Gene transfer to ovine corneal endothelium.
OBJECTIVE
Modification of a donor cornea by gene therapy has potential to modulate irreversible rejection, the major cause of corneal graft failure. The sheep is a useful model for the human in this respect, as ovine endothelial cells are amitotic. The aim of the study was to investigate the ability of various non-viral and viral agents to transfer a reporter gene to ovine corneal endothelium.


METHODS
The non-viral agents Transfectin-10, Transfectin-20, Transfectin-50, SuperFect, Effectene and CLONfectin were used to deliver the reporter gene, Escherichia coli lacZ, to ovine corneal endothelium in vitro. A Herpes simplex virus-1 and an adenoviral vector each encoding E. coli lacZ were similarly tested. Infected corneas were organ-cutured for up to 7 days in vitro to allow transfection efficiency, duration of gene expression and toxicity attributable to each vector to be compared.


RESULTS
Scattered single or clusters of endothelial cells expressing the reporter gene were observed after transfection with CLONfectin, Transfectin-10, Transfectin-20 and Transfectin-50. SuperFect and Effectene were virtually ineffective. At best, the absolute number of infected cells per endothelial monolayer after 3 or 7 days of organ culture was estimated as < 0.01%. The Herpes simplex virus-1 vector also failed to transduce ovine corneal endothelium efficiently. In contrast, transfection rates of up to 70% of endothelial cells were observed with the adenoviral vector.


CONCLUSIONS
Non-viral vectors and Herpes simplex virus-1 are unlikely to be suitable for gene therapy of corneal endothelium, because the efficiency of transfection is low compared with the rates achieved with adenoviral vectors.
Klebe
S
S
Department of Ophthalmology, Flinders University of South Australia, Bedford Park, Australia.
Sykes
P J
PJ
Coster
D J
DJ
Bloom
D C
DC
Williams
K A
KA
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002452:Cell Count; D003673:Defective Viruses; D004728:Endothelium, Corneal / Q000378:metabolism*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics; D007763:Lac Operon; D009924:Organ Culture Techniques; D012756:Sheep; D001616:beta-Galactosidase / Q000378:metabolism
D001616:beta-Galactosidase