Enhanced short-term plasmid transfection of filtration surgery tissues.
OBJECTIVE
To quantify and localize plasmid transfection of filtration surgery tissues using two delivery techniques.


METHODS
Full-thickness filtering procedures were performed on eyes of New Zealand albino rabbits. In 10 eyes, naked plasmid DNA in saline was either injected beneath Tenon's capsule at the filtration site or absorbed into a collagen shield that was then placed external to the sclerostomy and under the Tenon's capsule. Forty-eight hours after surgery, levels of the reporter gene, chloramphenicol acetyltransferase (CAT) were measured in samples of ocular tissues. In two additional eyes, the ss-galactosidase (ss-GAL:) reporter gene expression was localized histologically.


RESULTS
Injection of plasmid DNA in saline vehicle into the filtration bleb produced readily detectable CAT activity in bleb tissue (conjunctiva, Tenon's capsule, and sclera) whereas CAT activity was nearly undetectable in samples of the cornea, iris-ciliary body, and tissues located opposite the bleb site. Delivery of the plasmid DNA into the bleb through a collagen shield increased CAT activity 30-fold over injection of plasmid in saline (2711 +/- 567 mU/mg versus 92 +/- 38 mU/mg). ss-Gal activity was imaged only in the region of the bleb, and microscopic examination showed ss-Gal activity localized to Tenon's capsule fibroblasts, with minimal ss-Gal activity observed in inflammatory cells or scleral fibroblasts.


CONCLUSIONS
Transfection of filtration tissues is enhanced by absorption of naked DNA into a collagen shield. Furthermore, transfection is localized to the fibroblasts and inflammatory cells of the filtration bleb site. Gene therapy using naked plasmid DNA and a simple collagen shield delivery vehicle may be useful for regulating wound healing after glaucoma surgery.
Angella
G J
GJ
Departments of Ophthalmology and. Obstetrics and Gynecology, University of Florida, Gainesville. Karolinska Institute, Stockholm, Sweden.
Sherwood
M B
MB
Balasubramanian
L
L
Doyle
J W
JW
Smith
M F
MF
van Setten
G
G
Goldstein
M
M
Schultz
G S
GS
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003228:Conjunctiva / Q000378:metabolism*; D003238:Connective Tissue / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D018463:Filtering Surgery; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics*; D011817:Rabbits; D012590:Sclera / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase