Transferrin-polyethylenimine conjugate, FuGENE6 and TransIT-LT as nonviral vectors for gene transfer to the corneal endothelium.
OBJECTIVE
To investigate the efficacy and pathogenicity of three commercially available nonviral DNA vectors for gene transfer to the corneal endothelium.


METHODS
Corneas obtained from New Zealand White rabbits were cultured ex vivo. For cell culture, the corneal endothelial cells were removed and cultured in vitro under standard conditions. Using the vectors, culture cells or ex vivo corneas were transfected with plasmid DNA coding for green fluorescent protein (GFP). Expression of the transduced gene was monitored by fluorescence microscopy. Transfection efficiency was estimated as the percentage of GFP-positive cells identified. The viability and morphology of the endothelium were also examined.


RESULTS
Transferrin-polyethylenimine conjugate was effective in vitro but not ex vivo. FuGENE6 and TransIT-LT mediated the transfer of GFP gene both in in vitro and in ex vivo culture. Their efficiency estimated at day 3 was 28.8% and 38.8% in vitro, and 8.1% and 8.8% ex vivo, respectively. Viability staining revealed no dead cells. Morphological study showed no apparent alteration.


CONCLUSIONS
FuGENE6 and TransIT-LT are safe, simple to use, and may be useful alternative methods for gene transfer to the corneal endothelium, avoiding certain side effects of viral vectors. As the efficiency could be enhanced, these nonviral vectors may be promising for practical application.
Nguyen
Thanh Ha
TH
Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
Murakami
Akira
A
Fujiki
Keiko
K
Kanai
Atsushi
A
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008055:Lipids / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D011094:Polyethyleneimine / Q000494:pharmacology; D011817:Rabbits; D014162:Transfection / Q000379:methods*; D014168:Transferrin / Q000235:genetics*
C411955:FuGene; D008055:Lipids; D008164:Luminescent Proteins; D014168:Transferrin; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine