Abstract
Purpose: :
To study the effect on intraocular pressure (IOP) in New Zealand White rabbits of siRNA topical administration targeting carbonic anhydrases, to find a treatment for ocular hypertension and glaucoma.
Methods: :
We have designed several siRNAs for the following carbonic anhydrases genes: CA2, CA4 and CA12. After an in vitro validation, we tested in vivo the effect on IOP levels using the following sequences: siRNA 004 silencing CA2; siRNA 003 silencing CA4 and siRNAs 092 and 146 targeting CA12. siRNAs were administered in saline solution (0.9% w/v) to a final volume of 40ul in one eye during four consecutive days and the opposite eye was taken as a control applying 40ul of sterile saline (0.9% w/v). Two rabbits were used in each experiment. IOP levels were measured from the beginning of the experiment several times a day along ten days. The siRNAs 004 (CA2) and 003 (CA4) have also been used to perform time–dosing kinetic assays and to check different application protocols. Also, we performed some in vivo studies using a CA2 Low Score siRNA and a CA2 High Score siRNA, to validate the predictive algorithm designed by GENOMICA SAU to select the siRNA sequences, following the protocol described above.
Results: :
The CA2 siRNA 004 (n=4) reduced IOP in 28.38 ± 1.67 % during 110 hours; the CA4 siRNA 003 (n=3) caused a decrease in IOP of 18.05 ± 1.13 % lasting 78 hours; and CA12 siRNA 092 (n=2) and 146 (n=2) reduced IOP in 21.38 ± 2.92 and 11.40 ± 1.12, for 65 and 50 hours respectively. As a control of IOP reduction we used Xalatan, which reduced IOP 23.40 ± 2.36 % during 5 to 6 hours. The time–dosing kinetics and the application protocols assays using siRNAs 004 (CA2) and 003 (CA4), showed that the most effective protocol consists on applying the highest dose in several applications. Also, the algorithm validation, done with a CA2 Low Score siRNA and a CA2 High Score siRNA, provides some interesting data, confirming that our software can be used in order to discard those non effective siRNA sequences but is not able to distinguish the best option between those siRNA that work.
Conclusions: :
The reduction of IOP by siRNAs targeting carbonic anhydrases is comparable with that produced by commercial drugs. Moreover, siRNA treatment shows a generalized long lasting effect when compared to commercial drugs.
Keywords: intraocular pressure • gene transfer/gene therapy • pharmacology