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(Investigative Ophthalmology and Visual Science. 2000;41:4158-4162.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Enhanced Short-Term Plasmid Transfection of Filtration Surgery Tissues

Guy J. Angella1, Mark B. Sherwood1, Lakshmi Balasubramanian1, J. William Doyle1, Mary F. Smith1, Gysbert van Setten2, Michael Goldstein1 and Gregory S. Schultz3

1 From the Departments of Ophthalmology and 2 Obstetrics and Gynecology, University of Florida, Gainesville; and the 3 Karolinska Institute, Stockholm, Sweden.

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 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.




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T. Borras, C. R. Brandt, R. Nickells, and R. Ritch
Gene Therapy for Glaucoma: Treating a Multifaceted, Chronic Disease
Invest. Ophthalmol. Vis. Sci., August 1, 2002; 43(8): 2513 - 2518.
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