Efficiency of cytokine gene transfer in corneal endothelial cells and organ-cultured corneas mediated by liposomal vehicles and recombinant adenovirus.
OBJECTIVE
Gene transfer of immunoregulatory cytokines could contribute to reduce rejection of corneal grafts. The aim of our study was to examine the gene transfer efficiency of liposomal vehicles compared to adenoviral vectors for transferring the Epstein-Barr-virus-derived interleukin 10 homologue (viral IL-10, vIL-10) into corneal endothelial cells and organ-cultured human corneas (HC) in vitro.


METHODS
To test liposomal efficiency, 2 lipid formulations (SP-Chol/DOPE 20/80 and DDAB/DOPE 30/70 in various concentrations) were complexed with a plasmid containing the vIL-10 cDNA in an eukaryotic expression vector (pcDSRalpha-BCRF-I). The complexes were transferred to (1) subconfluent bovine corneal endothelial cells (BCEC) after 1 passage and to (2) HC stored in organ culture. In addition, BCEC and HC were transduced with the recombinant adenoviral vector encoding for vIL-10 (AdvIL-10). Secretion of vIL-10 in the supernatants from both transfected BCEC and HC was measured by specific ELISA.


RESULTS
For gene transfer in BCEC, both transfection methods (liposomes and adenovirus) led to high secretion of vIL-10 [>2 ng/ml (liposomes) and <150 ng/ml (adenovirus) per 5,000 initially planted BCEC]. Expression levels in BCEC were dependent on the concentration of applied liposomes. For gene transfer in HC, only the adenoviral transduction technique achieved a high production of vIL-10, whereas liposomal transfection led only to low vIL-10 secretion (4.8 microg/ml vs. 95 pg/ml per quarter of cornea).


CONCLUSIONS
For transfection of corneal endothelial cells in culture, liposomes can be considered as a safe and useful alternative method of gene transfer avoiding side-effects of viral vectors. However, for transfection of organ-cultured HC, adenoviral vectors are superior to liposomal vehicles.
Bertelmann
Eckart
E
Department of Ophthalmology, Charité, Humboldt University, Berlin, Germany. EBertelmann@gmx.de
Ritter
Thomas
T
Vogt
Katrin
K
Reszka
Regina
R
Hartmann
Christian
C
Pleyer
Uwe
U
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002417:Cattle; D003315:Cornea / Q000166:cytology; D046508:Culture Techniques; D016207:Cytokines / Q000235:genetics; D004728:Endothelium, Corneal / Q000502:physiology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics*; D008081:Liposomes; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D016207:Cytokines; D008081:Liposomes; D011994:Recombinant Proteins; D016753:Interleukin-10