Biologic response of human intervertebral disc cells to gene therapy cocktail

seonghwan moon, Kotaro Nishida, Lars G. Gilbertson, Hwan Mo Lee, Hyang Kim, Ronald A. Hall, Paul D. Robbins, James D. Kang

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Study Design. In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs. Objective. To examine the biologic effect of "cocktail" therapeutic gene transfer to human IVD cells in three-dimensional cultures. Summary of Background Data. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted. Methods. Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immunosorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads. Results. Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control. Conclusion. Combination or "cocktail" gene therapy offers a promising mechanism for maximizing matrixsynthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.

Original languageEnglish
Pages (from-to)1850-1855
Number of pages6
JournalSpine
Volume33
Issue number17
DOIs
Publication statusPublished - 2008 Aug 1

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Intervertebral Disc
Genetic Therapy
Genes
Insulin-Like Growth Factor I
Proteoglycans
Adenoviridae
Transgenes
Poisons
Therapeutic Uses
Luciferases

All Science Journal Classification (ASJC) codes

  • Orthopedics and Sports Medicine
  • Clinical Neurology

Cite this

moon, S., Nishida, K., Gilbertson, L. G., Lee, H. M., Kim, H., Hall, R. A., ... Kang, J. D. (2008). Biologic response of human intervertebral disc cells to gene therapy cocktail. Spine, 33(17), 1850-1855. https://doi.org/10.1097/BRS.0b013e31817e1cd7
moon, seonghwan ; Nishida, Kotaro ; Gilbertson, Lars G. ; Lee, Hwan Mo ; Kim, Hyang ; Hall, Ronald A. ; Robbins, Paul D. ; Kang, James D. / Biologic response of human intervertebral disc cells to gene therapy cocktail. In: Spine. 2008 ; Vol. 33, No. 17. pp. 1850-1855.
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abstract = "Study Design. In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs. Objective. To examine the biologic effect of {"}cocktail{"} therapeutic gene transfer to human IVD cells in three-dimensional cultures. Summary of Background Data. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted. Methods. Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immunosorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads. Results. Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control. Conclusion. Combination or {"}cocktail{"} gene therapy offers a promising mechanism for maximizing matrixsynthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.",
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moon, S, Nishida, K, Gilbertson, LG, Lee, HM, Kim, H, Hall, RA, Robbins, PD & Kang, JD 2008, 'Biologic response of human intervertebral disc cells to gene therapy cocktail', Spine, vol. 33, no. 17, pp. 1850-1855. https://doi.org/10.1097/BRS.0b013e31817e1cd7

Biologic response of human intervertebral disc cells to gene therapy cocktail. / moon, seonghwan; Nishida, Kotaro; Gilbertson, Lars G.; Lee, Hwan Mo; Kim, Hyang; Hall, Ronald A.; Robbins, Paul D.; Kang, James D.

In: Spine, Vol. 33, No. 17, 01.08.2008, p. 1850-1855.

Research output: Contribution to journalArticle

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AU - moon, seonghwan

AU - Nishida, Kotaro

AU - Gilbertson, Lars G.

AU - Lee, Hwan Mo

AU - Kim, Hyang

AU - Hall, Ronald A.

AU - Robbins, Paul D.

AU - Kang, James D.

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N2 - Study Design. In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs. Objective. To examine the biologic effect of "cocktail" therapeutic gene transfer to human IVD cells in three-dimensional cultures. Summary of Background Data. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted. Methods. Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immunosorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads. Results. Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control. Conclusion. Combination or "cocktail" gene therapy offers a promising mechanism for maximizing matrixsynthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.

AB - Study Design. In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs. Objective. To examine the biologic effect of "cocktail" therapeutic gene transfer to human IVD cells in three-dimensional cultures. Summary of Background Data. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted. Methods. Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immunosorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads. Results. Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control. Conclusion. Combination or "cocktail" gene therapy offers a promising mechanism for maximizing matrixsynthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.

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moon S, Nishida K, Gilbertson LG, Lee HM, Kim H, Hall RA et al. Biologic response of human intervertebral disc cells to gene therapy cocktail. Spine. 2008 Aug 1;33(17):1850-1855. https://doi.org/10.1097/BRS.0b013e31817e1cd7