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

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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

Equipment

Iain Fraser Cytometry Centre

Andrea Holme (Manager), Linda Duncan (Senior Application Scientist), Ailsa Laird (Technician) & Kate Burgoyne (Technician)

Institute of Medical Sciences

Research Facilities: Facility

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