Outflow Facility Enhancement By Caldesmon Gene Therapy In Organ–cultured Human And Monkey Eyes

Abstract

Abstract: : Purpose: To determine the effect of caldesmon gene therapy on outflow facility (OF) in human and monkey organ cultured anterior segments. Methods:An adenoviral vector encoding a GFP–caldesmon fusion protein driven by a CMV5 promoter–enhancer was constructed by homologous recombination (AdGFPCald). Six human and five rhesus or cynomolgus monkey paired anterior segments were mounted on organ culture dishes and perfused with DMEM at a constant rate of 2.5µl/min. For human eyes, following 24 hours of equilibration, baseline OF was calculated as the flow divided by the intraocular pressure (IOP). Human anterior segments were injected with a single dose of 10(7) pfu of AdGFPCald to one eye; vehicle to the opposite eye. IOP was monitored continuously for 66 hours and average OF calculated every 6 hours. For monkey segments, baseline OF was determined by two–level constant pressure perfusion for 45–60 min after overnight equilibration. Segments were then injected via the infusion tubing with 20ul containing 7.5x10(8)pfu/ml AdGFP to one eye; AdGFPCald to the opposite eye. Post–treatment OF was monitored at days 2, 5–6, and in some cases up to 9 days after injection. Human and monkey segments were embedded in OCT and examined for the presence of fluorescence Results: Baseline OF (µl/min/mmHg) was no different between the paired eyes, and averaged (mean±sem): human, 0.20±0.03 (n=11); monkey, 0.45±0.06 (n=10). In humans, the IOP began to decrease in AdGFPCald segments within 10 hours after the injection and continued to decrease for the duration of the 66 hours. The percent change of final OF from baseline was 49.0±24.8% (AdGFPCald) (p<0.09) and 0.6±7.8% (vehicle) (p<0.9). In monkey segments, the OF increase was not detected until 5 or 6 days after the initial injection in most cases (4 of 5). When all eyes were considered, the mean maximum OF increase in AdGFPCald vs AdGFP eyes corrected for baseline was 102±30% (p<0.05). However, most segments appeared to be contaminated by days 5–6. Fluorescence was present in both paired segments of monkey eyes and in the AdGFPCald segment of human eyes. Conclusions:Caldesmon gene therapy can increase outflow facility in the human and monkey anterior segments in organ culture and has the potential to be used in vivo to control IOP in humans.

Keywords: anterior segment • gene transfer/gene therapy • outflow: trabecular meshwork