Minimizing side effects of ballistic gene transfer into the murine corneal epithelium.
BACKGROUND
The beneficial effect of modulating an allospecific immune response by ballistic IL-4 and CTLA4 gene transfer to deliver minimalistic immunologically defined gene expression (MIDGE) vectors into the corneal epithelium was demonstrated in corneal transplantation. However, side effects reduced graft survival in control animals after ballistic transfer without DNA.


METHODS
An adapter was constructed for the gene gun apparatus to enlarge and keep constant the distance between the gun and the cornea. Mice were treated by ballistic transfer of luciferase- or IL-10 -encoding MIDGE vectors using gold particles different in quantity, size and size uniformity. Levels of protein expression were determined. Treated corneas were observed under the scanning electron microscope and immunohistologically. Three groups of Balb/c (H-2d) mice received a C3H (H-2 k) corneal graft and two of them had gold particles delivered into the corneal epithelium by gene gun.


RESULTS
Using the gene gun and the distance piece, scanning electron microscopy did not reveal morphological differences of the corneal surface compared with untreated corneas on day 2 and 5. Sagittal histological sections of the central cornea did not show an invasion of macrophages 24 h after treatment. The expression of luciferase and IL-10 was not reduced when a smaller amount of gold (0.1 mg instead of 0.5 mg) was employed. Ballistic gold treatment did not reduce graft survival.


CONCLUSIONS
Ballistic gene transfer into the corneal epithelium allows high cytokine expression in the cornea without measurable side effects if an apparatus is used that is adapted for this specific purpose.
Zhang
Er-Ping
EP
Augenklinik und Poliklinik, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Germany.
Muller
Anja
A
Schulte
Felix
F
König
Merediz Sven A
MS
Sack
Florian
F
Junghans
Claas
C
Wittig
Burghardt
B
Hoffmann
Friedrich
F
D000818:Animals; D019470:Biolistics; D016039:Corneal Transplantation / Q000502:physiology; D003587:Cytomegalovirus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D005822:Genetic Vectors; D006046:Gold; D016753:Interleukin-10 / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008855:Microscopy, Electron, Scanning; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection / Q000379:methods
D011993:Recombinant Fusion Proteins; D016753:Interleukin-10; D006046:Gold; D008156:Luciferases