Robbins, Paul D.

AU - Evans, Christopher H

AU - Kang, James D.

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N2 - Study Design. Human intervertebral disc cells were cultured in monolayer and treated with adenovirus-containing marker genes to determine the susceptibility of the cells to adenovirus-mediated gene transfer. Objectives. To test the efficacy of the adenovirus-mediated gene transfer technique for transferring exogenous genes to human intervertebral disc cells in vitro. Summary of Background Data. Upregulated proteoglycan synthesis after direct in vivo adenovirus-mediated transfer of growth factor genes to the rabbit intervertebral disc has previously been reported. Before contemplating extending this approach to the treatment of human disc disease, it is necessary to demonstrate that human intervertebral disc cells are indeed susceptible to adenovirus-mediated gene transduction. Methods. Human intervertebral disc cells were isolated from disc tissue obtained from 15 patients during surgical disc procedures. The cells were cultured in monolayer and treated with saline containing five different doses of adenovirus carrying the lacZ gene (Ad/CMV-lacZ), saline containing adenovirus carrying the luciferase gene (Ad/CMV-luciferase), or saline alone. Transgene expression was analyzed by 5-bromo-4-chloro-3-indolyl-β-galactosidase (X-Gal) staining and luciferase assay. Results. Adenovirus efficiently transferred lacZ and luciferase marker genes to cells from degenerated discs as well as to cells from nondegenerated discs. A minimum dose of 150 MOI Ad/CMV-lacZ was found to be sufficient to achieve transduction of approximately 100% of disc cells-regardless of patient age, sex, surgical indication, disc level, and degeneration grade. No statistically significant difference in the luciferase activities could be detected in disc cell cultures from degenerated and nondegenerated discs treated with Ad/CMV-luciferase. Conclusions. In vitro transducibility of human intervertebral disc cells by adenovirus is relatively insensitive to disc degeneration grade. Because the rate-limiting step for successful gene therapy is the ability to transfer genesefficiently to the target tissue, the achievement of efficient gene transfer to human intervertebral disc cells (using a direct, adenovirus-mediated approach) is an important and necessary step in the development of gene therapy strategies for the management of human intervertebral disc disorders.

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KW - Degenerative disc disease

KW - Human cells

KW - Intervertebral disc

KW - Intradiscal gene therapy

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