Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy.

PURPOSE: Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas. METHODS: rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken beta-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen microl rAV at 10(7) plaque forming units per 1 microl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5-32 days. rAAV were added at 1.2-7.8x10(10) vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14-23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x10(11) vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry. RESULTS: By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent. CONCLUSIONS: rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.

Delayed suprachoroidal hemorrhage after glaucoma filtration procedures.

OBJECTIVES: To determine the incidence of, risk factors for, and outcomes of delayed suprachoroidal hemorrhage (DSCH) after glaucoma filtration surgery. DESIGN: Retrospective case-control study. PARTICIPANTS: All patients undergoing glaucoma filtration procedures between 1986 and 2000 at Indiana University who were diagnosed postoperatively with suprachoroidal hemorrhage. A total of 66 patients with DSCH were identified. These were compared with a randomly selected group of patients who underwent similar procedures but did not have suprachoroidal hemorrhage. METHODS: Total cases of DSCH were initially compared with the total number of glaucoma surgeries to determine the overall incidence and the incidence in the different procedures. Subsequently, a case-control study was performed comparing the group with hemorrhage to the control group to identify risk factors. Finally, outcomes and prognostic factors were determined by comparing vision preoperatively and postoperatively and parameters of patients with good and poor outcomes. MAIN OUTCOME MEASURES: Incidence of DSCH, risk factors associated with its occurrence, visual outcomes, and factors important for prognosis. RESULTS: Of a total of 2285 glaucoma filtration procedures, 66 (2.9%) cases of DSCH were identified. It developed in 9 of 615 (1.5%) trabeculectomies without antimetabolite, 30 of 1248 (2.4%) trabeculectomies with antimetabolite, 2 of 72 (2.8%) valved tube shunt implantations, and 25 of 350 (7.1%) nonvalved tube shunt implantations. The increased incidence of DSCH after tube shunts compared with trabeculectomy-associated DSCH was significant (P < 0.0001) with an odds ratio of 3.2. The risk factors for DSCH after glaucoma surgery include white race (P = 0.012), anticoagulation (P = 0.034), severe postoperative hypotony (P = 0.033), and aphakia/anterior chamber intraocular lens (P = 0.002). The visual outcomes of patients with hemorrhage were poor, with a decrease in logarithm of the minimum angle of resolution visual acuity from 0.72 to 1.36, which was statistically significant compared with the controls (P < 0.009). CONCLUSIONS: Delayed suprachoroidal hemorrhage occurs more frequently after tube shunt implantation than after trabeculectomy. Caution should be exercised when operating on patients with known risk factors, because the visual outcomes after DSCH are poor.