Ex vivo adenovirus mediated gene transfection of human conjunctival epithelium.
OBJECTIVE
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 anti-inflammatory 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.


CONCLUSIONS
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.
Shen
J
J
Department of Ophthalmology, University of Aberdeen Medical School, Foresterhill, AB25 2ZD, UK.
Taylor
N
N
Duncan
L
L
Kovesdi
I
I
Bruder
J T
JT
Forrester
J V
JV
Dick
A D
AD
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002455:Cell Division; D002478:Cells, Cultured; D003233:Conjunctivitis, Allergic / Q000628:therapy*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins; D006801:Humans; D016753:Interleukin-10 / Q000032:analysis / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000166:cytology; D014161:Transduction, Genetic / Q000379:methods*
D017077:Culture Media, Conditioned; D008164:Luminescent Proteins; D016753:Interleukin-10; D049452:Green Fluorescent Proteins