Antisense-oligonucleotides as inhibitors of tgf-r signaling

Abstract

The present invention relates to antisense-oligonucleotides having a length of at least 10 nucleotides, wherein at least two of the nucleotides are LNAs, their use as inhibitors of TGF-R signaling, pharmaceutical compositions containing such antisense-oligonucleotides and the use for prophylaxis and treatment of neurological, neurodegenerative, fibrotic and hyperproliferative diseases.

1 - 21 . (canceled) 
     
     
         22 . An antisense-oligonucleotide consisting of 10 to 28 nucleotides and at least two of the 10 to 28 nucleotides are LNAs, wherein the antisense-oligonucleotide has a gapmer structure with 1 to 5 LNA units at the 3′ terminal end and 1 to 5 LNA units at the 5′ terminal end and the antisense-oligonucleotide hybridizes with a region of the gene encoding the TGF-R II  or with a region of the mRNA encoding the TGF-R II , wherein the region of the gene encoding the TGF-R II  or the region of the mRNA encoding the TGF-R II  comprises the sequence TGGTCCATTC (Seq. ID No. 4), and the antisense-oligonucleotide comprises a sequence hybridizing with said sequence TGGTCCATTC (Seq. ID No. 4) and salts and optical isomers of said antisense-oligonucleotide, wherein the antisense-oligonucleotide inhibits the expression of TGF-R II . 
     
     
         23 . The antisense-oligonucleotide according to  claim 22 , wherein the antisense-oligonucleotide hybridizes selectively only with the sequence TGGTCCATTC (Seq. ID No. 4) of the region of the gene encoding the TGF-R II  or of the region of the mRNA encoding the TGF-R II . 
     
     
         24 . The antisense-oligonucleotide according to  claim 22 , wherein the antisense-oligonucleotide has a length of 12 to 20 nucleotides and/or wherein the antisense-oligonucleotide has phosphate, phosphorothioate and/or phosphorodithioate as internucleotide linkages. 
     
     
         25 . The antisense-oligonucleotide according to  claim 22 , wherein the antisense-oligonucleotide is represented by the following general formula (S6): 
       
         
           
                 
               
                   (Seq. ID No. 100) 
                 
                 
                 
               
                     
                   5′-N 7 - AATGGACC -N 8 -3′ 
                 
             
                
               
            
             
                
               
            
           
         
         wherein
 N 7  represents: TGAATCTTGAATATCTCATG- (Seq. ID No. 583), GAATCTTGAATATCTCATG- (Seq. ID No. 584), AATCTTGAATATCTCATG- (Seq. ID No. 585), ATCTTGAATATCTCATG- (Seq. ID No. 586), TCTTGAATATCTCATG- (Seq. ID No. 587), CTTGAATATCTCATG- (Seq. ID No. 588), TTGAATATCTCATG- (Seq. ID No. 589), TGAATATCTCATG- (Seq. ID No. 590), GAATATCTCATG- (Seq. ID No. 591), AATATCTCATG- (Seq. ID No. 592), ATATCTCATG- (Seq. ID No. 593), TATCTCATG-, ATCTCATG-, TCTCATG-, CTCATG-, TCATG-, CATG-, ATG-, TG-, or G-; and 
 N8 is selected from: AGTATTCTAGAAACTCACCA, (Seq. ID No. 594), AGTATTCTAGAAACTCACC, (Seq. ID No. 595), AGTATTCTAGAAACTCAC, (Seq. ID No. 596), AGTATTCTAGAAACTCA, (Seq. ID No. 597), AGTATTCTAGAAACTC, (Seq. ID No. 598), AGTATTCTAGAAACT, (Seq. ID No. 599), AGTATTCTAGAAAC, (Seq. ID No. 600), AGTATTCTAGAAA, (Seq. ID No. 601), AGTATTCTAGAA, (Seq. ID No. 602), AGTATTCTAGA, (Seq. ID No. 603), AGTATTCTAG, (Seq. ID No. 604), AGTATTCTA, AGTATTCT, AGTATTC, AGTATT, AGTAT, AGTA, AGT, AG, or A. 
 
       
     
     
         26 . The antisense-oligonucleotide according to  claim 22 , wherein the last 2 to 4 nucleotides at the 5′ terminal end are LNA nucleotides and the last 2 to 4 nucleotides at the 3′ terminal end are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the LNA nucleotides at the 3′ terminal end at least 6 consecutive nucleotides are present which are non-LNA nucleotides or which are DNA nucleotides. 
     
     
         27 . The antisense-oligonucleotide according to  claim 25 , wherein the last 2 to 4 nucleotides at the 5′ terminal end are LNA nucleotides and the last 2 to 4 nucleotides at the 3′ terminal end are LNA nucleotides and between the LNA nucleotides at the 5′ terminal end and the LNA nucleotides at the 3′ terminal end at least 6 consecutive nucleotides are present which are non-LNA nucleotides or which are DNA nucleotides. 
     
     
         28 . The antisense-oligonucleotide according to  claim 22 , wherein the LNA nucleotides are linked to each other through a phosphorothioate group or a phosphorodithioate group or wherein all nucleotides are linked to each other through a phosphate group or a phosphorothioate group or a phosphorodithioate group. 
     
     
         29 . The antisense-oligonucleotide according to  claim 25 , wherein the LNA nucleotides are linked to each other through a phosphorothioate group or a phosphorodithioate group or wherein all nucleotides are linked to each other through a phosphate group or a phosphorothioate group or a phosphorodithioate group. 
     
     
         30 . The antisense-oligonucleotide according to  claim 22 , wherein the LNA nucleotides are selected from the group consisting of: 
       
         
           
           
               
               
           
         
         wherein 
         IL′ represents —X″—P(═X′)(X − )—; 
         X′ represents ═O or ═S; 
         X represents —O − , —OH, —OR H , —NHR H , —N(R H ) 2 , —OCH 2 CH 2 OR H , —OCH 2 CH 2 SR H , —BH 3   − , —R H , —SH, —SR H , or —S − ; 
         X″ represents —O—, —NH—, —NR H —, —CH 2 —, or —S—; 
         Y is —O—, —NH—, —NR H —, —CH 2 — or —S—; 
         R C  and R H  are independently of each other selected from hydrogen and C 1-4 -alkyl; and 
         B represents a nucleobase selected from the group consisting of: 
         adenine, thymine, guanine, cytosine, uracil, 5-methylcytosine, 5-hydroxymethyl cytosine, N 4 -methylcytosine, xanthine, hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 6-ethyladenine, 6-ethylguanine, 2-propyladenine, 2-propylguanine, 6-carboxyuracil, 5,6-dihydrouracil, 5-propynyl uracil, 5-propynyl cytosine, 6-aza uracil, 6-aza cytosine, 6-aza thymine, 5-uracil, 4-thiouracil, 8-fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine, 8-bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-trifluoromethyluracil, 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, 5-iodocytosine, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine. 
       
     
     
         31 . The antisense-oligonucleotide according to  claim 25 , wherein the LNA nucleotides are selected from the group consisting of: 
       
         
           
           
               
               
           
         
         wherein 
         IL′ represents —X″—P(═X′)(X − )—; 
         X′ represents ═O or ═S; 
         X represents —O − , —OH, —OR H , —NHR H , —N(R H ) 2 , —OCH 2 CH 2 OR H , —OCH 2 CH 2 SR H , —BH 3   − , —R H , —SH, —SR H , or —S − ; 
         X″ represents —O—, —NH—, —NR H —, —CH 2 —, or —S—; 
         Y is —O—, —NH—, —NR H —, —CH 2 — or —S—; 
         R C  and R H  are independently of each other selected from hydrogen and C 1-4 -alkyl; and 
         B represents a nucleobase selected from the group consisting of: 
         adenine, thymine, guanine, cytosine, uracil, 5-methylcytosine, 5-hydroxymethyl cytosine, N 4 -methylcytosine, xanthine, hypoxanthine, 7-deazaxanthine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 6-ethyladenine, 6-ethylguanine, 2-propyladenine, 2-propylguanine, 6-carboxyuracil, 5,6-dihydrouracil, 5-propynyl uracil, 5-propynyl cytosine, 6-aza uracil, 6-aza cytosine, 6-aza thymine, 5-uracil, 4-thiouracil, 8-fluoroadenine, 8-chloroadenine, 8-bromoadenine, 8-iodoadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-fluoroguanine, 8-chloroguanine, 8-bromoguanine, 8-iodoguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-trifluoromethyluracil, 5-fluorocytosine, 5-bromocytosine, 5-chlorocytosine, 5-iodocytosine, 5-trifluoromethylcytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 3-deazaguanine, 3-deazaadenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine. 
       
     
     
         32 . The antisense-oligonucleotide according to  claim 22  having one of the following gapmer structures selected from the group consisting of: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, and 4-11-3. 
     
     
         33 . The antisense-oligonucleotide according to  claim 25  having one of the following gapmer structures selected from the group consisting of: 3-8-3, 4-8-2, 2-8-4, 3-8-4, 4-8-3, 4-8-4, 3-9-3, 4-9-2, 2-9-4, 4-9-3, 3-9-4, 4-9-4, 3-10-3, 2-10-4, 4-10-2, 3-10-4, 4-10-3, 4-10-4, 2-11-4, 4-11-2, 3-11-4, and 4-11-3. 
     
     
         34 . The antisense-oligonucleotide according to  claim 22 , wherein the antisense oligonucleotides bind with 100% complementarity to the mRNA encoding TGF-R II  and do not bind to any other region in the human transcriptome. 
     
     
         35 . The antisense-oligonucleotide according to  claim 25 , wherein the antisense oligonucleotides bind with 100% complementarity to the mRNA encoding TGF-R II  and do not bind to any other region in the human transcriptome. 
     
     
         36 . A method for promoting regeneration and functional reconnection of damaged nerve pathways and/or for treatment and compensation of age induced decreases in neuronal stem cell renewal comprising administering to a patient an antisense-oligonucleotide according to  claim 22 . 
     
     
         37 . A method for promoting regeneration and functional reconnection of damaged nerve pathways and/or for treatment and compensation of age induced decreases in neuronal stem cell renewal comprising administering to a patient an antisense-oligonucleotide according to  claim 25 . 
     
     
         38 . A method for prophylaxis and treatment of a disease selected from the group consisting of neurodegenerative diseases, neuroinflammatory disorders, traumatic or posttraumatic disorders, neurovascular disorders, hypoxic disorders, postinfectious central nervous system disorders, fibrotic diseases, hyperproliferative diseases, cancer, tumors, presbyakusis and presbyopia comprising administering to a patient an antisense-oligonucleotide according to  claim 22 . 
     
     
         39 . A method for prophylaxis and treatment of a disease selected from the group consisting of neurodegenerative diseases, neuroinflammatory disorders, traumatic or posttraumatic disorders, neurovascular disorders, hypoxic disorders, postinfectious central nervous system disorders, fibrotic diseases, hyperproliferative diseases, cancer, tumors, presbyakusis and presbyopia comprising administering to a patient an antisense-oligonucleotide according to  claim 25 . 
     
     
         40 . The method according to  claim 38 , wherein the neurodegenerative diseases and neuroinflammatory disorders are selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Creutzfeldt Jakob disease, new variant of Creutzfeldt Jakobs disease, Hallervorden Spatz disease, Huntington's disease, multisystem atrophy, dementia, frontotemporal dementia, motor neuron disorders, amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebellar atrophies, schizophrenia, affective disorders, major depression, meningoencephalitis, bacterial meningoencephalitis, viral meningoencephalitis, CNS autoimmune disorders, multiple sclerosis, acute ischemic/hypoxic lesions, stroke, CNS and spinal cord trauma, head and spinal trauma, brain traumatic injuries, arteriosclerosis, atherosclerosis, microangiopathic dementia, Binswanger' disease, retinal degeneration, cochlear degeneration, macular degeneration, cochlear deafness, AIDS related dementia, retinitis pigmentosa, fragile X associated tremor/ataxia syndrome, progressive supranuclear palsy, striatonigral degeneration, olivopontocerebellear degeneration, Shy Drager syndrome, age dependant memory deficits, neurodevelopmental disorders associated with dementia, Down's Syndrome, synucleinopathies, superoxide dismutase mutations, trinucleotide repeat disorders, trauma, hypoxia, vascular diseases, vascular inflammations, and CNS ageing and wherein the fibrotic diseases are selected from the group consisting of: pulmonary fibrosis, cystic fibrosis, hepatic cirrhosis, endomyocardial fibrosis, old myocardial infarction, atrial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid, systemic sclerosis, arthrofibrosis, Peyronie's disease, Dupuytren's contracture, and residuums after Lupus erythematodes. 
     
     
         41 . The method according to  claim 39 , wherein the neurodegenerative diseases and neuroinflammatory disorders are selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Creutzfeldt Jakob disease, new variant of Creutzfeldt Jakobs disease, Hallervorden Spatz disease, Huntington's disease, multisystem atrophy, dementia, frontotemporal dementia, motor neuron disorders, amyotrophic lateral sclerosis, spinal muscular atrophy, spinocerebellar atrophies, schizophrenia, affective disorders, major depression, meningoencephalitis, bacterial meningoencephalitis, viral meningoencephalitis, CNS autoimmune disorders, multiple sclerosis, acute ischemic/hypoxic lesions, stroke, CNS and spinal cord trauma, head and spinal trauma, brain traumatic injuries, arteriosclerosis, atherosclerosis, microangiopathic dementia, Binswanger' disease, retinal degeneration, cochlear degeneration, macular degeneration, cochlear deafness, AIDS related dementia, retinitis pigmentosa, fragile X associated tremor/ataxia syndrome, progressive supranuclear palsy, striatonigral degeneration, olivopontocerebellear degeneration, Shy Drager syndrome, age dependant memory deficits, neurodevelopmental disorders associated with dementia, Down's Syndrome, synucleinopathies, superoxide dismutase mutations, trinucleotide repeat disorders, trauma, hypoxia, vascular diseases, vascular inflammations, and CNS ageing and wherein the fibrotic diseases are selected from the group consisting of: pulmonary fibrosis, cystic fibrosis, hepatic cirrhosis, endomyocardial fibrosis, old myocardial infarction, atrial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, nephrogenic systemic fibrosis, Crohn's Disease, keloid, systemic sclerosis, arthrofibrosis, Peyronie's disease, Dupuytren's contracture, and residuums after Lupus erythematodes. 
     
     
         42 . A pharmaceutical composition comprising at least one antisense-oligonucleotide according to  claim 22  together with at least one pharmaceutically acceptable carrier, excipient, adjuvant, solvent or diluent.