Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium

J. Shen, N. Taylor, Linda Duncan, I. Kovesdi, J. T. Bruder, John Vincent Forrester, A. D. Dick

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Aim-To investigate the efficacy of "ex vivo" adenoviral vector mediated gene transfection of human conjunctival epithelial cell as a possible route for gene therapy for the distribution of antiinflammatory agents for the potential treatment of immune mediated ocular inflammatory disorders.

Methods-Human conjunctival cells (HCs) were cultured with various concentrations of recombinant adenoviral vectors carrying a reporter gene LacZ, GFP, or an immunomodulating cytokine vIL-10. VIL-10 in culture supernatant was detected by sandwich ELISA and biological activity was assessed by suppression of ConA stimulated splenocyte proliferation. X-gal and GFP expression was assessed by histochemistry.

Results-The extent of adenoviral vector mediated transfer of both reporter genes and vIL-10 was dose dependent. LacZ expression could be detected for at least 50 day after infection with multiple of infection (MOI) 200. Following AdCMVvIL-10 transduction, vIL-10 protein expression occurred between 4-6 days post-transduction, and was maintained at a detectable level for at least 1 month. Secreted VIL-10 showed biological activity, significantly inhibiting Con A induced splenocyte proliferation. Additionally, transfection of HCs with two Adv vectors, one carrying LacZ and the other carrying GFP, resulted in co-expression within a single cell.

Conclusion-These results confirm previous successful adenoviral vector mediated gene transfer to HCs and further show that expression can be maintained. Furthermore the data show HCs can secrete biologically active VIL-10 that could be developed as a strategy to suppress immune mediated disorders. The successful co-transduction of HCs as described for other tissues, opens avenues to develop a multiple target gene therapy locally.

Original languageEnglish
Pages (from-to)861-867
Number of pages6
JournalBritish Journal of Ophthalmology
Volume85
Issue number7
DOIs
Publication statusPublished - 2001

Keywords

  • OCULAR INFLAMMATORY DISEASE
  • COLLAGEN-INDUCED ARTHRITIS
  • VIRAL IL-10
  • TRANSGENE EXPRESSION
  • HUMAN CORNEA
  • IN-VIVO
  • THERAPY
  • MOUSE
  • DELIVERY
  • VECTOR

Cite this

Shen, J., Taylor, N., Duncan, L., Kovesdi, I., Bruder, J. T., Forrester, J. V., & Dick, A. D. (2001). Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium. British Journal of Ophthalmology, 85(7), 861-867. https://doi.org/10.1136/bjo.85.7.861

Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium. / Shen, J.; Taylor, N.; Duncan, Linda; Kovesdi, I.; Bruder, J. T.; Forrester, John Vincent; Dick, A. D.

In: British Journal of Ophthalmology, Vol. 85, No. 7, 2001, p. 861-867.

Research output: Contribution to journalArticle

Shen, J, Taylor, N, Duncan, L, Kovesdi, I, Bruder, JT, Forrester, JV & Dick, AD 2001, 'Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium', British Journal of Ophthalmology, vol. 85, no. 7, pp. 861-867. https://doi.org/10.1136/bjo.85.7.861
Shen, J. ; Taylor, N. ; Duncan, Linda ; Kovesdi, I. ; Bruder, J. T. ; Forrester, John Vincent ; Dick, A. D. / Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium. In: British Journal of Ophthalmology. 2001 ; Vol. 85, No. 7. pp. 861-867.
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AU - Duncan, Linda

AU - Kovesdi, I.

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AU - Forrester, John Vincent

AU - Dick, A. D.

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AB - Aim-To investigate the efficacy of "ex vivo" adenoviral vector mediated gene transfection of human conjunctival epithelial cell as a possible route for gene therapy for the distribution of antiinflammatory agents for the potential treatment of immune mediated ocular inflammatory disorders.Methods-Human conjunctival cells (HCs) were cultured with various concentrations of recombinant adenoviral vectors carrying a reporter gene LacZ, GFP, or an immunomodulating cytokine vIL-10. VIL-10 in culture supernatant was detected by sandwich ELISA and biological activity was assessed by suppression of ConA stimulated splenocyte proliferation. X-gal and GFP expression was assessed by histochemistry.Results-The extent of adenoviral vector mediated transfer of both reporter genes and vIL-10 was dose dependent. LacZ expression could be detected for at least 50 day after infection with multiple of infection (MOI) 200. Following AdCMVvIL-10 transduction, vIL-10 protein expression occurred between 4-6 days post-transduction, and was maintained at a detectable level for at least 1 month. Secreted VIL-10 showed biological activity, significantly inhibiting Con A induced splenocyte proliferation. Additionally, transfection of HCs with two Adv vectors, one carrying LacZ and the other carrying GFP, resulted in co-expression within a single cell.Conclusion-These results confirm previous successful adenoviral vector mediated gene transfer to HCs and further show that expression can be maintained. Furthermore the data show HCs can secrete biologically active VIL-10 that could be developed as a strategy to suppress immune mediated disorders. The successful co-transduction of HCs as described for other tissues, opens avenues to develop a multiple target gene therapy locally.

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KW - IN-VIVO

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