Abstract
Purpose:
To investigate whether a safer, long term/low toxicity self-complementary AAV2 vector, scAAV2.GRE.MMP1, would also reduce IOP in the sheep model. To optimize the model by comparing sheep age, breed and steroid administrations.
Methods:
The glucocorticoid-inducible expression cassette was cloned into the scAAV plasmid vector pHpa-trs-SK (mutated terminal repeat downstream from cassette) (pMG21). Plasmid pMG21 was used by the UNC core facility to generate the scAAV2.GRE.MMP1 virus. Corriedale (3 y old) and Katahdin (7 mo and 3 y) received Prednisolone, Durezol or sterile PBS drops 3X daily for 1-2 wks. One sheep received a subtenon Kenalog injection. After pressure elevation, sheep were IC-injected with 2-7 X10^12 scAAV2.GRE.MMP1 and control scAAV2.GFP viral genomes. TonoVet IOP measurements were performed 1 wk prior to corticosteroid treatment (baseline) and 2-3X a wk 1-2 h post-steroid treatment. Animals were euthanized 2-3 wks post-injection. Their anterior segments were processed for morphological and fluorescence evaluation.
Results:
A total of 7 sheep were studied. The mean IOPs at baseline were 12.5±0.8 mmHg in 7 mo Katahdin (n=4 eyes) and 15.5±0.5 mmHg in both 3 y old Katahdin and Corriedale (n=10 eyes). Young Katahdin eyes treated with Prednisolone did not experience IOP elevation while older Corriedale showed a 3.6 mmHg increase 1 wk post treatment. Eyes treated with Durezol showed a significant increase in IOPs up to 18.0±1.7 mmHg and 21.4±1.8 mmHg at 3 d (p=0.007 & p=0.0005) and 24.0±1.7 mmHg and 25.0±2.4 mmHg at 3 wks (p=0.0006 & p=0.001) in 3 yr old Corriedale and Katahdin respectively. An injection of Kenalog increased IOP to 17.3±0.9 2 wks post-injection. All 4 sheep injected with MMP1 (Ad.GRE.MMP1 n=1; scAAV2.GRE.MMP1 n=3 eyes) saw significant decrease in IOP. In a representative experiment, scAAV2.GRE.MMP1 injected eyes decreased steroid-induced IOP at 3 wks from 23.5±2.1 to 18±1.4 mmHg (n=2 eyes, p<0.05). Fluorescence microscopy showed positive GFP transduction in the TM of the Ad.GFP and scAAV2.GFP injected eyes.
Conclusions:
Steroid type and age influenced IOP response, while sheep breeds did not. Gene transfer of MMP1 by injection of scAAV2.GRE.MMP1 into the anterior chamber of living sheep lowers steroid-induced elevated IOP making it a great candidate for long term treatment of steroid glaucoma. This gene transfer strategy will be very useful for the evaluation of lowering IOP TM drugs.
Keywords: 538 gene transfer/gene therapy •
735 trabecular meshwork •
421 anterior segment