Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops.
BACKGROUND
The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies.


METHODS
The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR.


RESULTS
The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range.


CONCLUSIONS
Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.
Toropainen
Elisa
E
Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland. elisa.toropainen@uku.fi
Hornof
Margit
M
Kaarniranta
Kai
K
Johansson
Pinja
P
Urtti
Arto
A
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000737:chemistry / Q000378:metabolism*; D005123:Eye / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D006801:Humans; D008081:Liposomes / Q000008:administration & dosage*; D008156:Luciferases / Q000235:genetics; D009883:Ophthalmic Solutions; D010539:Permeability; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010957:Plasmids / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011817:Rabbits; D013666:Tears / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019076:Transgenes
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D009883:Ophthalmic Solutions; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D012333:RNA, Messenger; D008156:Luciferases; D000469:Alkaline Phosphatase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane