RNAi-mediated inhibition of RHO kinase for treatment of ocular disorders
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 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, an antisense nucleotide strand, and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides;
wherein the antisense strand hybridizes under physiological conditions to a portion of RNA corresponding to SEQ ID NO:1 or SEQ ID NO:2 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, respectively,
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 is at risk of developing ocular hypertension.
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, 3781, 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 . A method of treating ocular hypertension in a subject in need thereof, 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, an antisense nucleotide strand, and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides;
wherein the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, respectively,
wherein the ocular hypertension is treated thereby.
9 . A method of attenuating expression of Rho kinase mRNA of a subject, comprising:
administering to 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, 3781, 3782, 998, 1132, 1200, 1648, 1674, 1708, or 2077, and the interfering RNA has a region of at least near-perfect 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 near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:2,
wherein the expression of Rho kinase mRNA is thereby attenuated.
10 . A method of attenuating expression of an ocular hypertension target mRNA in a subject, the method comprising:
administering to 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 an mRNA corresponding to any one of SEQ ID NO:3, and SEQ ID NO:9-SEQ ID NO:79
wherein the expression of the ocular hypertension target mRNA is attenuated thereby.
11 . The method of claim 10 wherein the ocular hypertension target 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 SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79.
12 . The method of claim 10 wherein the ocular hypertension target 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, and SEQ ID NO:9-SEQ ID NO:79.
16 . A method of treating ocular hypertension in a subject in need thereof, 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 an mRNA corresponding to any one of SEQ ID NO:3, and SEQ ID NO:9-SEQ ID NO:79,
wherein the ocular hypertension is treated thereby.
17 . The method of claim 16 wherein 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:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ I) NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79.
18 . The method of claim 16 wherein 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.
19 . The method of claim 16 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.
20 . The method of claim 16 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.
21 . The method of claim 16 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, and SEQ ID NO:9-SEQ ID NO:79.
22 . The method of claim 16 wherein the subject has glaucoma.
23 . The method of claim 1 wherein the sense nucleotide strand and the antisense nucleotide strand are connected by a hairpin loop.
24 . The method of claim 8 wherein the sense nucleotide strand and the antisense nucleotide strand are connected by a hairpin loop.
25 . The method of claim 10 wherein the interfering RNA is an shRNA.
26 . The method of claim 10 wherein the interfering RNA is an siRNA.
27 . The method of claim 10 wherein the interfering RNA is an miRNA.
28 . The method of claim 16 wherein the interfering RNA is an shRNA.
29 . The method of claim 16 wherein the interfering RNA is an siRNA.
30 . The method of claim 16 wherein the interfering RNA is an miRNA.
31 . The method of claim 8 wherein the composition is administered via a topical, intravitreal, transcleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route.
32 . The method of claim 8 wherein the composition is administered via in vivo expression from an interfering RNA expression vector.
33 . The method of claim 16 wherein the composition is administered via a topical, intravitreal, transcleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route.
34 . The method of claim 16 wherein the composition is administered via in vivo expression from an interfering RNA expression vector.
35 . A method of treating ocular hypertension in a subject in need thereof, comprising:
administering to the subject a composition comprising a double stranded siRNA molecule that down regulates expression of a ROCK1 or ROCK2 gene via RNA interference,
wherein:
each strand of the siRNA molecule is independently about 19 to about 27 nucleotides in length; and
one strand of the siRNA molecule comprises a nucleotide sequence having substantial complementarity to an mRNA corresponding to the ROCK1 or ROCK2 gene, respectively, so that the siRNA molecule directs cleavage of the mRNA via RNA interference.
36 . The method of claim 35 wherein the composition is administered via an aerosol, buccal, dermal, intradermal, inhaling, intramuscular, intranasal, intraocular, intrapulmonary, intravenous, intraperitoneal, nasal, ocular, oral, otic, parenteral, patch, subcutaneous, sublingual, topical, or transdermal route.
37 . The method of claim 35 wherein the interfering RNA is administered via in vivo expression from an expression vector capable of expressing the interfering RNA.
38 . The method of claim 35 wherein the interfering RNA is an miRNA.
39 . The method of claim 35 wherein each strand of the siRNA molecule is independently about 19 nucleotides to about 25 nucleotides in length.
40 . The method of claim 35 wherein each strand of the siRNA molecule is independently about 19 nucleotides to about 21 nucleotides in length.
41 . A composition comprising an interfering RNA having a length of 19 to 49 nucleotides and comprising a nucleotide sequence corresponding to any one of SEQ ID NO:3, and SEQ ID NO:9-SEQ ID NO:79, or a complement thereof, and a pharmaceutically acceptable carrier.
42 . The composition of claim 41 wherein the interfering RNA is an shRNA.
43 . The composition of claim 41 wherein the interfering RNA is an siRNA.
44 . The composition of claim 41 wherein the interfering RNA is an miRNA.
45 . A composition comprising a double stranded siRNA molecule that down regulates expression of a ROCK1 or ROCK2 gene via RNA interference, wherein:
each strand of the siRNA molecule is independently about 19 to about 27 nucleotides in length; and
one strand of the siRNA molecule comprises a nucleotide sequence having substantial complementarity to an mRNA corresponding to the ROCK1 or ROCK2 gene, respectively, so that the siRNA molecule directs cleavage of the mRNA via RNA interference.
46 . The composition of claim 45 wherein each strand of the siRNA molecule is independently about 19 nucleotides to about 25 nucleotides in length.
47 . The composition of claim 45 wherein each strand of the siRNA molecule is independently about 19 nucleotides to about 21 nucleotides in length.