RNAi-Mediated Inhibition of RHO Kinase for Treatment of Ocular Disorders

Abstract

RNA interference is provided for inhibition of Rho kinase mRNA expression for treating patients with ocular disorders, particularly for treating intraocular pressure, ocular hypertension and glaucoma. Rho kinase mRNA targets include mRNA for ROCK1 and ROCK2.

1 . A method of attenuating expression of Rho kinase mRNA of a subject, comprising:
 administering to an eye of the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising:
 a sense nucleotide strand and an antisense nucleotide strand 
 wherein the antisense strand:
 comprises a ribonucleotide sequence consisting of the base sequence of SEQ ID NO: 3, 9-28, or 30-79 with uridine bases substituted for thymidine bases; 
 is substantially complementary to the sense strand; and 
 hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO: 2; 
 
   wherein the interfering RNA directs RISC-mediated cleavage of Rho kinase mRNA, and wherein the expression of Rho kinase mRNA is attenuated thereby.   
     
     
         2 . The method of  claim 1  wherein the subject is a human and the human has ocular hypertension. 
     
     
         3 . The method of  claim 1  wherein the subject is a human and the human has glaucoma. 
     
     
         4 . The method of  claim 1  wherein the composition is administered via a topical, intravitreal, transcleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route. 
     
     
         5 . The method of  claim 1  wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:1 comprising nucleotide 605, 653, 659, 1248, 1562, 1876, 2266, 2474, 2485, 2740, 2808, 2834, 3007, 3146, 3199, 3245, 3379, 3453, 3511, 3513, 3519, 3782, 998, 1132, 1200, 1648, 1674, 1708, or 2077. 
     
     
         6 . The method of  claim 1  wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:2 comprising nucleotide 1102, 1865, 2000, 2229, 2514, 2584, 2738, 3305, 4111, 4652, 5184, 5187, 5255, 5315, 5439, 5450, 5578, 5579, 5611, 5625, 5795, 6000, 6228, 6264, 584, 1337, 1678, 2773, 2814, 2941, 3357, 3398, 3481, 3633, 3644, 3645, 3767, 3836, 4023, 4097, 5202, or 5440. 
     
     
         7 . The method of  claim 1  further comprising administering to the subject a second interfering RNA having a length of 19 to 49 nucleotides, and comprising
 a sense nucleotide strand, an antisense nucleotide strand, and a region of at least near-perfect complementarity of at least 19 nucleotides; 
 wherein the antisense strand of the second interfering RNA hybridizes under physiological conditions to a second portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2 and the antisense strand has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the second hybridizing portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, respectively. 
 
     
     
         8 . The method of  claim 1  wherein the sense nucleotide strand and the antisense nucleotide strand are connected by a hairpin loop. 
     
     
         9 . A method of attenuating expression of Rho kinase mRNA of a subject, comprising:
 administering to an eye of the subject a composition comprising an effective amount of single-stranded interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier,
 wherein the single-stranded interfering RNA hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide 605, 653, 659, 1248, 1562, 1876, 2266, 2474, 2485, 2740, 2808, 2834, 3007, 3146, 3199, 3245, 3379, 3453, 3511, 3513, 3519, 3782, 998, 1132, 1200, 1648, 1674, 1708, or 2077, and the interfering RNA has a region of at least 80% to 100 contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1, 
 or 
 wherein the single-stranded interfering RNA hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:2 comprising nucleotide 1102, 1865, 2000, 2229, 2514, 2584, 2738, 3305, 4111, 4652, 5184, 5187, 5255, 5315, 5439, 5450, 5578, 5579, 5611, 5625, 5795, 6000, 6228, 6264, 584, 1337, 1678, 2773, 2814, 2941, 3357, 3398, 3481, 3633, 3644, 3645, 3767, 3836, 4023, 4097, 5202, or 5440 and the interfering RNA has a region of at least 80% to 100% contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:2, 
   wherein the interfering RNA directs cleavage of Rho kinase mRNA, and wherein the expression of Rho kinase mRNA is thereby attenuated.   
     
     
         10 . A method of attenuating expression of Rho kinase mRNA in a subject, the method comprising:
 administering to an eye of the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising:
 a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of a ribonucleotide sequence consisting of the base sequence of 3, 9-28, or 30-79, with uridine bases substituted for thymidine bases 
   wherein the interfering RNA directs RISC-mediated cleavage of Rho kinase mRNA, and wherein the expression of the Rho kinase mRNA is attenuated thereby.   
     
     
         11 . The method of  claim 10  wherein the Rho kinase mRNA is ROCK1 mRNA and the interfering RNA comprises:
 a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of an mRNA corresponding to 3, 9-28, 30, or 73-79. 
 
     
     
         12 . The method of  claim 10  wherein the Rho kinase mRNA is ROCK2 mRNA and the interfering RNA comprises:
 a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of an mRNA corresponding to SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72. 
 
     
     
         13 . The method of  claim 10  wherein the interfering RNA comprises a region of at least 14 contiguous nucleotides having at least 85% sequence complementarity to, or at least 85% sequence identity with, the penultimate 14 nucleotides of the 3′ end of an mRNA corresponding to the sequence identified by the sequence identifier. 
     
     
         14 . The method of  claim 10  wherein the interfering RNA comprises a region of at least 15, 16, 17, or 18 contiguous nucleotides having at least 80% sequence complementarity to, or at least 80% sequence identity with, the penultimate 15, 16, 17, or 18 nucleotides, respectively, of the 3′ end of an mRNA corresponding to the sequence identified by the sequence identifier. 
     
     
         15 . The method of  claim 10  wherein the composition further comprises a second interfering RNA having a length of 19 to 49 nucleotides and comprising a region of at least 13 contiguous nucleotides having at least 90% complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of a second mRNA corresponding to any one of SEQ ID NO: 3, 9-28, or 30-79. 
     
     
         16 . The method of  claim 10 , wherein the subject has ocular hypertension. 
     
     
         17 . The method of  claim 10 , wherein the subject has glaucoma. 
     
     
         18 . The method of  claim 10  wherein the composition is administered via a topical, intravitreal, transcleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route. 
     
     
         19 . The method of  claim 10  wherein the composition is administered via in vivo expression from an interfering RNA expression vector.