Purchase this article with an account.
LaKisha K. Buie, Juan Carabana, M. Grazia Spiga, Teresa Borras; Anterior Chamber Delivery of Dominant-negative RhoA by an scAAV Vector Prevents Nocturnal IOP Elevation in Living Rats. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2486.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We had previously shown that inhibiting the RhoA pathway by delivering dominant-negative RhoA (T19N) with an Adenoviral vector (Ad) increased outflow facility in perfused human anterior segments. Because we have now shown that self-complementary AAV (scAAV) vectors have longer duration of transgene expression and lower immune response than Ads, we constructed an scAAV2.RhoA2 viral vector and investigated its ability to lower IOP in living rats.
The dominant-negative human RhoA cDNA with its own Kozak sequence was placed under the control of a new translation-enhanced CMV5 promoter. The expression cassette was cloned into scAAV plasmid vector pHpa-trs-SK (mutated terminal repeat downstream from cassette) (pMG27). Plasmid pMG27 was used by the UNC core facility to generate the scAAV2.RhoA2 virus. Its effect on HTM cells’ actin cytoskeleton was assessed by phalloidin staining. Its effect on nocturnal IOP elevation was assessed on Wistar rats, on a 12 h-white light/12 h-2 lux red light, 9pm-9am reverse light cycle model. Rats in this model require 3 wks adaptation to exhibit nocturnal elevated IOP. A total of 2-7 X1011 scAAV2.RhoA2 and control scAAV2.GFP viral particles were intracamerally injected into the rats’ OD and OS prior to observing the nocturnal increase. Tonolab measurements were performed 2-3 h after onset of the nocturnal cycle and 1-2 times/wk post-injection. Values obtained at pre-injection were used as the baselines. Animals were euthanized 7 wks post-injection.
HTM cells infected with scAAV2.RhoA2 exhibited the actin stress fibers reduction typical of inhibiting the RhoA pathway. scAAV2.RhoA2-injected eyes maintained a baseline IOP during the dark cycle up to 7 wks post-injection. Eyes injected with scAAV.GFP showed a significantly higher nocturnal IOP elevation than the baseline. In a first experiment (n=7 rats), the mean IOPs of the scAAV2.RhoA2-injected eyes were 11.5±0.6 mmHg at baseline, 11.8±0.2 mmHg at 4 wks and 13.1±0.2 mmHg at 7 wks (p=0.5 and 0.01 from baseline). For the same time periods, IOPs of the scAAV.GFP injected eyes were 13.1±0.4 mmHg at baseline, 18.9±0.7 mmHg at 4 wks and 20.5±0.6 mmHg at 7 wks (p=0.0005 and 0.0001 from baseline). IOP differences between treated and control eyes during the dark cycle were significant at all time-points with values of -1.6±0.05 mmHg at baseline (p=0.06), -12.3±4.3 at 4 wks (p=0.0004) and -7.5±1.8 at 7 wks (p=0.001).
This is the first example of a gene transfer affecting nocturnal IOP elevation in living animals. Gene therapy with dominant-negative RhoA carried by a safe long-term vector could be an important tool for the control of elevated nocturnal IOP.
This PDF is available to Subscribers Only