Effect of intraviteal injection of AAV9.TGFβ2 on the expression of anterior segment and intraocular pressure in rabbits

Abstract

Purpose: TGFβ2 levels are elevated in aqueous humor (AH) of primary open angle glaucoma patients and have been associated with ultrastructural changes to trabecular meshwork (TM) leading to elevated intraocular pressure (IOP). This study is to explore viral transduction and overexpression of TGFβ2 on IOP in New Zealand rabbits.

Methods: Eight rabbits received unilateral intravitreal injection of 50µl of AAV9-mut446-sc-smCBA-hTGFβ2 (1.25E12 vg/mL) and other four received AAV9-mut446-sc-smCBA-hGFP (6.4E12 vg/mL). The injections were repeated one week following the initial injection. IOP was measured at day 0 and once or twice per week using a pneumatonometer on day 6-62 post second-injection. Ocular inflammation including flare, cells and fibrin in anterior chamber and GFP expression were examined by slit lamp (SL) beginning one day after injection. The expression of GFP in the anterior segment of rabbits was assessed by immunohistochemistry (IHC). Total and bioactive TGFβ2 in rabbit AH were detected by ELISA 62 days post-injection.

Results: GFP expression was detected in 75% of four AAV9.GFP treated rabbits by SL beginning 14 days post-injection and 50% of these injected eyes showed a positive expression of GFP at 49 and 62 days post-injection by IHC in anterior segment of eye. Compared with uninjected (UI) eyes, total TGFβ2 in AH showed an increasing trend (P=0.1) in AAV9.TGFβ2 (2314 ± 316 vs UI eye: 1664 ± 198 pg/mL), while was significantly different in AAV9.GFP treated group (2412 ± 246 vs UI eye: 1972 ± 185 pg/mL; p=0.035). Bioactive TGFβ2 levels in AAV9.TGF β2 eyes were 41 ± 9 pg/mL (UI eye: 42 ± 9 pg/mL) while that in AAV9.GFP eyes was 16 ± 8 pg/mL (UI eye: 41 ± 21 pg/mL). No statistically significant difference in total and bioactive TGFβ2 levels were found between AAV9.TGFβ2 and AAV9.GFP injected groups. Inflammation could be detected in 50% of eyes in both groups lasting for 2-3 weeks. Compared with pre-injection baseline, IOP was not significantly increased in either AAV9.TGFβ2 (BL: 20 ± 0.8 mmHg; Day 62: 23 ± 0.5 mmHg) or AAV9.GFP treated eyes (BL: 17 ± 0.5 mmHg; Day 62: 19 ± 1.5 mmHg).

Conclusions: Our results indicate that AAV9.GFP can be transduced in the ocular anterior segment of rabbits. However, TGFβ2 levels in AH did not result in elevated IOP in rabbits and could be due to species differences and/or insufficient expression of TGFβ2 with these vectors.

Keywords: 538 gene transfer/gene therapy • 421 anterior segment • 568 intraocular pressure