A nonviral vector system for efficient gene transfer to corneal endothelial cells via membrane integrins.
BACKGROUND
Genetic manipulation of allografts to suppress their ability to induce rejection is a promising approach for controlling rejection responses. A key to this approach is the development of appropriate DNA vectors. We are developing nonviral DNA vector systems based on synthetic peptides containing an integrin-binding segment for cellular targeting and a polylysine segment for DNA binding.


METHODS
Two such peptides have been tested for their ability to deliver the beta-galactosidase reporter gene to the corneal endothelial cells of the rabbit, pig, and man. One peptide was derived from a phage display library, the other from the integrin-binding moiety of the toxin from the American pit viper, Crotalus molossus molossus. Corneas were cultured overnight and then exposed to the DNA/peptide vector under a variety of conditions involving different DNA concentrations, chloroquine concentrations, times of exposure, presence of serum, and presence of polyanion buffers. Expression of the beta-galactosidase gene was determined after 3 additional days in culture. Effects of the treatment on the viability of the endothelium were examined by confocal microscopy.


RESULTS
We report that approximately 30% of corneal endothelial cells can be transfected with our optimal protocol using the molossin-based vector. Transfection is dependent on the presence of chloroquine and is inhibited by polyanion buffers such as HEPES. Viability of the corneal endothelium was excellent, except if corneas were incubated at high concentrations of chloroquine (0.5 mM) for prolonged periods (24 hr).


CONCLUSIONS
Synthetic peptides containing both an integrin targeting and a DNA-binding moiety are promising as simple and highly versatile DNA vectors for use in corneal transplantation.
Shewring
L
L
Transplantation Biology Unit, Institute of Child Health, London, England.
Collins
L
L
Lightman
S L
SL
Hart
S
S
Gustafsson
K
K
Fabre
J W
JW
D000818:Animals; D002738:Chloroquine / Q000633:toxicity; D017077:Culture Media, Conditioned; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005312:Fetal Blood / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016023:Integrins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009842:Oligopeptides / Q000235:genetics; D011485:Protein Binding; D011817:Rabbits; D014161:Transduction, Genetic; D014162:Transfection; D019172:Transplantation Conditioning; D014757:Viper Venoms / Q000235:genetics
D017077:Culture Media, Conditioned; D016023:Integrins; D009842:Oligopeptides; D014757:Viper Venoms; D002738:Chloroquine