Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium

Aim-To investigate the efficacy of "ex vivo" adenoviral vector mediated gen e transfection of human conjunctival epithelial cell as a possible route fo r gene therapy for the distribution of antiinflammatory agents for the pote ntial treatment of immune mediated ocular inflammatory disorders. Methods-Human conjunctival cells (HCs) were cultured with various concentra tions 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 suppress ion of ConA stimulated splenocyte proliferation. X-gal and GFP expression w as 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. Follo wing 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 in hibiting 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 medi ated gene transfer to HCs and further show that expression can be maintaine d. Furthermore the data show HCs can secrete biologically active VIL-10 tha t could be developed as a strategy to suppress immune mediated disorders. T he successful co-transduction of HCs as described for other tissues, opens avenues to develop a multiple target gene therapy locally.