title: Biologic response of human intervertebral disc cells to gene therapy cocktail.
abstract: METHODS
  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.
  
  
  BACKGROUND
  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-beta1 gene (Ad/TGF-beta1), 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-beta1, 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-beta1, 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-beta1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Seong-Hwan
authors.initials: SH
authors.lastname: Moon
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
authors.forename: Lars G
authors.initials: LG
authors.lastname: Gilbertson
authors.forename: Hwan-Mo
authors.initials: HM
authors.lastname: Lee
authors.forename: Hyang
authors.initials: H
authors.lastname: Kim
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Hall
authors.forename: Paul D
authors.initials: PD
authors.lastname: Robbins
authors.forename: James D
authors.initials: JD
authors.lastname: Kang
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000328:Adult; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000502:physiology*; D008875:Middle Aged; D011509:Proteoglycans / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011509:Proteoglycans