Adenovirus-mediated gene therapy using human p21WAF-1/Cip-1 to prevent wound healing in a rabbit model of glaucoma filtration surgery.
OBJECTIVE
To determine if adenovirus-mediated p21(WAF-1/Cip-1) (p21) gene therapy can prevent fibroproliferation and wound healing in a rabbit model of glaucoma filtration surgery.


METHODS
In vitro studies were performed using rabbit Tenon fibroblasts harvested from fresh tissue. In vivo studies were conducted in New Zealand white rabbits. A full-thickness sclerotomy was performed under a limbal-based conjunctival flap. Reagents tested included a replication-deficient recombinant adenovirus containing the human p21 gene (rAd.p21); the nonspecific marker gene for green fluorescent protein or beta-galactosidase; mitomycin, 0.5 mg/mL; and balanced saline solution. Each treatment was applied episclerally for 5 minutes before the sclerotomy using a soaked cellulose sponge placed under the surgically created conjunctival flap. Independent experiments were conducted to (1) monitor changes in intraocular pressure during a 30-day period after treatment and examine surgical site histological features, (2) examine changes in bleb morphologic features over 30 days, (3) determine outflow facility 14 days after treatment, and (4) examine the localization and persistence of rAd.p21 expression between 3 and 60 days after treatment.


RESULTS
Treatment of Tenon fibroblasts with rAd.p21 resulted in a dose-dependent inhibition of DNA synthesis and cell growth in vitro. In vivo, rAd.p21 inhibited wound healing and fibroproliferation after filtration surgery, comparably to mitomycin. Mitomycin caused notable thinning of the bleb wall. In addition, 2 of the 5 mitomycin-treated eyes exhibited an abscess with hypopyon and hyalitis 30 days after surgery, which was not observed in any of the rAd.p21-treated eyes. None of the treatments resulted in a significantly sustained decrease in intraocular pressure during the 30-day period, although mitomycin treatment resulted in a significant (P =.02) increase in outflow facility 2 weeks after surgery in separate animals. Mitomycin- and rAd.p21-treated eyes had functioning blebs at the end of the experiment based on slitlamp examination.


CONCLUSIONS
Mitomycin and rAd.p21 were effective in preventing fibroproliferation and wound healing in a rabbit model of glaucoma surgery. Mitomycin treatment increased outflow facility in normal-pressure eyes.


CONCLUSIONS
Gene therapy with rAd.p21 may provide an effective antiproliferative for glaucoma filtration surgery, without the complications associated with mitomycin.
Perkins
Todd W
TW
Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, 1300 University Ave, Room 6640 MSC, Madison, WI 53706-1532, USA.
Faha
Barbara
B
Ni
Ming
M
Kiland
Julie A
JA
Poulsen
Gretchen L
GL
Antelman
Doug
D
Atencio
Isabella
I
Shinoda
Jeremy
J
Sinha
Dinesh
D
Brumback
Lyndia
L
Maneval
Daniel
D
Kaufman
Paul L
PL
Nickells
Robert W
RW
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002455:Cell Division; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000502:physiology; D003673:Defective Viruses; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005901:Glaucoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D049452:Green Fluorescent Proteins; D007429:Intraocular Pressure; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D023421:Models, Animal; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012599:Sclerostomy; D014162:Transfection; D014945:Wound Healing; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D004247:DNA; D001616:beta-Galactosidase