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Guy J. Angella, Mark B. Sherwood, Lakshmi Balasubramanian, J. William Doyle, Mary F. Smith, Gysbert van Setten, Michael Goldstein, Gregory S. Schultz; Enhanced Short-Term Plasmid Transfection of Filtration Surgery Tissues. Invest. Ophthalmol. Vis. Sci. 2000;41(13):4158-4162.
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purpose. 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 β-galactosidase (β-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). β-Gal activity was imaged only in the region of the
bleb, and microscopic examination showed β-Gal activity localized to
Tenon’s capsule fibroblasts, with minimal β-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
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