Mediated phki inhibition of rho-kinase for treating ophthalmic disorders

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and concerns mediated PHKi inhibition of RHO-kinase for treating ophthalmic disorders. The substance of the invention involves RNA-interference for mRNA Rho-kinase expression inhibition for treating patients with ophthalmic disorders, especially for treating intraocular pressure, eye hypertension and glaucoma The mRNA Rho-kinase targets include gene ROCK 1 mRNA.

EFFECT: creation of the agent exhibiting improved properties.

56 cl, 2 ex, 4 dwg, 1 tbl

 

The technical field to which the invention relates

The invention relates to the field of compositions interfering RNA for inhibiting the expression of mRNA targets of Rho-kinase with eye disorders, in particular, to reduce intraocular pressure in the treatment of ocular hypertension and glaucoma.

Background of the invention

Glaucoma is a heterogeneous group of optic neuropathy with some common clinical signs. Loss of vision in glaucoma is due to selective death of ganglion cells in the retina neural retina, which is clinically diagnosed by characteristic changes in visual field defects, nerve fiber layer and progressive excavation of the optic disc (optic nerve disc). One of the main risk factors for glaucoma is the presence of ocular hypertension (OHT), i.e., increased intraocular pressure (IOP). Normal IOP is essential for maintaining the shape of the eye and to ensure that the pressure gradient for flow watery moisture to deprived vessels of the cornea and the crystalline lens. IOP can also participate in the pathogenesis of normal pressure glaucoma (NTG), which is proved by the presence of patients with improvement after reducing IOP medicines. After amendments to the Central thickness of the cornea indicators VG is in patients with NTG detected, many of these patients present ocular hypertension.

Increased IOP associated with glaucoma, occurs as a result of increased resistance to aqueous outflow in the trabecular network (TC), a small number of specialized tissue located in the rainbow-corneal angle of the anterior chamber. Glaucomatous changes in vehicle include loss of cells in the TS and the deposition and accumulation of extracellular sediment, including protein blackebrry material. In addition, there are also changes in glaucoma in the optic nerve disc. In the eyes of glaucoma occur morphological and volatile changes in glial cells of the optic nerve disc. In response to elevated IOP and/or transient ischemic strokes, changing the composition of the extracellular matrix of the optic nerve disc and changes in the morphology of glial cells and axons of the ganglion cells of the retina.

Primary glaucoma are the result of violations in the current intraocular fluids that have anatomical or physiological basis. Secondary glaucoma as a result of damage or injury to the eye or preceding disease. Primary open-angle glaucoma (POAG), also known as or chronic simple glaucoma, represents the majority of all primary glaucoma. POAG is characterized by degeneration of trabecular the second network, leading to abnormally high resistance to the outflow of fluid from the eye. The consequence of this resistance is the increase in IOP, which is necessary for passing normally produced by the eyes of the fluid at the increased resistance.

Associated with Rho containing the double helix protein kinase, also known as Rho-kinase or ROCK, are effectors of the Rho family of small GTP-binding proteins (Rho-GTPase). There is evidence that the transmission signal Rho-GTPase plays a role in the regulation of aqueous outflow, for example, by changing the organization of the cytoskeleton of the trabecular network (TC) and/or cells of the ciliary muscle (CM). Low molecular weight inhibitors of Rho-kinase cause reversible changes in the morphology and organization of the cytoskeleton of cells TC, reduce the contractility of the isolated tissue of the CM and increase the ease of aqueous outflow in organ culture (Waki M. et al., Curr Eye Res. 22:470-4 (2001); M. Honjo et al, Invest Ophthalmol Vis Sci. 42:137-44 (2001); Rao PV. et al., Mol. Vis. 11:288-97 (2005); Rao PV. et al., Invest Ophthalmol. Vis Sci. 42:1029-37 (2001)). Similar effects occur when the expression of dominant-negative Rho-binding domain. However, treatment with low molecular weight inhibitors of Rho-kinase also causes vasodilatation and hyperemia of the conjunctiva. In addition, the efficiency based on low molecular weight compounds of drugs I have is a relatively short, requiring dose for each day, and in some cases efficiency over time is reduced.

Due to the importance of ocular hypertension in glaucoma and the side effects of existing treatments, it would be desirable to have an improved method of treating ocular hypertension.

The invention

The present invention relates to an interfering RNA that reduces the expression of mRNA of Rho-kinase, reducing, thus, the intraocular pressure in patients with ocular hypertension or glaucoma or at risk of developing hypertension or glaucoma. Target Rho-kinase include ROCK1 (also known as ROCKI, ROKP or p160ROCK) and ROCK2 (also known as ROCKII or ROKα). Interfering RNA according to the invention is effective for the treatment of patients with ocular hypertension or glaucoma, such as normal pressure glaucoma and open-angle glaucoma.

One of the embodiments of the present invention relates to a method of reducing the mRNA expression of Rho-kinase in the individual. The method includes the administration to an individual a composition comprising an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier. In one embodiment, the implementation of the introduction is carried out in the eyes of the individual to reduce expression of the target in ocular hypertension in humans.

One option is to carry out the invention interfering RNA contains the semantic chain of nucleotides, chain antisense nucleotide and a region of at least near-absolute contiguous complementarity of at least 19 nucleotides. In addition, the antisense chain under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, which are semantic sequences of cDNA encoding ROCK1 and ROCK2, respectively (GenBank accession numbers NM_005406 and NM_004850, respectively). The antisense chain contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, respectively. The introduction of such composition reduces the expression of Rho-kinase in the individual.

In one variant of the invention, the interfering RNA is designed so that its purpose was to mRNA corresponding to SEQ ID NO:1 containing the 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. In another embodiment of the invention, the interfering RNA is designed so that its purpose was to mRNA corresponding to SEQ ID NO:2, containing the 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.

The present invention also relates to the introduction the human individual in addition to the first and second interfering RNA interfering RNA. The method includes the administration to an individual of the second interfering RNA with a length of 19 to 49 nucleotides and contains the semantic chain of nucleotides, chain antisense nucleotide and a region of at least near-absolute complementarity of at least 19 nucleotides; where the antisense chain of the second interfering RNA under physiological conditions hybridizes with the second part of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, and the antisense chain contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the second hybridization portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, respectively. The second interfering RNA may target the same mRNA, the first interfering RNA, or may have another target mRNA. Besides, this way you can enter the third, fourth, or fifth, etc. interfering RNA.

Another variant implementation of the invention is a method of reducing the expression of Rho-kinase in the individual, including the introduction of individual compositions containing an effective amount of single-stranded interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier.

To reduce the expression of ROCK1 single-stranded interfering RNA under physiological conditions hybridizes part mRNA, SEQ ID NO:1, containing the 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 interfering RNA contains a region of at least near-absolute complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1. Therefore, reduced expression of ROCK1.

To reduce the expression of ROCK2 single-stranded interfering RNA under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:2, containing the 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 interfering RNA contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:2. Therefore, reduced expression of ROCK2.

Another variant implementation of the invention is a method of treating ocular hypertension or glaucoma in a patient. The method includes introducing into the eyes of the individual compositions containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains the semantic chain of nucleotides, antisense chain of nucleotides and the region at IU the e almost absolute continuous complementarity, at least 19 nucleotides. The antisense chain under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, and contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, respectively. Thus the treatment of ocular hypertension or glaucoma.

Another variant implementation of the invention is a method of treating ocular hypertension or glaucoma in a patient, where the method includes introducing into the eyes of the individual compositions containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA containing the region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'end of an mRNA corresponding to any of SEQ ID NO:3 and SEQ ID NO:9 - SEQ ID NO:79 where therefore the treatment of ocular hypertension.

Another variant implementation of the invention is a method of reducing expression of a target mRNA Rho-kinase in the individual, including the introduction of individual compositions containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides, pharmaceutical and the Eski acceptable carrier, where interfering RNA contains a region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'end of an mRNA corresponding to any of SEQ ID NO:3 and SEQ ID NO:9 - SEQ ID NO:79, as described below.

If the target mRNA Rho-kinase is a ROCK1 mRNA, interfering RNA contains a region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'-end of 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.

If the target mRNA Rho-kinase is a ROCK2 mRNA, interfering RNA contains a region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'-end of 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 NO60, 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.

In an additional embodiment of the present invention, the scope of consecutive nucleotides represents a region of at least 14 consecutive nucleotides, at least 85% complementary, or at least 85% identical to the penultimate 14 nucleotides of the 3'-end of mRNA corresponding to a sequence with the sequence identifier. In another embodiment of the invention field of consecutive nucleotides is an area, usually from 15, 16, 17 or 18 consecutive nucleotides, at least 80% complementary, or at least 80% identical to the penultimate 15, 16, 17 or 18 nucleotides, respectively, 3'-end of mRNA corresponding to a target sequence defined by the sequence identifier.

An additional variant embodiment of the invention is a method of treating ocular hypertension in a patient, where the method includes the administration to an individual a composition comprising a molecule of double-stranded siRNAs that reduce the expression of ROCK1 or ROCK2 due to RNA interference, where the length of each chain molecules siRNAs independently is from about 19 to about 27 nucleotides; and one of the chain of mo is ecoli siRNAs contains a nucleotide sequence with substantial complementarity to the mRNA, the corresponding gene ROCK1 or ROCK2, respectively, so that the molecule siRNAs leads to cleavage of the mRNA due to RNA interference.

The embodiment of the present invention are compositions containing interfering RNA with a length of 19 to 49 nucleotides and nucleotide sequence of any of SEQ ID NO:3 and SEQ ID NO:9 - SEQ ID NO:79 or complementary to them and a pharmaceutically acceptable carrier. In one embodiment, the implementation of the interfering RNA is selected. The term "isolated" indicates that interfering RNA does not contain its natural environment.

Another embodiment of the invention are compositions containing a molecule of double-stranded siRNAs that reduce the expression of ROCK1 or ROCK2 due to RNA interference, where the length of each chain molecules siRNAs independently is from about 19 to about 27 nucleotides; and one of the chain molecules siRNAs contains a nucleotide sequence with substantial complementarity to an mRNA corresponding to the gene ROCK1 or ROCK2, respectively, so that the molecule siRNAs leads to cleavage of the mRNA due to RNA interference.

The present invention has the advantage compared to low molecular weight inhibitors of Rho-kinase, as an unwanted side effect of modern molecular medicine the funds, for example, blood can be separated from the desirable effect of reducing intraocular pressure.

Any of the embodiments, as described herein, in getting medicines to reduce the mRNA expression ROCK1 or ROCK2 is also a variant implementation of the present invention.

Brief description of drawings

Figure 1 is provided "Western"blot ROCK1 cell GTM-3, transfected with siRNAs ROCK1 № 1, № 2, № 3 and № 4; siRNAs ROCK2 № 1, № 2, № 3 and № 4; group siRNAs ROCK1; omnidirectional control siRNAs; control buffer (without siRNAs). The concentration of siRNAs was 100 nm. Arrows indicate the position of the strips 160 kDa protein ROCK1 and 42 kDa protein actin.

In figure 2, is provided "Western"blot ROCK1 cell GTM-3, transfected with siRNAs ROCK1 № 1, № 2, № 3 and № 4, and directional control siRNAs, each at 10 nm, 1 nm and 0.1 nm, and a control buffer (without siRNAs). Arrows indicate the position of the strips 160 kDa protein ROCK1 and 42 kDa protein actin.

Figure 3 is provided "Western"blot ROCK2 cell GTM-3, transfected with siRNAs ROCK2 № 1, № 2, № 3 and № 4, a ROCK1 pool, and directional control siRNAs, each at 100 nm, and a control buffer (without siRNAs). Arrows indicate the position of the strips 160 kDa protein ROCK2 and 42 kDa protein actin.

Figure 4 provided "Western"blot ROCK2 cell GTM-3, transfected with siRNAs ROCK2 № 1, № 2, № 3 and № 4, and NEAP Alanna control siRNAs, each at 10 nm, 1 nm, and 0.1 nm, and a control buffer (without siRNAs). Arrows indicate the position of the strips 160 kDa protein ROCK2 and 42 kDa protein actin.

Detailed description of the invention

RNA interference (RNC) is a process by which double-stranded RNA (ds) is used to suppress gene expression. Although the need to be bound by theory no, RNC begins with the cleavage of longer ds small interfering RNA (siRNAs) is similar to the RNase III enzyme, daystrom. SiRNAs are ds, the length of which generally ranged from approximately 19 to 28 nucleotides, or from 20 to 25 nucleotides, or from 21 to 22 nucleotides and which often contain "sticky" 3'-ends of the 2 nucleotides and 5'phosphate and 3'-hydroxyl ends. One strand siRNAs built into ribonucleoprotein complex known as induced RNA inhibitory complex (RISC). RISC uses this circuit siRNAs to identify mRNA molecules that are at least partially complementary integrated circuit siRNAs, and then splits this mRNA target or inhibits its translation. Thus, the circuit siRNAs, which is incorporated into the RISC, known as the chain guide or antisense chain. The other strand siRNAs, known as the " chain-passenger or meaning circuit, is removed from siRNAs, and she, at least partially homologous to the mRNA target. Experts who am in this area is clear, that, in principle, be incorporated into RISC and to function as a guide chain, every chain siRNAs. However, the design of siRNAs (e.g., reduced stability of duplex siRNAs at the 5'end of the antisense chain) may help to embed in RISC antisense chain.

Mediated RISC cleavage of the mRNA sequence at least partially complementary to the chain guide, leads to lower steady state level of this mRNA and the corresponding protein encoded by this mRNA. Alternatively, RISC can also reduce the expression of the corresponding protein due to repression of translation without cleavage of the mRNA target. With RISC can also interact and suppress the expression of other genes, RNA molecules, and such RNA molecules. Examples of other RNA molecules that interact with RISC include korotkosrochnye RNA (CSRC), single-stranded siRNAs, microRNAs (MCMC) and is a substrate of disera 27-membered duplexes. As used herein, unless otherwise indicated, the term "siRNAs" refers to double-stranded interfering RNA. Examples of such RNA molecules that can interact with RISC include RNA molecules containing one or more chemically modified nucleotides, one or more deoxyribonucleotides and/or one or more links that is different from f spadefish. For purposes of this discussion, all RNA molecules or such RNA molecules that can interact with RISC and participate in RISC mediated changes in gene expression, referred to as the "interfering RNA". Thus, siRNAs, CSRC, MCMC and which is the substrate of disera 27-membered duplexes are subgroups "interfering RNA".

It is proved that interfering RNA according to variants of the invention the catalytically acts on the cleavage of the mRNA target, i.e. interfering RNA capable of inhibiting mRNA target in substochiometric quantities. Compared with the treatments with application of antisense structures, to ensure a therapeutic effect in such conditions, the splitting should be significantly less than interfering RNA.

The present invention relates to the use of interfering RNA for inhibition of the mRNA expression of Rho-kinase (ROCK), thereby reducing intraocular pressure in patients with glaucoma. There are two isoforms of Rho-kinase: ROCK1 (also known as ROCKI, ROKβ or p160ROCK) and ROCK2 (also known as ROCKII or ROKα). As described herein, in accordance with the present invention interfering RNA provided exogenously or expressed endogenously, especially effective for suppressing mRNA ROCK.

Discobolus the s ROCK inhibitors cause reversible changes in the morphology and organization of the cytoskeleton of the cells of the trabecular network, reduce the contractility of the isolated tissue of the ciliary muscles and increase the ease of aqueous outflow in organ culture. Similar effects occur when the expression of dominant-negative Rho-binding domain. Processing of low molecular weight inhibitors ROCK reduces IOP, however, proved that this treatment also causes hyperaemia. Low molecular weight inhibitors of ROCK, tested to date, in addition to ROCK1 and ROCK2 inhibit multiple kinases. Expect that the use of interfering RNA of the present invention with specificity for mRNA ROCK1 or ROCK2 will allow you to separate the desirable effect of reducing IOP from unwanted effect processing hyperemia.

Unless otherwise specified in the present document nucleic acid sequence recorded in the direction from 5' to 3'. As used herein, the term "nucleic acid" refers to, or to DNA or RNA, or modified forms containing purine or pyrimidine bases present in DNA (adenine A, cytosine C, guanine "G," thymine "T") or RNA (adenine A, cytosine C, guanine G, uracil "U"). Provided hereunder interfering RNA may contain base "T", especially at the 3'-ends, despite the fact that the Foundation of the "T" is not found in RNA in nature. "Nucleic acid includes the terms "oligonucleotide" and "polynucleotide" and can refer to single-stranded molecule or to a double-stranded molecule. Double-stranded molecule is formed by mating grounds for the Watson-Crick between the bases A and T bases C and G and between the bases A and U. the Chain double-stranded molecules can be partially, substantially or fully complementary to each other and can form a hybrid duplex, the strength of binding depends on the nature and degree of complementary base sequence.

The sequence of an mRNA is easily deduced from the sequence of the corresponding DNA sequence. For example, SEQ ID NO:1 represents the semantic chain DNA sequence corresponding to the mRNA ROCK1. The mRNA sequence identical to the sequence of the sense DNA strand replacing grounds "T" at the base of the "U". Thus, the sequence of ROCK1 mRNA is known from SEQ ID NO:1, and the sequence ROCK2 mRNA is known from SEQ ID NO:2.

mRNA Rho kinase (ROCK1 and ROCK2): associated with Rho containing the double helix protein kinase, also known as Rho-kinase or ROCK, are effectors of the Rho family of small GTP-binding proteins (Rho-GTPase). There is evidence that the transmission signal Rho-GTPase plays a role in the regulation of aqueous outflow, for example, by changing the organization of the cytoskeleton of the trabecular network (TC) and/or cells of the ciliary muscle (CM).

ROCK are serine/Trets the NIN protein kinase, which are activated by GTP-bound Rho. The ROCK activation leads to the phosphorylation of several substrates involved in Assembly of actin filaments and airway cells, including, for example, light chain of myosin, light chain phosphatase myosin, LIM-kinase, adducin, ERM. Thus, ROCK regulate a wide variety of cellular processes, including the formation of fiber tension, contraction, adhesion, migration, phagocytosis, apoptosis and cytokines. The two ROCK isoforms are ROCK1 (also known as ROCKI, ROKβ or p160ROCK) and ROCK2 (also known as ROCKII or ROKα). Two isoforms are very similar, especially in their kinase domains (92% identity at the amino acid level), however, they show differences in tissue distribution and subcellular localization, which indicates that they can have a special, not duplicate functions. And ROCK1 and ROCK2 are expressed in the anterior part of the human eye.

In the GenBank database of the National Center for Biotechnology Information at ncbi.nlm.nih.gov given the DNA sequence for ROCK1 as inventory numbers NM_005406 contained in the List of sequences as SEQ ID NO:1. SEQ ID NO:1 is a sequence of semantic chain DNA corresponding to the mRNA that encodes ROCK1 (except for the replacement of the base of the "T" at the base of the "U"). The coding sequence of ROCK1 comprise nucleotides 1-4065.

Equivalent to the above sequence ROCK1 mRNA are forms of alternative splicing, allelic forms, isozyme or related forms. Related is ROCK1 mRNA of other mammalian species that is homologous to SEQ ID NO:1 (ortholog).

In the database of GenBank presents the DNA sequence for ROCK2 in the form of inventory number NM_ 004850 contained in the List of sequences as SEQ ID NO:2. SEQ ID NO:2 is the sequence of semantic chain DNA corresponding to the mRNA that encodes ROCK2 (except for the replacement of the base of the "T" at the base of the "U"). The coding sequence ROCK2 comprise nucleotides 450-4616.

Equivalents of the above sequence ROCK2 mRNA are forms of alternative splicing, allelic forms, isozyme or related forms. Related is ROCK2 mRNA of other mammalian species that is homologous to SEQ ID NO:2 (ortholog).

The decrease in the mRNA expression:As used herein, the phrase "decrease in the expression of mRNA" means the introduction or expression of the number of interfering RNA (e.g., siRNAs) for reduction of the mRNA of the target protein or by cleavage of the mRNA, or by direct inhibition of translation. Decrease in the expression of mRNA of the target or the corresponding protein is usually referred to as "knockdown" and are relative to the levels that exist after the introduction or expression of directional control RNA (for example, nenapirali the Naya control siRNAs). Options for implementation under this document provides knockdown expression in size from 50% to 100% inclusive. However, in the framework of the present invention is not necessary to have achieved such levels of knockdown. In one of the embodiments to reduce IOP enter one interfering RNA directed to one of the targets of Rho-kinase. In other embodiments implement to reduce IOP enter two or more interfering RNA directed to the same target Rho-kinase (for example, ROCK1). In other embodiments implement to reduce IOP enter two or more interfering RNA directed to both targets of Rho-kinases (for example, ROCK1 and ROCK2).

As a rule, knockdown assessed by measuring the levels of mRNA using amplification when quantitative polymerase chain reaction (qPCR) or by measuring the levels of protein for Western-blot or enzyme-linked immunosorbent analysis (ELISA). Analysis of the protein level provides an estimate of the cleavage of the mRNA and inhibiting translation. Additional ways to measure knockdown include hybridization of RNA solution, protection from nucleases, "Northern blot"hybridization, monitoring gene expression using microarray, antibody binding, radioimmunoassay analysis and fluorescence activated cell.

Inhibition of ROCK1 or ROCK2 you can also define ain vitr assessing the levels of mRNA targets or levels of the protein target, for example, in TC cells after transfection interfering RNA for ROCK1 or ROCK2, as described below.

Specified herein inhibition targets also assume in human and mammal when observing improve symptoms of glaucoma, such as, for example, improved intraocular pressure, improvements in the loss of field of view or improvements in the changes of the optic nerve.

Interfering RNA:In one variant of the invention, the interfering RNA (e.g., siRNAs) contains a sense chain and antisense chain, and sense and antisense chains contain a region of at least near-absolute contiguous complementarity of at least 19 nucleotides. In an additional embodiment of the invention interfering RNA (e.g., siRNAs) contains a sense chain and antisense chain and antisense chain contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the sequence of the target mRNA ROCK1 or ROCK2, but the semantic chain contains a region of at least near-absolute continuous identity, at least 19 nucleotides with the sequence of the target mRNA ROCK1 or ROCK2, respectively. In an additional embodiment, Khujand is the implementation of the invention interfering RNA contains a region of at least 13, 14, 15, 16, 17 or 18 consecutive nucleotides with a certain percentage of complementarity of sequences or with a certain percentage of sequence identity with, the penultimate 13, 14, 15, 16, 17 or 18 nucleotides, respectively, 3'-end corresponding to a target sequence within the mRNA.

The length of each chain interfering RNA ranges from 19 to 49 nucleotides and may be 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides.

The antisense chain siRNAs is an active guiding factor siRNAs in which the antisense chain is built into RISC, thus allowing RISC to identify mRNA target at least partial complementarity to the antisense chain siRNAs for cleavage or repression of translation.

In the variants of implementation of the present invention to a target sequence of the interfering RNA (e.g., a target sequence siRNAs) within a target sequence of mRNA is chosen using available design tools. Then interfering RNA corresponding to the target sequence ROCK1 or ROCK2, tested by transfection of cells expressing mRNA target, and the subsequent evaluation of knockdown as described above.

Methods selection of sequences are targets for siRNAs provide the s in Tuschl, T. et al., "The siRNA User Guide", revised may 6, 2004, available on the website of the Rockefeller University; Technical Bulletin No. 506, "siRNA Design Guidelines", Ambion Inc. on the website Ambion; and others available on the web site design tools, for example, on web sites Invitrogen, Dharmacon, Integrated DNA Technologies, Genscript or Proligo. The initial search parameters may include the content of G/C from 35% to 55% and the length of siRNAs from 19 to 27 nucleotides. The sequence of the target may be in a coding region or 5'- or 3'-untranslated regions of mRNA.

An implementation option 19-nucleotide sequence of the target DNA to mRNA ROCK1 is in terms of nucleotide SEQ ID NO:1 from 605 to 623:

5'-ATAACATGCTGCTGGATAA-3' SEQ ID NO:3.

siRNAs according to the invention, directed to the corresponding mRNA sequence SEQ ID NO:3 and containing 21-nucleotide chain and 2-nucleotide "sticky" 3'-ends, represents:

5'-AUAACAUGCUGCUGGAUAANN-3' SEQ ID NO:4

3'-NNUAUUGUACGACGACCUAUU-5' SEQ ID NO:5.

Each residue "N" can be any nucleotide (A, C, G, U, T) or modified nucleotide. 3'-end may contain some remnants of "N" between and including 1, 2, 3, 4, 5 and 6. The remains of the "N" on each chain may be the same residue (e.g., UU, AA, CC, GG or TT) or they may be different (for example, AC, AG, AU, CA, CG, CU, GA, GC, GU, UA, UC, or UG). "Sticky" 3'-ends can be the same or they may be different. In one embodiment, the wasp is estline both chains contain "sticky" 3'-ends UU.

siRNAs according to the invention, directed to the corresponding mRNA sequence SEQ ID NO:3 and containing 21-nucleotide chain and "sticky" 3'-ends UU on each chain represents:

5'-AUAACAUGCUGCUGGAUAAUU-3' SEQ ID NO:6

3'-UUUAUUGUACGACGACCUAUU-5' SEQ ID NO:7.

Interfering RNA may also contain a "sticky" nucleotide 5'-end, or it may have blunt ends. siRNAs according to the invention, directed to the corresponding mRNA sequence SEQ ID NO:3 and containing 19-nucleotide chain and blunt ends, represents:

5'-AUAACAUGCUGCUGGAUAA-3' SEQ ID NO:80

3'-UAUUGUACGACGACCUAUU-5' SEQ ID NO:81.

Chain double-stranded interfering RNA (e.g., siRNAs) can connect to the formation of a hairpin or structure of the stem-loop (for example, CSRC). CSRC according to the invention, directed to the corresponding mRNA sequence SEQ ID NO:2 and containing double-stranded region of the stem 19 P.N. and "sticky" 3'-end of the UU represents:

N is nucleotide A, T, C, G, U or modified form known to the person skilled in the art. The number of N nucleotides in the loop is a number from 3 to 23, or from 5 to 15, or from 7 to 13, or from 4 to 9, or 9 to 11, inclusive, or the number of nucleotides N is 9. Some of the nucleotides in the loop may be involved in interactions with the formation of pairs of nucleotide the other nucleotides of the loop. Examples of oligonucleotide sequences that can be used to form a loop, include 5'-UUCAAGAGA-3' (Brummelkamp, T.R. et al (2002) Science 296:550) and 5'-UUUGUGUAG-3' (Castanotto, D. et al. (2002) RNA 8:1454). The person skilled in the art it is clear that the resulting single-stranded oligonucleotide forms the structure of the stem-loop or hairpin containing double-stranded region, interacting with the mechanism RNC.

Sequence-target siRNAs indicated above, can be extended at the 3'-end to facilitate the construction of which is the substrate of disera 27-membered duplexes. The lengthening of the 19-nucleotide sequence of the target DNA (SEQ ID NO:3)shown in the DNA sequence of ROCK1 (SEQ ID NO:1) of 6 nucleotides, 25-nucleotide sequence of the target DNA, in terms of nucleotide SEQ ID NO:1 from 605 to 629;

5'-ATAACATGCTGCTGGATAAATCTGG-3' SEQ ID NO:82.

Which is the substrate of disera 27-membered duplex according to the invention, directed to the corresponding mRNA sequence SEQ ID NO:10, is:

5'-AUAACAUGCUGCUGGAUAAAUCUGG-3' SEQ ID NO:83

3'-UUUAUUGUACGACGACCUAUUUAGACC-5' SEQ ID NO:84.

Two nucleotides on the 3'-end of the sense circuit (i.e. the GG nucleotides of SEQ ID NO:83) can be deoxynucleotide for improved processing. The design which is the substrate of disera 27-membered duplexes from sequences of target DL is Noah 19-21 nucleotides, such as is provided in this document is discussed further on the web site Integrated DNA Technologies (IDT) and Kim, D.-H. et al., (February 2005) Nature Biotechnology 23:2; 222-226.

When interfering RNA receive chemical synthesis, phosphorylation at position 5' of the nucleotide at the 5'end of one or both chains (when present) can increase the efficiency and specificity of siRNAs associated RISC complex, but it is not necessarily as phosphorylation can occur intracellularly.

The table lists examples of target sequence DNA ROCK1 and ROCK2 of SEQ ID NO:1 and SEQ ID NO:2, respectively, of which design siRNAs of the present invention mentioned above. ROCK1 and ROCK2 encode two isoforms of Rho-kinase, as described above.

40
Table
Target sequence ROCK1 and ROCK2 for siRNAs
Target sequence ROCK1No. starting nucleotide relative to SEQ ID NO:1SEQ ID NO:
ATAACATGCTGCTGGATAA6053
GTACTTGTATGAAGATGAA6539
GTATGAAGATGAATAAGGA59 10
TAGCTCCAATGCAGATAAA124811
ATCAGTTGGAAGACTTAAA156212
GACCTTCAAGCTCGAATTA187613
GAACATTTGACTGGAAATA226614
TAGCTCAGCTTACGAAACA247415
ACGAAACAGTATAGAGGAA248516
TTTGAATTGACGCAAGAAA274017
CACTGTTAGTCGGCTTGAA280818
ACAGCATGCTAACCAAAGA283419
GTTAACAAATTGGCAGAAA300720
ACCAGATGGTAGTGAAACA314621
GTAGAAGAATGTGCACATA319922
GCAAAGAGAGTGATATTGA 324523
GTACCAAATAGAGGAAATA337924
GTTCTATAATGACGAACAA345325
GATAAACTGTTTCACGTTA351126
TAAACTGTTTCACGTTAGA351327
GTTTCACGTTAGACCTGTA351928
TGTCGAAGATGCCATGTTA378129
GTCGAAGATGCCATGTTAA378230
AACGACATCTCTTCTTCAA99873
GAAGAAACATTCCCTATTC113274
TAGCAATCGTAGATACTTA120075
GCCAATGACTTACTTAGGA164876
GGACACAGCTGTAAGATTG167477
GAGATGAGCAAGTCAATTA 170878
GTAACCAAAGCTCGTTTAA207779
Target sequence ROCK2No. starting nucleotide relative to SEQ ID NO:2SEQ ID NO:
ACAACATGCTCTTGGATAA110231
TGTTAATACTCGCCTAGAA186532
GAAAGCTGATCATGAAGCA200033
CAGCTGGAATCTAACAATA222934
GATATGACATACCAACTAA251435
AGGCACGACTAGCAGATAA258436
ATTAGACTGTGACCTCAAA273837
GATGATGGCTAGACACAAA330538
CTAAAGAAATTCCAAGGAT411139
TCGTATTCTTCCAGTGAAA4652
TTGCAACTATGCACTTGTA518441
CAACTATGCACTTGTATAA518742
GTTGCATGTTCATGTTTAA525543
TTCCTAATGCTTCATGATA531544
CTAGCTTTGTGGAAGATAA543945
GAAGATAAATCGTGCACTA545046
CCTTGATGTCTGTCTATTA557847
CTTGATGTCTGTCTATTAT557948
TTTACAGACCTCAGTATTA561149
TATTAGTCTGTGACTACAA562550
TAAATATGATCCTCAGACA579551
CAGCAATGGTAAGCGTAAA600052
CTCCGTCTCTACCAATATA6228 53
TGATGGTGGTGGCCTGTAA626454
CTTGCTGGATGGCTTAAAT58455
GGATTCACTTGTAGGAACA133756
TCATCGGATTTACCTACTA167857
TAAATGAGCTCCTTAAACA277358
GTTAGAAACCTGACATTAA281459
ATAACCATCTCATGGAAAT294160
TCTCTTGAGGAAACTAATA335761
CAATCTTGCAAATGAGAAA339862
TAAGCGCAGCAGCTATTAA348163
GAGAATAGAAAGCTACATA363364
GCTACATATGGAGCTTAAA364465
CTACATATGGAGCTTAAAT3645 66
GATGACATTGGACAGTAAA376767
TCTGGATAGTTCCAGTATA383668
GAACAATCCAATCCTTACA402369
GTATAGAGCAGATGCTAAA409770
ATAAAGCCATAATGTTGGA520271
TAGCTTTGTGGAAGATAAA544072

As indicated in the examples above, the person skilled in the art can use the information to a target sequence, are listed in the table to construct interfering RNA with a length shorter or longer than the sequences presented in the table, and on the basis of the provisions of the sequence in SEQ ID NO:1 or SEQ ID NO:2 and adding or deleting nucleotides, complementary or nearly complementary to SEQ ID NO:1 or SEQ ID NO:2, respectively.

The reaction of cleavage of the RNA target-directed siRNAs and other forms of interfering RNA is highly specific in respect of the sequences. For specified herein inhibition of mRNA variants of implementation of the siRNAs are basically containing a chain of semantic nucleotide siRNAs, identical in sequence to part of the mRNA target and antisense chain of nucleotides that is completely complementary to part of the mRNA target. However, 100% complementarity of the sequences between the antisense chain siRNAs and mRNA target or between the antisense chain siRNAs and semantic chain siRNAs in the practical implementation of the present invention is not required. Thus, for example, the invention provides for variations in the sequence that can be expected due to genetic mutation engine, polymorphism chain or evolutionary divergence.

In one of the embodiments of the invention the antisense chain siRNAs has at least almost absolute contiguous complementarity of at least 19 nucleotides with the mRNA target. As used herein, "almost absolute" means that the antisense chain siRNAs "substantially complementary", and the semantic chain siRNAs "substantially identical" at least part of an mRNA target. As is known to the person skilled in the art, "identity" represents the degree of relatedness of the sequences from nucleotide sequences, as determined by the coincidence of the order and identity of the nucleotides in the sequences. In one of the embodiments the antisense chain siRNAs with 80% and from 80% to 100% complementary the spine, for example, 85%, 90% or 95% complementarity with the sequence of the target mRNA believe almost completely complementary and can be used in the present invention. "Absolute" continuous complementarity is a standard base pairing according to the Watson-Crick neighboring base pairs. As used herein, "at least almost absolute continuous complementarity includes absolute complementarity. To determine the highest degree of coincidence of the nucleotide sequences of the developed computer methods for determining identity or complementarity, for example, BLASTN (Altschul, S.F., et al. (1990) J. Mol. Biol. 215:403-410).

The term "percent identity" means the percentage of consecutive nucleotides of the first nucleic acid molecule, which coincide with the next consecutive nucleotides of the same length in the second nucleic acid molecule. The term "percentage of complementarity" refers to the percentage of consecutive nucleotides of the first nucleic acid molecule, which can be paired bases on Watson-Crick with a number of consecutive nucleotides of the second nucleic acid molecule.

The relationship between mRNA target (sense circuit) and one of the chains siRNAs (sense circuit) is a relation of identity. Semantic chain is siRNAs, if it is present, also referred to as chain-passenger. The relationship between mRNA target (sense circuit) and the other chain siRNAs (antisense chain) represents the ratio of complementarity. The antisense chain siRNAs also known guide chain.

The penultimate base in the nucleic acid sequence, which is written in the direction from 5' to 3', is following the last basis, i.e. the basis of the following base on the 3'-end. The penultimate 13 bases of the nucleic acid sequence recorded in the direction from 5' to 3'represent the last 13 bases of the sequence following the last base at the 3'end, and not including the base at the 3'-end. Similarly, the penultimate 14, 15, 16, 17, or 18 bases of the nucleic acid sequence recorded in the direction from 5' to 3'represent the last 14, 15, 16, 17, or 18 bases of a sequence, respectively, following the base at the 3'-end and not including the base at the 3'-end.

The phrase "region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'end of an mRNA corresponding to any one of (the IDs of the sequences)allows a single nucleotide substitution. Two substitutions nucleot the species (i.e. 11/13=85% identity/complementarity) are not included in this phrase.

In one of the embodiments of the invention field of consecutive nucleotides represents a region of at least 14 consecutive nucleotides, at least 85% complementary, or at least 85% identical to the penultimate 14 nucleotides of the 3'-end of mRNA corresponding to a sequence defined by each of the sequence identifier. Two substitutions of nucleotides (i.e 12/14=86% identity/complementarity) in the following sentence is included.

In an additional embodiment of the invention field of consecutive nucleotides represents an area of at least 15, 16, 17 or 18 consecutive nucleotides, at least 80% sequence complementarity to, or at least 80% sequence identity with, the penultimate 14 nucleotides of the 3'-end of mRNA corresponding to a sequence with the sequence identifier. In such a phrase included the replacement of three nucleotides.

The sequence of the target mRNA corresponding to SEQ ID NO:1 or SEQ ID NO:2, may be located in the 5'- or 3'-untranslated regions of mRNA, as well as in the coding region of mRNA.

One or both strands of double-stranded interfering RNA may contain "sticky" 3'-ends of a length of from 1 to 6 nucleotides that m is may be ribonucleotides or deoxyribonucleotides or a mixture. The nucleotide sticky ends are not coupled. In one of the embodiments of the invention interfering RNA contains "sticky" 3'-ends of the TT or UU. In another embodiment of the invention, the interfering RNA comprises at least one blunt end. The ends usually contain 5'-phosphate group or a 3'-hydroxyl group. In other embodiments, implementation of the antisense chain contains 5'-phosphate group, and a semantic chain contains a 5'-hydroxyl group. In other embodiments, implementation of the ends of the optionally modified by covalent addition of other molecules or functional groups.

Sense and antisense chain of the double-stranded siRNAs can be in duplex configuration of two single circuits, as described above, or can be a single molecule, where the region of complementarity to form a base pair and covalently linked by a hairpin loop with the formation of a single chain. I believe that the pin inside the cell is cleaved protein called daystrom, with the formation of interfering RNA of two separate RNA molecules with dual bases.

Interfering RNA may differ from naturally occurring RNA by the addition, deletion, substitution or modification of one or more nucleotides. With interfering RNA at the 5'-end 3'-end or isotrivial to be associated non-nucleotide material. Such modifications are intended primarily to increase the sustainability of interfering RNA to nucleases, improvement of cell capture, improvement of cell orientation, to facilitate detection of interfering RNA, to improve stability or to reduce the potential activation pathway of interferon. For example, the interfering RNA may contain purine nucleotides at the ends of the "sticky" ends. For example, the conjugation of cholesterol to the 3'end of the sense chain molecules siRNAs through pyrolidine linker also ensures the stability of siRNAs.

Additional modifications include, for example, the 3'-end Biotin molecule, a peptide known as having the properties of penetration into the cell, nanoparticles, peptidomimetic, a fluorescent dye or a dendrimer.

The nucleotides can be modified in the main part, sugar or phosphate part of their molecules, and they can function in the variants of implementation of the present invention. For example, modifications include replacement of alkyl, alkoxy, amino, death, halogen, hydroxyl, thiol groups or a combination thereof. The nucleotides can be replaced by analogues with greater stability, for example, as a replacement ribonucleotide deoxyribonucleotides, or bearing modification of sugars, such as 2'-OH g is PI, substituted 2'-amino, 2'-O-methyl groups, 2'-methoxyaniline groups or methylene bridge between the 2'-O and 4'-C. Examples of purine or pyrimidine nucleotide analogues include xanthine, gipoksantin, aspurin, methylthioadenosine, 7-deazaadenosine and O - and N-modified nucleotides. The phosphate group of the nucleotide can be modified by replacing one or more oxygen atoms of the phosphate group, nitrogen or sulphur (phosphorothioate). Modifications are useful, for example, to enhance function, improve stability or permeability, or to indicate the location or orientation.

In the circuit antisense interfering RNA can be a region or regions that are not complementary to a portion of SEQ ID NO:1 or SEQ ID NO:2. Complementary region may be located on the 3', 5' or both ends of the region of complementarity or between two complementary regions.

Interfering RNA can be obtained exogenous chemical synthesis, transcriptionin vitroor by cleavage of longer double-stranded RNA disera or other appropriate nuclease with similar activity. Chemically synthesized interfering RNA derived from protected ribonucleopeptide conventional synthesizer DNA/RNA can be obtained from commercial suppliers, such as Ambion Inc. (Austin, TX), Invitrogen (Carlsbad, CA) or Dharacon (Lafayette, CO). Interfering RNA purified, for example, extraction with a solvent or resin, precipitation, electrophoresis, chromatography or a combination thereof. Alternatively, the interfering RNA can be used without special purification or no it in order to avoid losses due to sample processing.

Interfering RNA can also Express endogenous with plasmid or virus expressing vectors or with minimal expressing cassettes, for example, obtained by means of PCR fragments containing one or more promoters and the corresponding matrix or matrix interfering RNA. Examples of commercially available based plasmids expressing vectors for CSRC include representatives of the series pSilencer (Ambion, Austin, TX) and pCpG-siRNAs (InvivoGen, San Diego, CA). Viral vectors for the expression of the interfering RNA can be obtained from a variety of viruses, including adenovirus, adeno-associated virus, lentivirus (e.g., HIV, FIV and EIAV) and the herpes virus. Examples of commercially available viral vectors for the expression of CSRC include pSilencer adeno (Ambion, Austin, TX) and pLenti6/BLOCK-iT™-DEST (Invitrogen, Carlsbad, CA). The choice of viral vectors methods of expression of interfering RNA with vector and methods of delivery of viral vectors well-known specialist in this field. Examples of sets to obtain formed during PCR ekspressiruyushchie CSRC include Silencer Express (Ambion, Austin, TX) and siXpress (cool simple point pointers rubber, Madison, WI). The first interfering RNA can be entered by the expressionin vivofirst expressing vector capable of expression of the first interfering RNA, and the second interfering RNA can be entered by the expressionin vivofrom the second expressing vector capable of expression of the second interfering RNA, or both interfering RNA can be entered by the expressionin vivowith one expressing vector capable of expression of both interfering RNA.

Interfering RNA can be expressed from a variety of eukaryotic promoters known to specialists in this field, including the promoters of pol III, such as promoters U6 or H1, or the promoters of pol II, such as the promoters of cytomegaloviruses. Specialists in this field it is clear that these promoters can also be adapted to provide induced expression of interfering RNA.

Hybridization under physiological conditions:In certain embodiments of the implementation of the present invention the antisense chain interfering RNA hybridizes with mRNAin vivoas part of the RISC complex.

"Hybridization" refers to the process in which single-stranded nucleic acids with complementary or nearly complementary sequence of bases interact with education is the use of hydrogen bonded complexes, called hybrids. The hybridization reaction is sensitive and selective.In vitrothe specificity of hybridization (i.e. severity) control, for example, the concentration of salt or formamide in solutions for prehybridization and hybridization and hybridization temperature; such methods are well known in this field, in particular the severity increases with decrease in the concentration of salt, increasing the concentration of formamide or raising the hybridization temperature.

For example, conditions of high stringency can occur in about 50% formamide at a temperature of from 37°C to 42°C. Conditions with reduced severity can occur in approximately 35% to 25% formamide at a temperature from 30°C to 35°C. Examples of conditions of stringency for hybridization are provided in Sambrook, J., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. further examples of stringent conditions include hybridization 400 mm NaCl, 40 mm PIPES pH of 6.4, 1 mm EDTA, 50°C or 70°C for 12-16 hours followed by washing, or hybridization at 70°C in 1×SSC or 50°C in 1×SSC, 50% formamide followed by washing at 70°C in 0.3×SSC, or hybridization at 70°C in 4×SSC or 50°C in 4×SSC, 50% formamide followed by washing at 67°C in 1×SSC. The hybridization temperature is about 5-10°C less than the melting temperature (Tm) of the hybrid, where Tmdetermine hibri the impressive length of 19 to 49 base pairs using the following calculation: T m°C=81,5+16,6(log10[Na+])+0,41(% G+C)-(600/N), where N represents the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer.

The above analysis of hybridizationin vitroprovides a way to predict whether the binding between the candidate siRNAs and target specific. However, in the area of the RISC complex can also occur specific cleavage of the antisense chain that does not see high stringency hybridizationin vitro.

Single-stranded interfering RNA:As stated above, interfering RNA, ultimately functioning as a single chain. It is revealed that single-stranded (o/C) interfering RNA carries the suppression of mRNA, albeit less efficiently than double-stranded siRNAs. Thus, embodiments of the present invention also relates to the introduction of the o/C interfering RNA, which in physiological conditions hybridizes with a part of SEQ ID NO:1 or SEQ ID NO:2 and contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of SEQ ID NO:1 or SEQ ID NO:2, respectively. O/C interfering RNA has a length of 19 to 49 nucleotides, and d/C siRNAs shown above. O/C interfering RNA contains a 5'-phosphate or fosfauriliruetsain situorin vivo in position 5'. "phosphorylated at the 5'-end" is used, for example, to describe polynucleotides or oligonucleotides with a phosphate group attached via ester bonds to the C5-hydroxyl of the sugar (e.g. ribose, desoxyribose or their equivalent) at the 5'end of polynucleotide or oligonucleotide.

O/C interfering RNA synthesized chemically or by transcriptionin vitroor Express endogenous with vectors, or expressing cassettes, and for d/C interfering RNA. 5'-phosphate groups can be added using the kinase or 5'-phosphate may be a result of nuclease cleavage of RNA. Delivery is made as to d/C interfering RNA. In one embodiment, implementation o/C interfering RNA-protected ends and modifications for resistance to nucleases injected to suppress. O/C interfering RNA can be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts for inhibiting annealing or for stabilization.

Hairpin interfering RNA:Hairpin interfering RNA is a separate molecule (for example, separate oligonucleotide chain), containing both sense and antisense chain interfering RNA in a structure of the stem-loop or hairpin (for example, CSRC). For example, CSRC can Express is encoded with a vector DNA, in which oligonucleotides DNA encoding semantic interfering chain RNA by a short spacer connected with DNA oligonucleotides encoding the back of the complementary chain antisense interfering RNA. If you are selected for expressing the vector, you can add a 3'-terminal T and the nucleotides forming the areas of restriction. The resulting RNA transcript folds himself with the formation of the structure of the stem-loop.

Route of administration:interfering RNA can be delivered, for example, aerosol, buccal, dermal, intradermal, inhalation, intramuscular, intranasal, intraocular, intra-lungs, intravenous, intraperitoneally, nazalnam, ocular, oral, ear, parenteral, focal, subcutaneous, sublingual, local or transdermal way of introduction.

Interfering RNA can be delivered directly into the eye through an injection in the eye tissues, such as periocular, conjunctival, subtenancy, cell, intravitreal, intraocular, subretinal, subconjunctival, or retro-bulbar vnutrikvartalniy injection; by direct application to the eye using a catheter or other device application, such as a retinal pellet, intraocular insert, suppose the oriy or implant, containing porous or non-porous gelatin material; by topical ocular drops or ointments or through devices with a slow release in a blind pouch or implanted next to the sclera (transscleral) or in the eye. Cell injection can be performed through the cornea into the anterior chamber to allow the tool to reach the trabecular network. Vnutrikapilliarnuu injection can be performed in venous collective tubules, draining schlemmov channel or schlemmov channel.

Individual:The individual with the necessity of treatment of ocular hypertension or at risk of developing ocular hypertension is a human or other mammal with ocular hypertension or risk of ocular hypertension associated with undesirable or inappropriate expression or activity specified in this document targets, i.e. ROCK1 or ROCK2. The structure of the eye associated with such violations may include, for example, the eye, the retina of the eye choroid, lens, cornea, trabecular network, iris, optic nerve, optic nerve, sclera, anterior or posterior segments or ciliary body. The target may also be a cell of the eye, a cell culture eye, organ, or organ or tissue of the eyeex vivo.

The composition and dosages:Formats ticheskie formulations contain interfering RNA or a salt thereof according to the invention to 99 wt.%, mixed with a physiologically acceptable carrier medium, such as water, buffer, saline, glycine, hyaluronic acid, mannitol, etc.

Interfering RNA according to the present invention is administered in the form of solutions, suspensions or emulsions. The following represents examples of possible compositions used in this invention.

The amount in wt.%.
Interfering RNAto 99; 0,1-99; 0,1-50; 0,5-10,0
The hypromellose0,5
Sodium chloride0,8
The benzalkonium chloride0,01
EDTA0,01
NaOH/HClin quantity, sufficient for pH 7,4
Purified water (without RNase)in quantity, sufficient for 100 ml

The amount in wt.%.
Interfering RNAto 99; 0,1-99; 0,1-50; 0,5-10,0
Phosphate-saline buffer1,0
The benzalkonium chloride0,01
Polysorbate 800,5
Purified water (without RNase)quantity sufficient for 100%

The amount in wt.%.
Interfering RNAto 99; 0,1-99; 0,1-50; 0,5-10,0
Monobasic sodium phosphate0,05
Dibasic sodium phosphate (anhydrous)0,15
Sodium chloride0,75
EDTA sodium0,05
Cremophor EL0,1
The benzalkonium chloride0,01
HCl and/or NaOHpH 7.3 to 7.4
Purified water (without RNase)quantity sufficient for 100%

The amount in wt.%.
Interfering RNAto 99; 0,1-99; 0,1-50; 0,5-10,0
Phosphate-saline buffer1,0
Hydroxypropyl-β-cyclodextrin4,0
Purified water (without RNase)quantity sufficient for 100%

Typically, an effective amount of interfering RNA according to variants of the invention results in an extracellular concentration at the surface of target cells from 100 PM to 1 μm, or from 1 nm to 100 nm, or from 5 nm to approximately 50 nm, or about 25 nm. The dose required to achieve this local concentration varies depending on a number of factors, including method of delivery, parcel delivery, number of cell layers between the parcel delivery and cell-target or target-tissue, whether local or systemic delivery, etc. are the Concentration at the site of delivery may be significantly greater than on the surface of target cells or target tissue. Topical compositions are delivered to the surface of the target organ from one to four times per day or extended delivery Protocol, such as the hedgehog the day, weekly, biweekly, monthly, or longer, in accordance with the appropriate choice of a qualified clinical medical doctor. the pH of the composition is from about pH 4 to pH 9 or pH 4.5 to a pH of 7.4.

Expect therapeutic treatment of patients interfering RNA directed against mRNA ROCK1 or ROCK2, will have an advantage over treatment with low molecular weight compounds due to the increased duration of action, thereby allowing less frequent dosing and greater patient compliance with the treatment regimen.

An effective amount of the composition may depend on such factors as, for example, age, race and gender of the individual, the severity of ocular hypertension, the velocity of transcript/protein of the target genes, the efficiency of the interfering RNA and the stability of the interfering RNA. In one embodiment, the implementation of the interfering RNA is delivered to the target organ tapicerki and she reaches containing mRNA ROCK1 or ROCK2 tissue, such as trabecular network, the retina or the optic nerve disc in therapeutic dose, thereby improving associated with ocular hypertension pathological process.

Acceptable media:Acceptable media refers to media that the biggest cause from slight irritation of the mucous membrane of the eye to the lack of irritation, if the well is but provide suitable preservation and deliver one or more interfering RNA according to the present invention in a homogenous dosage. Acceptable media for the introduction of interfering RNA on the modalities for the implementation of the present invention includes cationic reagents for lipid transfection using TransIT®-TKO (cool simple point pointers rubber Corporation, Madison, WI), lipofectin (LIPOFECTIN®), lipofectamine (Lipofectamine), oligofectamine (mRNA for™) (Invitrogen, Carlsbad, CA) or dharmafect (DHARMAFECT™) (Dharmacon, Lafayette, CO); polycation, such as polyethyleneimine; cationic peptides, such as Tat, polyalanine or penetratin (Antp peptide); or liposomes. Liposomes are formed from standard form vesicles of lipids and Sterol, such as cholesterol, and may include, for example, the guide molecule such as a monoclonal antibody with affinity binding to surface antigens of endothelial cells. In addition, liposomes can be a pegylated liposomes.

Interfering RNA can be delivered in solution, suspension or in bioerodible or aboratively media. Interfering RNA can be delivered separately or as complexes of certain covalent conjugates. Interfering RNA can also form complexes with cationic lipids, cationic peptides or cationic polymers, complexes with proteins, fusion proteins or belcoville with the binding properties of nucleic acids (e.g., Protamine); or they can be encapsulated in nanoparticles or liposomes. You can make tone - or cell-specific delivery by incorporating the guiding groups, such as the antibody or antibody fragment.

For delivery to the eye interfering RNA can be combined with ophthalmologist acceptable preservatives, auxiliary solvents, surfactants, amplifiers viscosity, amplifiers suction, buffers, sodium chloride or water with the formation of aqueous, sterile ophthalmic suspension or solution. Composition of solutions can be obtained by dissolving interfering RNA in a physiologically acceptable isotonic aqueous buffer. In addition, the solution can include acceptable surfactant to assist in dissolving the inhibitor. To improve retention of connection to the compositions of the present invention can be added thickening agents such as hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose, polyvinylpyrrolidone or the like.

To obtain sterile ophthalmic ointment compositions interfering RNA is combined with a preservative in an appropriate medium such as mineral oil, liquid lanolin, or medical vaseline. Sterile ophthalmic gel the composition which can be obtained by suspension interfering RNA in a hydrophilic base, for example, derived from a combination of carbopol-940 (CARBOPOL®-940) (BF Goodrich, Charlotte, NC) or the like, known in the field of ways. For example, intraocular injection, you can use viscosit (VISCOAT®) (Alcon Laboratories, Inc., Fort Worth, TX). Other compositions of the present invention may contain amplifiers suction, such as cremophor and TWEEN® 80 (polyoxyethylene sorbitan monolaurate, Sigma Aldrich, St. Louis, MO), in the case when the interfering RNA worse absorbed in the eye.

Sets:In embodiments implementing the present invention is provided a kit comprising reagents for suppression of expression of mRNA in the cell, as described above. Set contains expressing siRNAs or CSRC vector. For expressing siRNAs vectors and expressing CSRC non-viral vectors the set also contains a reagent for transfection or other suitable delivery medium. For expressing CSRC viral vectors set may contain viral vector and/or required to obtain a viral vector components (for example, packing cell line and the vector containing the matrix of viral vector and additional vectors-helpers for packaging). The kit may also contain positive and negative control expressing siRNAs or CSRC vectors (e.g., omnidirectional control siRNAs or siRNAs directed at pastor NYY mRNA). The kit may also contain reagents for determination of knockdown of a specified gene target (for example, primers and probes for quantitative PCR for detection of mRNA of the target and/or antibodies to the corresponding protein for Western blotting). An alternative set may contain a sequence of siRNAs or sequence CSRC and instructions and materials necessary to obtain siRNAs through transcriptionin vitroor to construct expressing CSRC vector.

Additionally in the form of a kit is provided a pharmaceutical combination which is Packed kit includes bearing means adapted for maintaining the container means in a closed them together with the shell, and the first container means containing composition interfering RNA and an acceptable carrier. As can be easily understood by experts in this field, such sets, if desired, can additionally include one or more of various conventional components of pharmaceutical kits, such as, containers with one or more pharmaceutically acceptable carriers, additional containers, etc. In the set is also in the form of tabs or labels may contain printed instructions indicating the number of components for introduction; guidelines for the introduction and/or instructions for mixing the components.

<> The ability of interfering RNA to inhibit the levels of endogenous expression of the target genes, for example, in the cells of the trabecular network (TC) of a person appreciatein vitroas described below. Transformed cells TC man, for example, cell lines, denoted by the GTM-3 or HTM-3 (see, Pang, I.H. et at., 1994. Curr. Eye Res. 13:51-63), 24 hours before transfection were seeded in standard culture medium (for example, DMEM, supplemented with 10% fetal calf serum). The transfection performed using Dharmafect 1 (Dharmacon, Lafayette, CO) according to the manufacturer's instructions at a concentration of interfering RNA in the range from 0.1 nm to 100 nm. As negative and positive controls using omnidirectional siRNAs SiCONTROL™ No. 1 and siRNAs of cyclophilin B siCONTROL™ (Dharmacon), respectively. The levels of MRNA targets and levels of cyclophilin B mRNA (PPIB, NM_000942) assess via qPCR 24 hours after transfection using, for example, forward and reverse primers TAQMAN® and a set of probes, which preferably captures plot-target (Applied Biosystems, Foster City, CA). When the efficiency of transfection with 100% positive control siRNAs leads essentially to a complete knockdown of mRNA cyclophilin B mRNA. Thus, knockdown of mRNA target correct for transfection efficiency relative to the mRNA level of cyclophilin B in TC cells, transfected with siRNAs of cyclophilin B. Levels of Bel is and target estimate approximately 72 hours after transfection (the actual time depends on the velocity of the protein), for example, by Western blotting. Standard methods for isolating RNA and/or protein from the cultured cells are well known to specialists in this field. To reduce the likelihood of non-specific not determined target effects, use the lowest possible concentration of interfering RNA, which gives the desired level of knockdown of the expression of the target genes.

The ability of interfering RNA of the present invention to suppress the levels of expression of protein Rho-kinase is additionally illustrated in examples 1 and 2, as set forth below.

Example 1

Interfering RNA for specific suppression of ROCK1 in the cells of the trabecular network

In the present study validates the ability of interfering RNA to inhibit ROCK1 levels of endogenous ROCK1 expression in cultured glaucomatous cells of the trabecular network (TC) of a person.

Transfection of cells GTM-3 (Pang, I.H., et al. 1994 Curr Eye Res. 13:51-63) was performed using standard concentrations of siRNAs ROCK1 or ROCK2 or omnidirectional control siRNAsin vitro(100 nm) and reagent for transfection DHARMAFECT® No. 1 (Dharmacon, Chicago, IL). All siRNAs were dissolved in 1× buffer siRNAs, an aqueous solution of 20 mm KCl, 6 mm HEPES (pH 7.5), 0.2 mm MgCl2. The expression of ROCK1 protein was assessed through analysis of Western blotting 72 h after transfection. siRNAs ROCK1 are dwuhtsepochechny the th interfering RNA with specificity to the following targets: siROCK1 No. 1, directed to SEQ ID NO:23; siROCK1 No. 2, directed to SEQ ID NO:29; siROCK1 No. 3, directed to SEQ ID NO:10; siROCK1 No. 4, directed to SEQ ID NO:9. Sequence siROCK2 shown in example 2 below. As shown by data of the Western blot in figure 1, at 100 nm each of the four siRNAs ROCK1 reduced the expression of ROCK1 relatively omnidirectional control siRNAs. Directed to SEQ ID NO:29 siROCK1 No. 2 and directed to SEQ ID NO:10 siROCK1 No. 3 are particularly effective. siRNAs ROCK2 had a small, if not provided, the effect on the expression of ROCK1, confirming the specificity of siRNAs ROCK2 target ROCK2.

Conducted additional research using siRNAs with lower concentrations. Cell GTM-3 was transfusional siRNAs ROCK1 or omnidirectional control siRNAs at 10 nm, 1 nm and 0.1 nm, and the expression of the target genes was assessed through analysis of Western blotting 72 h after transfection. Control samples contained the control buffer in which the amount of siRNAs was replaced by an equal volume of 1× buffer siRNAs (without siRNAs). As shown by the data of figure 2, each of the four siRNAs ROCK1 significantly reduced the expression of ROCK1 protein at 10 nm and 1 nm, however, siROCK1 No. 2 is also relatively efficiently suppressed the expression of ROCK1 protein at 0.1 nm.

Example 2

Interfering RNA for specific suppression of ROCK2 in the cells of the trabecular network

In the present study validates the FPIC of the institutional capacity interfering RNA ROCK2 suppress the levels of endogenous expression of ROCK2 in cultured glaucomatous cells of the trabecular network (TC) of a person.

Transfection of cells GTM-3 (Pang, I.H., et al., 1994 Curr Eye Res. 13:51-63) was performed using standard concentrations of siRNAs ROCK1 or ROCK2 or omnidirectional control siRNAsin vitro(100 nm) and reagent for transfection DHARMAFECT® No. 1 (Dharmacon, Chicago, IL). The expression of ROCK2 protein was assessed through analysis of Western blotting 72 h after transfection. siRNAs ROCK2 are double-stranded interfering RNA with specificity to the following targets: siROCK2 No. 1, directed to SEQ ID NO:33; siROCK2 No. 2, directed to SEQ ID NO:38; siROCK2 No. 3, directed to SEQ ID NO:34; siROCK2 No. 4, directed to SEQ ID NO:39. As shown by data of the Western blot in figure 3, at 100 nm each of the four siRNAs ROCK2 reduced expression of ROCK2 relatively omnidirectional control siRNAs pool and relatively specific for ROCK1 siRNAs. A pool of siRNAs ROCK1 had a small, if not provided, the effect on the expression of ROCK2, confirming the specificity of siRNAs ROCK1 target ROCK1.

Conducted additional research using siRNAs with lower concentrations. Cell GTM-3 was transfusional siRNAs ROCK2 or omnidirectional control siRNAs at 10 nm, 1 nm and 0.1 nm, and the expression of the target genes was assessed through analysis of Western blotting 72 h after transfection. Control samples contained the control buffer in which the amount of siRNAs was replaced by an equal volume of 1× buffer siRNAs (without siRNAs). As shown by means of the CMV data figure 4, each of the four siRNAs ROCK2 significantly reduced the expression of ROCK2 protein at 10 nm and 1 nm, with siROCK2 No. 3, demonstrating greater than other efficiency.

Herein, references in those cases where they have provided for illustrative methodological or other details supplementary to those herein specifically incorporated by reference.

Specialists in this field taking into account the present description will understand that it is possible to carry out the obvious modifications of the embodiments described herein without deviating from the essence and scope of the invention. All options for the implementation described in this document, it is possible given the present description to carry out and perform without undue experimentation. The full scope of this invention are given in the description and is equivalent variants of implementation. The description should not be construed to excessive narrowing of the full scope of protection to which the present invention relates.

1. The method of reducing the expression of Rho-kinase by using interfering RNA, aimed at ROCK1 gene Rho-kinase for the treatment of eye diseases in the individual, including:
introduction to the individual a composition containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where Cerveira RNA contains:
the semantic chain of nucleotides, chain antisense nucleotide and a region of at least near-absolute contiguous complementarity of at least 19 nucleotides;
where the antisense chain under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:1, and contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1,
where thus reduce the mRNA expression of Rho-kinase.

2. The method according to claim 1, where the individual is a person and the person is ocular hypertension.

3. The method according to claim 1, where the individual is a human and humans there is a risk of ocular hypertension.

4. The method according to claim 1, where the composition is administered by local, intravitreal, transscleral, periocular, conjunctival, subtenons, cell, subretinal, subconjunctival, retro-bulbar or vnutrikorovogo way of introduction.

5. The method according to claim 1, where the antisense chain is designed for mRNA target corresponding to SEQ ID NO:1 containing the 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 according to claim 1, additionally including the introduction of individual second interferirajuce the RNA with a length of 19 to 49 nucleotides and contains the semantic chain of nucleotides, chain antisense nucleotide and a region of at least near-absolute complementarity of at least 19 nucleotides;
where the antisense chain of the second interfering RNA under physiological conditions hybridizes with the second part of mRNA corresponding to SEQ ID NO:1, and the antisense chain contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the second hybridization portion of mRNA corresponding to SEQ ID NO:1.

7. A method of treating ocular hypertension in a patient using interfering RNA, aimed at ROCK1 gene Rho-kinase, including:
introduction in the eyes of the patient a composition containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains:
the semantic chain of nucleotides, antisense chain of nucleotides, and a region of at least near-absolute contiguous complementarity of at least 19 nucleotides;
where the antisense chain under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:1, and contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1,
so treat eye hyperten the AI.

8. The way to reduce the mRNA expression of Rho-kinase in the individual by using interfering RNA, aimed at ROCK1 gene Rho-kinase for the treatment of eye diseases in the individual, including:
introduction to the individual a composition containing an effective amount of single-stranded interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier,
where single-stranded interfering RNA under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:1 containing the 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 interfering RNA contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with part hybridization of mRNA corresponding to SEQ ID NO:1, which thus reduces the mRNA expression of Rho-kinase.

9. The method of reducing the expression of mRNA target in ocular hypertension in individuals using interfering RNA, aimed at ROCK1 gene Rho-kinase, where the method includes:
introduction to the individual a composition containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains:
region of at least 13 consecutive nucleotides of at least 90% complementary, or minicamera 90% identical to the penultimate 13 nucleotides of the 3'-end of mRNA, corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79, where thus reduce the expression of mRNA target in ocular hypertension.

10. The method according to claim 9, where the mRNA target in ocular hypertension is a ROCK1 mRNA and interfering RNA contains: area of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'-end of 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.

11. The method according to claim 9, where the interfering RNA contains a region of at least 14 consecutive nucleotides, at least 85% complementary, or at least 85% identical to the penultimate 14 nucleotides of the 3'-end of mRNA corresponding to a sequence defined by the sequence identifier.

12. The method according to claim 9, where the interfering RNA contains an area of at least 15, 16, 17 or 18 consecutive nucleotides, at least 80% sequence complementarity to, or at least 80% sequence identity with, the penultimate 15, 16, 17 or 18 nucleotides, respectively, 3 konza mRNA, the corresponding sequence defined by the sequence identifier.

13. The method according to claim 9, where the composition further comprises a second interfering RNA with a length of 19 to 49 nucleotides and contains a region of at least 13 consecutive nucleotides, at least 90% complementarity, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3'-end of the mRNA corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79.

14. A method of treating ocular hypertension in a patient using interfering RNA, aimed at ROCK1 gene Rho-kinase, where the method includes:
introduction in the eyes of the individual compositions containing an effective amount of interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains:
region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'end of an mRNA corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79, so the treatment of ocular hypertension.

15. The method according to 14, where interfering RNA contains:
region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the pre the last 13 nucleotides of the 3'-end of 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.

16. The method according to 14, where interfering RNA contains a region of at least 14 consecutive nucleotides, at least 85% complementary, or at least 85% identical to the penultimate 14 nucleotides of the 3'-end of mRNA corresponding to a sequence defined by the sequence identifier.

17. The method according to 14, where interfering RNA contains an area of at least 15, 16, 17 or 18 consecutive nucleotides, at least 80% sequence complementarity to, or at least 80% sequence identity with, the penultimate 15, 16, 17 or 18 nucleotides, respectively, 3'-end of mRNA corresponding to a sequence defined by the sequence identifier.

18. The method according to 14, where the composition further comprises a second interfering RNA with a length of 19 to 49 nucleotides and contains a region of at least 13 consecutive nucleotides, at least 90% complementarity, or at least 90% sequence identity with, the penultimate 13 n what cleotide 3'-end of the mRNA, corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79.

19. The method according to 14, where the individual is present glaucoma.

20. The method according to claim 1, where the semantic chain of nucleotides and the antisense chain of nucleotides linked by a hairpin loop.

21. The method according to claim 7, where the semantic chain of nucleotides and the antisense chain of nucleotides linked by a hairpin loop.

22. The method according to claim 9, where the interfering RNA is CSRC.

23. The method according to claim 9, where the interfering RNA is a siPHK.

24. The method according to claim 9, where the interfering RNA is MCMC.

25. The method according to 14, where the interfering RNA is CSRC.

26. The method according to 14, where the interfering RNA is a siPHK.

27. The method according to 14, where the interfering RNA is MCMC.

28. The method according to claim 7, where the composition is administered by local, intravitreal, transscleral, periocular, conjunctival, subtenons, cell, subretinal, subconjunctival, retro-bulbar or vnutrikorovogo way of introduction.

29. The method according to claim 7, where the composition is administered by expression in vivo expressing interfering RNA vector.

30. The method according to 14, where the composition is administered by local, intravitreal, transscleral, periocular, conjunctival, subtenon is about, chamber, subretinal, subconjunctival, retro-bulbar or vnutrikorovogo way of introduction.

31. The method according to 14, where the composition is administered by expression in vivo expressing interfering RNA vector.

32. A method of treating ocular hypertension in a patient using interfering RNA, aimed at ROCK1 gene Rho-kinase, including:
introduction to the individual a composition containing a molecule of double-stranded siPHK reducing the expression of ROCK1 gene due to RNA interference, where:
the length of each chain molecules siPHK is independently about 19 to about 27 nucleotides; and one of the chain molecules siPHK contains a nucleotide sequence with substantial complementarity to an mRNA corresponding to the gene ROCK1, so that the molecule siPHK causes cleavage of mRNA due to RNA interference.

33. The method according to p, where the composition is administered aerosol, buccal, dermal, intradermal, inhalation, intramuscular, intranasal, intraocular, intra-lungs, intravenous, intraperitoneally, nazalnam, ocular, oral, ear, parenteral, focal, subcutaneous, sublingual, local or transdermal way of introduction.

34. The method according to p, where the interfering RNA is administered by the expression in vivo expressing vector, able is about to expression of interfering RNA.

35. The method according to p, where the interfering RNA is MCMC.

36. The method according to p, where the length of each chain molecules siPHK independently is from about 19 nucleotides to about 25 nucleotides.

37. The method according to p, where the length of each chain molecules siPHK independently is from about 19 nucleotides to about 21 nucleotides.

38. Application to obtain a composition for reducing expression of mRNA with ocular hypertension in the individual an effective amount of interfering RNA targeted at ROCK1 gene Rho-kinase, with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains:
region of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'end of an mRNA corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79.

39. Used to produce drugs for the treatment of ocular hypertension in the individual, the composition containing:
an effective amount of interfering RNA targeted to ROCK1 gene Rho-kinase, with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains: area of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% and anticol with the penultimate 13 nucleotides of the 3'-end of mRNA, corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79, so the treatment of ocular hypertension.

40. The application of § 38 or 39, where interfering RNA contains: area of at least 13 consecutive nucleotides, at least 90% complementary, or at least 90% identical to the penultimate 13 nucleotides of the 3'end of an mRNA corresponding to any of SEQ ID NO:3, SEQ ID NO:9-SEQ ID NO:30 and SEQ ID NO:73-SEQ ID NO:79.

41. The application of § 38 or 39, where interfering RNA contains a region of at least 14 consecutive nucleotides, at least 85% complementary, or at least 85% identical to the penultimate 14 nucleotides of the 3'-end of mRNA corresponding to a sequence defined by the sequence identifier.

42. The application of § 38 or 39, where interfering RNA contains an area of at least 15, 16, 17 or 18 consecutive nucleotides, at least 80% sequence complementarity or at least 80% sequence identity with, the penultimate 15, 16, 17 or 18 nucleotides, respectively, 3'-end of mRNA corresponding to a sequence defined by the sequence identifier.

43. The application of § 38 or 39, where the composition further comprises a second interfering RNA with a length of 19 to 49 nucleotides which contains a region of at least 13 consecutive nucleotides, at least 90% complementarity, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3'-end of the mRNA corresponding to any of SEQ ID NO:3 and SEQ ID NO:9-SEQ ID NO:79.

44. Used to produce drugs for the treatment of ocular hypertension in the individual, the composition comprising: an effective amount of interfering RNA targeted at ROCK1 gene Rho-kinase, with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA contains the semantic chain of nucleotides, antisense chain of nucleotides, and a region of at least near-absolute contiguous complementarity of at least 19 nucleotides;
where the antisense chain under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ ID NO:1, and contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1.

45. Application to obtain a composition for reducing the expression of mRNA of the gene ROCK1 Rho-kinase in the individual an effective amount of single-stranded interfering RNA with a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier,
where single-stranded interfering RNA under physiological conditions hybridizes with a portion of mRNA corresponding to SEQ IDNO:1, containing the 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 interfering RNA contains a region of at least near-absolute contiguous complementarity of at least 19 nucleotides with the hybridization part of the mRNA corresponding to SEQ ID NO:1.

46. The application of item 44, where the semantic chain of nucleotides and the antisense chain of nucleotides linked by a hairpin loop.

47. Use PP, 39 or 44, where the interfering RNA is CSRC.

48. Use PP, 39 or 44, where the interfering RNA is a siPHK.

49. Use PP, 39 or 44, where the interfering RNA is MCMC.

50. Application by § 39 or 44, where the composition is to get local, intravitreal, transscleral, periocular, conjunctival, subtenons, cell, subretinal, subconjunctival, retro-bulbar or vnutrikorovogo way of introduction.

51. Application by § 39 or 44, where the composition comprises expressing vector capable of expression of interfering RNA.

52. Application by § 39 or 44, where the drug is intended for treatment of ocular hypertension.

53. Used to produce drugs for the treatment of ocular hypertension in the individual compositions, with whom containing a series:
the double-stranded molecule siPHK reducing the expression of ROCK1 gene due to RNA interference, where:
the length of each chain molecules siPHK is independently about 19 to about 27 nucleotides; and
one of the chains of molecules siPHK contains a nucleotide sequence with substantial complementarity to an mRNA corresponding to the gene ROCK1, so that the molecule siPHK causes cleavage of mRNA due to RNA interference.

54. The application of item 53, where the composition was prepared for aerosol, buccal, dermal, intradermal, inhalation, intramuscular, intranasal, intraocular, intra-lungs, intravenous, intraperitoneal, nasal, ocular, oral, ear, parenteral, focal, subcutaneous, sublingual, local or transdermal route of administration.

55. The application of item 53, where the composition comprises expressing vector capable of expression of interfering RNA.

56. The application of item 53, where the interfering RNA is MCMC.



 

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EFFECT: strain has strong structural properties.

16 cl, 4 dwg, 6 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: method of producing cyclopropyl-condensed inhibitors of dipeptidyl peptidase IV involves using BOC-protected amine with structural formula (3) , obtained through reductive amination of acid with formula (1) by treating the said acid with ammonium formate, nicotinamide adenine dinucleotide, dithiothreitol and partially purified concentrate of phenyl alanine dehydrogenase and formate dehydrogenase (PDH/FDH) enzymes and without separation - by treating the obtained amine of formula (2) with ditertbutyl dicarbonate, obtaining BOC-protected amine.

EFFECT: cutting on costs.

13 cl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetic therapy and concerns nucleotide sequence that codes insulin-like human growth factor IGF-1 presented with a synthetic gene including a sequence SEQ ID NO:1, recombinant plasmid DNA, containing this sequence, an eukaryotic cell containing recombinant plasmid DNA, construction for genetic therapy and a pharmaceutical composition for genetic therapy with regenerative and wound healing action.

EFFECT: advantage of the invention consists in decreased doses and introduction rate of injected preparations.

5 cl, 2 ex, 2 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: present invention concerns to immunostimulating phosphorothioate CpG-oligonucleotides which can be used for stimulation of the immune response.

EFFECT: obtaining of CpG-oligonucleotides.

49 cl, 76 fig, 19 tbl, 34 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to ophthalmology. Ophthalmic fused protein formulations of a VEGF-specific antagonist are suitable for intravitreal injections. The ophthalmic formulation has stable liquid ingredients and lyophilizable ingredients. The ophthalmic formulation contains a protein antagonist, has amino acids 27-457 of the sequence SEQ ID NO:4, an organic solvent, a tonicity agent, a sodium phosphate buffer, and additionally, a stabilising agent.

EFFECT: invention provides stability of the ophthalmic formulation.

11 cl, 8 tbl, 4 dwg, 8 ex

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