Ex vivo adenovirus-mediated gene transfer to corneal graft endothelial cells in mice.
OBJECTIVE
Genetic modulation of donor tissue before corneal transplantation may have the potential to modulate alloimmunity and/or to prevent corneal endothelial cell death. This study was conducted to optimize adenovirus-mediated gene transfer to donor corneal endothelium and to delineate the kinetics of marker gene expression in syngeneic and allogeneic corneal grafts.


METHODS
BALB/c mouse corneas were incubated with replication-deficient adenovirus encoding green fluorescent protein (GFP) or empty vector ex vivo at a dose of 6 x 10(7) or 6 x 10(6) PFU at temperatures of 4 degrees C or 37 degrees C. After ex vivo infection, the donor corneas were transplanted orthotopically to BALB/c or C57BL/6 recipients. After transplantation, localization of GFP in the grafts was determined in cryosections of enucleated eyes, and GFP expression in the grafts was visualized in vivo by using epifluorescence microscopy over 12 weeks. All grafts were evaluated clinically by slit lamp biomicroscopy.


RESULTS
GFP expression was found to be restricted to the corneal endothelium. In vivo expression of GFP in syngeneic corneal grafts was demonstrated for up to 12 weeks. Syngeneic grafts incubated with the vector at 4 degrees C exhibited a more extensive and longer duration of expression of green fluorescence than grafts incubated at 37 degrees C. Moreover, the syngeneic grafts infected at 4 degrees C maintained their transparency, whereas those infected at 37 degrees C displayed a high degree of opacity. Corneal allogeneic grafts infected with a low dose of the vector displayed longer GFP expression and graft survival than the allogeneic grafts infected with a high dose of the viral vector.


CONCLUSIONS
Adenoviral vector can selectively and efficiently deliver exogenous gene(s) to the endothelium of corneal grafts during hypothermic organ preservation. Gene expression is retained in vivo in corneal syngeneic grafts for longer periods than are allogeneic grafts.
Qian
Ying
Y
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Leong
Fee-Lai
FL
Kazlauskas
Andrius
A
Dana
M Reza
MR
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D016039:Corneal Transplantation; D003673:Defective Viruses; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000637:transplantation; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins