Double stranded rna compounds to rhoa and use thereof

Abstract

The present invention relates to compounds, pharmaceutical compositions comprising same, methods of use thereof and kits for the down-regulation of RhoA gene. The compounds, compositions, methods and kits are useful in the treatment of subjects suffering from diseases or conditions and or symptoms associated with diseases or conditions in which RhoA expression has adverse consequences and for conferring neuroprotection.

That which is claimed is: 
     
         1 . A double stranded nucleic acid molecule comprising a sense strand and an antisense strand wherein the sense and antisense strands are selected from an oligonucleotide described as RHOA_48 (SEQ ID NOS: 79 and 113), RHOA_48u (SEQ ID NOS: 80 and 114), RHOA_50 (SEQ ID NOS: 82 and 116) or RHOA_58 (SEQ ID NOS: 143 and 157). 
     
     
         2 . A double stranded nucleic acid molecule of  claim 1  wherein the sequence of the antisense strand comprises SEQ ID NO:113 and the sequence of the sense strand comprises SEQ ID NO:79. 
     
     
         3 . A double stranded nucleic acid molecule of  claim 2 , wherein the antisense strand is SEQ ID NO:113 and includes (5′>3′) a mirror nucleotide or a 2′-5′ linked nucleotide in at least one of positions 5, 6, 7 or 8, and a non-nucleotide moiety covalently attached to the 3′ terminus; includes a mirror nucleotide or a 2′-5′ linked nucleotide in one or more of positions 5, 6, 7 or 8 (5′>3′), and a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus; and the sense strand is (SEQ ID NO:79) and includes 4 or 5 consecutive 2′-5′ linked nucleotides at the 3′ terminal or penultimate positions, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus, one or more 2′OMe sugar modified nucleotides, and a cap moiety covalently attached at the 5′ terminus. 
     
     
         4 . A double stranded nucleic acid molecule of  claim 3  wherein the antisense strand (SEQ ID NO:113) includes (5′>3′) 2′OMe sugar modified ribonucleotides at positions 1, 3, 11, 14, 15, 17 and 18, and a C3Pi-C3OH moiety covalently attached to the 3′ terminus; and the sense strand (SEQ ID NO:79) includes (5′>3′) 2′-5′ linked ribonucleotides at positions 15, 16, 17, 18 and 19, a 3′ terminal nucleotide or non-nucleotide overhang; and a cap moiety covalently attached at the 5′ terminus. 
     
     
         5 . A double stranded nucleic acid molecule of  claim 4  described as RHOA_48_S1833. 
     
     
         6 . A double stranded nucleic acid molecule of  claim 4  wherein the antisense strand further includes a 2′-5′ linked ribonucleotide at position 6, at position 7 or at positions 6 and 7. 
     
     
         7 . A double stranded nucleic acid molecule of  claim 6  described as RHOA_48_S1857. 
     
     
         8 . A double stranded nucleic acid molecule of  claim 6 , described as RHOA_48_S1873. 
     
     
         9 . A double stranded nucleic acid molecule of  claim 6  described as RHOA_48_S1856. 
     
     
         10 . A double stranded nucleic acid molecule of  claim 6  described as RHOA_48_S1872. 
     
     
         11 . A double stranded nucleic acid molecule of  claim 6  described as RHOA_48_S1858. 
     
     
         12 . A double stranded nucleic acid molecule of  claim 6  described as RHOA_48_S1859. 
     
     
         13 . A double stranded nucleic acid molecule of  claim 6  described as RHOA_48_S1860. 
     
     
         14 . A double stranded nucleic acid molecule of  claim 6  further comprising a Uridine (rU) to deoxyribothymidine (dT) substitution in position 1 of the antisense strand. 
     
     
         15 . A double stranded nucleic acid molecule of  claim 14  described as RHOA_48_S1884. 
     
     
         16 . A double stranded nucleic acid molecule of  claim 1  wherein the sequence of the antisense strand comprises SEQ ID NO:114 and the sequence of the sense strand comprises SEQ ID NO:80 
     
     
         17 . A double stranded nucleic acid molecule wherein the antisense strand (SEQ ID NO:114) includes (5′>3′) one or more 2′OMe sugar modified ribonucleotides, a mirror nucleotide or a 2′-5′ linked ribonucleotide in one or more of positions 5, 6, 7 or 8 (5′>3′), and a 3′ terminal nucleotide or non-nucleotide overhang; and the sense strand (SEQ ID NO:80) includes (5′>3′) 4 or 5 consecutive 2′-5′ linked nucleotides at the 3′ terminal or penultimate positions, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and a cap moiety covalently attached at the 5′ terminus. 
     
     
         18 . A double stranded nucleic acid molecule of  claim 17  described as RHOA_48u_S1812. 
     
     
         19 . A double stranded nucleic acid molecule of  claim 17  wherein the sense strand (SEQ ID NO:80) further includes one or more 2′OMe sugar modified ribonucleotides. 
     
     
         20 . A double stranded nucleic acid molecule of  claim 19  described as RHOA_48u_S1813. 
     
     
         21 . A double stranded nucleic acid molecule of  claim 19  described as RHOA_48u_S1870. 
     
     
         22 . A double stranded nucleic acid molecule of  claim 19  described as RHOA_48u_S1871. 
     
     
         23 . A double stranded nucleic acid molecule of  claim 1  wherein the sequence of the antisense strand comprises SEQ ID NO:116 and the sequence of the sense strand comprises SEQ ID NO:82. 
     
     
         24 . A double stranded nucleic acid molecule of  claim 23  wherein the antisense strand (SEQ ID NO:116) includes (5′>3′) a DNA, a mirror nucleotide or a 2′-5′ linked nucleotide in one or more of positions 5, 6, 7 or 8, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and one or more 2′OMe sugar modified nucleotides and wherein the sense strand (SEQ ID NO:82) includes (5′>3′) a mirror nucleotide or a 2′-5′ linked nucleotide in one or more of positions 9 or 10, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus, one or more 2′OMe sugar modified nucleotides, and a cap moiety covalently attached at the 5′ terminus. 
     
     
         24 . A double stranded nucleic acid molecule of  claim 24  described as RHOA_50_S1796. 
     
     
         25 . A double stranded nucleic acid molecule of  claim 24  described as RHOA_50_S1798. 
     
     
         26 . A double stranded nucleic acid molecule of  claim 24  described as RHOA_50_S1799. 
     
     
         27 . A double stranded nucleic acid molecule of  claim 24  described as RHOA_50_S1865. 
     
     
         28 . A double stranded nucleic acid molecule of  claim 24  described as RHOA_50_S1866. 
     
     
         29 . A double stranded nucleic acid molecule of  claim 1  wherein the sequence of the antisense strand comprises SEQ ID NO:157 and the sequence of the sense strand comprises SEQ ID NO:143. 
     
     
         30 . A double stranded nucleic acid molecule of  claim 29  wherein the antisense strand (SEQ ID NO:157) includes a mirror nucleotide or a 2′-5′ linked ribonucleotide in one or more of positions 5, 6, 7 or 8 (5′>3′), a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus and one or more 2′ OMe sugar modified nucleotides; and wherein the sense strand (SEQ ID NO:143) includes 4 or 5 consecutive 2′-5′ linked nucleotides at the 3′ terminal or penultimate positions or one or more 2′ OMe sugar modified nucleotides, a nucleotide or non-nucleotide moiety covalently attached at the 3′ terminus, and a cap moiety covalently attached at the 5′ terminus. 
     
     
         31 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1801. 
     
     
         32 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1804. 
     
     
         32 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1806. 
     
     
         33 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1861. 
     
     
         34 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1862. 
     
     
         35 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1877. 
     
     
         36 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1878. 
     
     
         37 . A double stranded nucleic acid molecule of  claim 30  described as RHOA_58_S1879. 
     
     
         38 . A double stranded nucleic acid molecule having the structure (A1): 
       
         
           
                 
                 
                 
                 
               
                     
                   (A1) 
                   5′ (N)x-Z 3′ 
                   (antisense strand) 
                 
                     
                     
                   3′ Z′-(N′)y-z″ 5′ 
                   (sense strand) 
                 
             
                
                
               
            
           
         
         wherein each N and N′ is a ribonucleotide which may be unmodified or modified, or an unconventional moiety; 
         wherein each of (N)x and (N′)y is an oligonucleotide in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond; 
         wherein each of Z and Z′ is independently present or absent, but if present independently comprises 1-5 consecutive nucleotides, 1-5 consecutive non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present; 
         wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of (N′)y; 
         each of x and y is independently an integer from 18 to 40; 
         wherein the sequence of (N′)y is complementary to the sequence of (N)x; and 
         wherein (N)x comprises an antisense sequence set forth in Table III. 
       
     
     
         39 . The double stranded nucleic acid molecule of  claim 38  wherein x=y=19. 
     
     
         40 . The double stranded nucleic acid molecule of  claim 38  or  39  wherein the sequence of the sense strand and the sequence of the antisense strand are selected from oligonucleotide pairs set forth in RHOA_31 (SEQ ID NOS:135 and 149), RHOA_33 (SEQ ID NOS:136 and 150), RHOA_37 (SEQ ID NOS:137 and 151), RHOA_38 (SEQ ID NOS:138 and 152), RHOA_43 (SEQ ID NOS:139 and 153), RHOA_52 (SEQ ID NOS:140 and 154), RHOA_56 (SEQ ID NOS:141 and 155), RHOA_57 (SEQ ID NOS:142 and 156), RHOA_58 (SEQ ID NOS:143 and 157), RHOA_68 (SEQ ID NOS:144 and 158), RHOA_69 (SEQ ID NOS:145 and 159), RHOA_70 (SEQ ID NOS:146 and 160), RHOA_73 (SEQ ID NOS:147 and 161) and RHOA_76 (SEQ ID NOS:148 and 162). 
     
     
         41 . A double stranded nucleic acid molecule of any one of  claims 38 - 40  wherein the molecule consists of unmodified ribonucleotides in the sense strand, in the antisense strand or in both the sense strand and the antisense strand. 
     
     
         42 . A double stranded nucleic acid molecule of any one of  claims 38 - 40  wherein the molecule comprises modified ribonucleotides and unconventional moieties in the sense strand, in the antisense strand or in both the sense strand and the antisense strand. 
     
     
         43 . A double stranded nucleic acid molecule having a structure (A2) set forth below: 
       
         
           
                 
                 
                 
                 
               
                     
                   (A2) 
                   5′ N1-(N)x-Z 3′ 
                   (antisense strand) 
                 
                     
                     
                   3′ Z′-N2-(N′)y-z″ 5′ 
                   (sense strand) 
                 
             
                
                
               
            
           
         
         wherein each N1, N2, N and N′ is independently an unmodified or modified nucleotide, or an unconventional moiety; 
         wherein each of (N)x and (N′)y is an oligonucleotide in which each consecutive N or N′ is joined to the adjacent N or N′ by a covalent bond; 
         wherein each of x and y is independently an integer between 17 and 39; 
         wherein N2 is covalently bound to (N′)y; 
         wherein N1 is covalently bound to (N)x and is mismatched to the target mRNA (SEQ ID NO:1) or is a complementary DNA moiety to the target mRNA; 
         wherein N1 is a moiety selected from the group consisting of natural or modified: uridine, deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or deoxyadenosine, an abasic ribose moiety and an abasic deoxyribose moiety; 
         wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′ terminus of N2-(N′)y; 
         wherein each of Z and Z′ is independently present or absent, but if present is independently 1-5 consecutive nucleotides, 1-5 consecutive non-nucleotide moieties or a combination thereof covalently attached at the 3′ terminus of the strand in which it is present; and 
         wherein the sequence of (N′)y is complementary to the sequence of (N)x; and 
         wherein the sequence of (N)x comprises an antisense sequence set forth in Table I. 
       
     
     
         44 . A double stranded nucleic acid molecule of  claim 43  wherein x=y=18. 
     
     
         45 . A double stranded nucleic acid molecule of  claim 43  or  44  wherein the sequence of the sense strand and the sequence of the antisense strand are selected from oligonucleotide pairs set forth in RHOA_32 (SEQ ID NOS:67 and 101), RHOA_34 (SEQ ID NOS:68 and 102), RHOA_35 (SEQ ID NOS:69 and 103), RHOA_36 (SEQ ID NOS:70 and 104), RHOA_39 (SEQ ID NOS:71 and 105), RHOA_40 (SEQ ID NOS:72 and 106), RHOA_41 (SEQ ID NOS:73 and 107), RHOA_42 (SEQ ID NOS:74 and 108), RHOA_44 (SEQ ID NOS:75 and 109), RHOA_45 (SEQ ID NOS:76 and 110), RHOA_46 (SEQ ID NOS:77 and 111), RHOA_47 (SEQ ID NOS:78 and 112), RHOA_48 (SEQ ID NOS:79 and 113), RHOA_48u (SEQ ID NOS:80 and 114), RHOA_49 (SEQ ID NOS:81 and 115), RHOA_50 (SEQ ID NOS:82 and 116) RHOA_51 (SEQ ID NOS:83 and 117), RHOA_53 (SEQ ID NOS:84 and 118), RHOA_54 (SEQ ID NOS:85 and 119), RHOA_55 (SEQ ID NOS:86 and 120), RHOA_59 (SEQ ID NOS:87 and 121), RHOA_60 (SEQ TD NOS:88 and 122), RHOA_61 (SEQ ID NOS:89 and 123), RHOA_61u (SEQ ID NOS:90 and 124), RHOA_62 (SEQ ID NOS:91 and 125), RHOA_63 (SEQ ID NOS:92 and 126) RHOA_64 (SEQ ID NOS:93 and 127), RHOA_65 (SEQ ID NO:94 and 128), RHOA_66 (SEQ ID NOS:95 and 129) RHOA_67 (SEQ ID NOS:96 and 130), RHOA_71 (SEQ ID NOS:97 and 131), RHOA_72 (SEQ ID NOS:98 and 132), RHOA_74 (SEQ ID NOS:99 and 133) and RHOA_75 (SEQ ID NOS:100 and 134). 
     
     
         46 . A double stranded nucleic acid molecule of any one of  claims 43 - 45  wherein the molecule consists of unmodified ribonucleotides in the sense strand, in the antisense strand or in both the sense strand and the antisense strand. 
     
     
         47 . A double stranded nucleic acid molecule of any one of  claims 43 - 45  wherein the molecule comprises modified ribonucleotides and unconventional moieties in the sense strand, in the antisense strand or in both the sense strand and the antisense strand. 
     
     
         48 . A double stranded nucleic acid molecule of  claim 38  or  43  selected from a molecule set forth in  FIG. 1   
     
     
         49 . A composition comprising a double stranded molecule of any one of  claims 1 - 48 ; and a pharmaceutically acceptable carrier. 
     
     
         50 . A composition comprising a double stranded molecule of any of the preceding claims packaged for use by a patient. 
     
     
         51 . The composition of  50 , wherein the composition includes a label or package insert that provides information about how said double stranded molecule of any of the preceding claims may be used. 
     
     
         52 . The composition of  claim 51 , wherein said label or package insert includes dosing information and/or indications for use. 
     
     
         53 . The composition of any of  claims 50 - 52 , wherein said label or package insert indicates that the double stranded molecule of any of the preceding claims is suitable for use in therapy. 
     
     
         54 . The composition of any of  claims 50 - 53 , wherein said label or package insert indicates that the double stranded molecule of any of the preceding claims is suitable for use in treating a patient suffering from a disease associated with RhoA. 
     
     
         55 . A double stranded nucleic acid molecule of any one of  claims 1 - 48  or a composition of  claim 49  for use in therapy. 
     
     
         56 . A double stranded nucleic acid molecule of any one of  claims 1 - 48  or a composition of  claim 49  for reducing the expression of RhoA in a cell. 
     
     
         57 . A method of reducing the expression of RhoA in a cell, comprising introducing into a cell a double stranded molecule of any of the preceding claims in an amount sufficient to reduce expression of RhoA. 
     
     
         58 . A method for treating an individual suffering from a disease associated with RhoA expression comprising administering to said individual a double stranded molecule of any of  claims 1 - 48  in an amount sufficient to reduce expression of RhoA. 
     
     
         59 . A method of treating a subject suffering from an injury, disease or disorder of the CNS comprising administering to the subject a double stranded molecule of any of  claims 1 - 48  which inhibits expression of RhoA in an amount effective to treat the disease or condition. 
     
     
         60 . A method for prevention, inhibition or treatment of neuronal degeneration caused by injury, disease, disorder or condition in the central nervous system (CNS) in an individual in need thereof, comprising administering to the individual an effective amount of a double stranded molecule of any of  claims 1 - 48 , thereby treating the injury, disease, disorder or condition. 
     
     
         61 . A method for effecting neuroprotection to a neuron damaged or at risk of damage by injury, disease, disorder or condition in the central nervous system (CNS) in an individual in need thereof, comprising administering to the individual an effective amount of a double stranded molecule of any of  claims 1 - 48 , thereby effecting neuroprotection. 
     
     
         62 . A method for effecting neural regeneration to a neuron damaged by injury, disease, disorder or condition in the central nervous system (CNS) in an individual in need thereof, comprising administering to the individual an effective amount of a double stranded molecule of any of  claims 1 - 48 , thereby effecting neural regeneration. 
     
     
         63 . The method of any one of  claims 60 - 62 , wherein said injury, disease, disorder or condition is selected from the group consisting of a neurological injury: neurodegenerative disease or disorder; brain damage caused by a cardiovascular event; cerebrovascular disorder or disease; dementia and hearing loss associated with neuronal degeneration. 
     
     
         64 . The method of  claim 63 , wherein said injury is a traumatic injury selected from the group consisting of traumatic brain injury (TBI) and spinal cord injury. 
     
     
         65 . The method of  claim 63 , wherein said injury, disease, disorder or condition is neurodegenerative disease or disorder. 
     
     
         66 . The method of  claim 63 , wherein said neurodegenerative disease or disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, facial nerve (Bell's) palsy, multiple sclerosis, glaucoma, Huntington's chorea, amyotrophic lateral sclerosis, Meniere's disease, glaucoma and macular degeneration. 
     
     
         67 . The method of  claim 63 , wherein said injury, disease, disorder or condition is brain damage caused by a cardiovascular event. 
     
     
         68 . The method of  claim 67 , wherein said cardiovascular event is cardiac arrest or cardiac surgery. 
     
     
         69 . The method of  claim 63 , wherein said injury, disease, disorder or condition is a cerebrovascular disorder or disease. 
     
     
         70 . The method of  claim 69 , wherein said cerebrovascular disorder is brain ischemia or stroke. 
     
     
         71 . The method of  claim 63 , wherein said injury, disease, disorder or condition is dementia. 
     
     
         72 . The method of  claim 71 , wherein said dementia is Alzheimer's disease or a non-Alzheimer's dementia selected from the group consisting of senile dementia, Lewy body dementia, vascular dementia and a dementia caused by Parkinson's disease, Huntington's chorea, Creutzfeld-Jacob disease (CJD) or HIV infection. 
     
     
         73 . A method for conferring neuroprotection to an individual afflicted with or at risk of developing neurodegeneration associated with a neurological disease, disorder, injury or condition, which comprises administering to said individual a double stranded molecule of any of  claims 1 - 48  or pharmaceutically acceptable salt thereof in an amount effective to ameliorate or prevent the neurodegeneration. 
     
     
         74 . The method of  claim 73  wherein the neurological disease is glaucoma, NAION or AMD. 
     
     
         75 . The method of  claim 73  wherein the neurodegeneration is neurodegeneration of the optic nerve. 
     
     
         76 . The method of  claim 73  wherein the neuroprotection conferred comprises the treatment of cerebral damage produced by thromboembolic (thrombotic) stroke or cranium-brain traumatic injuries. 
     
     
         77 . A method for conferring neuroprotection to the auditory nerve in an individual afflicted with Meniere's disease, which comprises administering to the individual a double stranded molecule of any of  claims 1 - 48  or pharmaceutically acceptable salt thereof in an amount effective to confer neuroprotection to the auditory nerve. 
     
     
         78 . A method for attenuating or preventing hearing loss in an individual afflicted with Meniere's disease, which comprises administering to said individual a double stranded molecule of any of  claims 1 - 48  or pharmaceutically acceptable salt thereof in an amount effective to ameliorate or prevent the hearing loss. 
     
     
         79 . The method of any of  claims 77 - 78  wherein the individual in need is afflicted with glaucoma or Meniere's disease. 
     
     
         80 . The method of any of  claims 77 - 79  wherein the compound is topically administered to the tympanic membrane of the individual. 
     
     
         81 . A cell comprising one or more of the a double stranded molecules of any of  claims 1 - 48 . 
     
     
         82 . A method for treating cancer or proliferative diseases in a subject in need thereof comprising administering to the subject a double stranded molecule of any of  claims 1 - 48  in an amount effective to treat the subject. 
     
     
         83 . A method for treating cancer, tumor growth or proliferative diseases in a subject in need thereof comprising administering to the subject a double stranded molecule of any of  claims 1 - 48  in an amount effective to treat the subject.