Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Brain derived neurotrophic factor (BDNF), in particular, by targeting natural antisense polynucleotides of Brain derived neurotrophic factor (BDNF). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of BDNF.

What is claimed is: 
     
         1 . A method of modulating a function of and/or the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in a biological system comprising: contacting said system with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a natural antisense of a Brain derived neurotrophic factor (BDNF) polynucleotide; thereby modulating a function of and/or the expression of the Brain derived neurotrophic factor (BDNF) polynucleotide, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded. 
     
     
         2 . A method of modulating a function of and/or the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in a biological system according to  claim 1  comprising: contacting said biological system with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within the natural antisense transcript nucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ TD NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded. thereby modulating a function of and/or the expression of the Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         3 . A method of modulating a function of and/or the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in patient cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the Brain derived neurotrophic factor (BDNF) polynucleotide; thereby modulating a function of and/or the expression of the Brain derived neurotrophic factor (BDNF) polynucleotide in patient cells or tissues in vivo or in vitro, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded. 
     
     
         4 . A method of modulating a function of and/or the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in patient cells or tissues according to  claim 3  comprising: contacting said biological system with at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within the natural antisense transcript nucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded, thereby modulating a function of and/or the expression of the Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         5 . A method of modulating a function of and/or the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in a biological system comprising: contacting said system with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Brain derived neurotrophic factor (BDNF) polynucleotide; thereby modulating a function of and/or the expression of the Brain derived neurotrophic factor (BDNF) polynucleotide, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded. 
     
     
         6 . The method of  claim 5 , wherein a function of and/or the expression of the Brain derived neurotrophic factor (BDNF) is increased in vivo or in vitro with respect to a control. 
     
     
         7 . The method of  claim 5 , wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         8 . The method of  claim 5 , wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of a Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         9 . The method of  claim 5 , wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         10 . The method of  claim 5 , wherein the at least one antisense oligonucleotide comprises one or more modifications selected from: at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof. 
     
     
         11 . The method of  claim 10 , wherein the one or more modifications comprise at least one modified sugar moiety selected from: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof. 
     
     
         12 . The method of  claim 10 , wherein the one or more modifications comprise at least one modified internucleoside linkage selected from: a phosphorothioate, 2′-Omethoxyethyl (MOE), 2′-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof. 
     
     
         13 . The method of  claim 10 , wherein the one or more modifications comprise at least one modified nucleotide selected from: a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), an analogue, a derivative, and combinations thereof. 
     
     
         14 . The method of  claim 1 , wherein the at least one oligonucleotide comprises at least one oligonucleotide sequences set forth as SEQ ID NOS: 12 to 49. 
     
     
         15 . A method of modulating a function of and/or the expression of a Brain derived neurotrophic factor (BDNF) gene in mammalian cells or tissues in vivo or in vitro comprising: contacting said cells or tissues with at least one short interfering RNA (siRNA) oligonucleotide 5 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for an antisense polynucleotide of a Brain derived neurotrophic factor (BDNF) polynucleotide, wherein said at least one siRNA oligonucleotide has at least 50% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense nucleic acid molecule of the Brain derived neurotrophic factor (BDNF) polynucleotide; and, modulating a function of and/or the expression of Brain derived neurotrophic factor (BDNF) in mammalian cells or tissues in vivo or in vitro. 
     
     
         16 . The method of  claim 15 , wherein said oligonucleotide has at least 80% sequence identity to a sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         17 . An oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within the natural antisense transcript nucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded, and optionally further comprising at least one modification wherein the at least one modification is selected from: at least one modified sugar moiety; at least one modified internucleotide linkage; at least one modified nucleotide, and combinations thereof; wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a Brain derived neurotrophic factor (BDNF) gene in vivo or in vitro as compared to a normal control. 
     
     
         18 . The oligonucleotide according to  claim 17  wherein said oligonucleotide is 5 to 30 nucleotides in length and has at least 50% sequence identity to the reverse complement of 5-30 consecutive nucleotides within a natural antisense transcript of the BDNF gene. 
     
     
         19 . The oligonucleotide of  claim 18 , wherein the at least one modification comprises an internucleotide linkage selected from the group consisting of: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof. 
     
     
         20 . The oligonucleotide of  claim 18 , wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage. 
     
     
         21 . The oligonucleotide of  claim 18 , wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages. 
     
     
         22 . The oligonucleotide of  claim 18 , wherein the oligonucleotide comprises at least one modified nucleotide, said modified nucleotide selected from: a peptide nucleic acid, a locked nucleic acid (LNA), analogue, derivative, and a combination thereof. 
     
     
         23 . The oligonucleotide of  claim 18 , wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof. 
     
     
         24 . The oligonucleotide of  claim 18 , wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified nucleotides selected from: peptide nucleic acids, locked nucleic acids (LNA), analogues, derivatives, and a combination thereof. 
     
     
         25 . The oligonucleotide of  claim 18 , wherein the oligonucleotide comprises at least one modified sugar moiety selected from: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof. 
     
     
         26 . The oligonucleotide of  claim 18 , wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise modified sugar moieties selected from: a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and a combination thereof. 
     
     
         27 . The oligonucleotide of  claim 18 , wherein the oligonucleotide is of at least about 5 to 30 nucleotides in length and hybridizes to an antisense and/or sense strand of a Brain derived neurotrophic factor (BDNF) polynucleotide wherein said oligonucleotide has at least about 60% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequences of the Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         28 . The oligonucleotide of  claim 18 , wherein the oligonucleotide has at least about 80% sequence identity to a complementary sequence of at least about five consecutive nucleic acids of the antisense and/or sense coding and/or noncoding nucleic acid sequence of the Brain derived neurotrophic factor (BDNF) polynucleotide. 
     
     
         29 . The oligonucleotide of  claim 18 , wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Brain derived neurotrophic factor (BDNF) polynucleotide in vivo or in vitro, as compared to a normal control. 
     
     
         30 . The oligonucleotide of  claim 18 , wherein the oligonucleotide comprises the sequences set forth as SEQ ID NOS: 12 to 49. 
     
     
         31 . A pharmaceutical composition comprising one or more oligonucleotides specific for one or more Brain derived neurotrophic factor (BDNF) polynucleotides according to  claim 17  and a pharmaceutically acceptable excipient. 
     
     
         32 . The composition of  claim 31 , wherein the oligonucleotides have at least about 40% sequence identity as compared to any one of the nucleotide sequences set forth as SEQ ID NOS: 12 to 49. 
     
     
         33 . The composition of  claim 31 , wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 12 to 49. 
     
     
         34 . The composition of  claim 33 , wherein the oligonucleotides set forth as SEQ ID NOS: 12 to 49 comprise one or more modifications or substitutions. 
     
     
         35 . The composition of  claim 34 , wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof. 
     
     
         36 . A method of preventing or treating a disease associated with at least one Brain derived neurotrophic factor (BDNF) polynucleotide and/or at least one encoded product thereof, comprising: administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide 5 to 30 nucleotides in length wherein said at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides within the natural antisense transcript nucleotides 1 to 1279 of SEQ ID NO: 3 or 1 to 1478 of SEQ ID NO: 4 or 1 to 1437 of SEQ ID NO: 5 or 1 to 2322 of SEQ ID NO: 6 or 1 to 2036 of SEQ ID NO: 7 or 1 to 2364 of SEQ ID NO: 8 or 1 to 3136 of SEQ ID NO: 9 or 1 to 906 of SEQ ID NO: 10 or 1 to 992 of SEQ ID NO: 11, with the proviso that the oligonucleotides having SEQ ID NOS 50-55 are excluded and that binds to a natural antisense sequence of said at least one Brain derived neurotrophic factor (BDNF) polynucleotide and modulates expression of said at least one Brain derived neurotrophic factor (BDNF) polynucleotide; thereby preventing or treating the disease associated with the at least one Brain derived neurotrophic factor (BDNF) polynucleotide and/or at least one encoded product thereof. 
     
     
         37 . The method of  claim 36 , wherein a disease associated with the at least one Brain derived neurotrophic factor (BDNF) polynucleotide is selected from: a disease or disorder associated with abnormal function and/or expression of BDNF, a neurological disease or disorder, a disease or a disorder associated with defective neurogenesis; a neurodegenerative disease or disorder (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis etc.); a neuropsychiatric disorder (depression, schizophrenia, schizofreniform disorder, schizoaffective disorder, and delusional disorder; anxiety disorders such as panic disorder, phobias (including agoraphobia), an obsessive-compulsive disorder, a posttraumatic stress disorder, a bipolar disorder, anorexia nervosa, bulimia nervosa), an autoimmune disorder (e.g., multiple sclerosis) of the central nervous system, memory loss, a long term or a short term memory disorder, benign forgetfulness, a childhood learning disorder, close head injury, an attention deficit disorder, neuronal reaction to viral infection, brain damage, narcolepsy, a sleep disorder (e.g., circadian rhythm disorders, insomnia and narcolepsy); severance of nerves or nerve damage, severance of cerebrospinal nerve cord (CNS) and a damage to brain or nerve cells, a neurological deficit associated with AIDS, a motor and tic disorder characterized by motor and/or vocal tics (e.g., Tourette's disorder, chronic motor or vocal tic disorder, transient tic disorder, and stereotypic movement disorder), a substance abuse disorder (e.g., substance dependence, substance abuse and the sequalae of substance abuse/dependence, such as substance-induced psychological disorder, substance withdrawal and substance-induced dementia or amnestic disorder), traumatic brain injury, tinnitus, neuralgia (e.g., trigeminal neuralgia) pain (e.g., chronic pain, chronic inflammatory pain, pain associated with arthritis, fibromyalgia, back pain, cancer-associated pain, pain associated with digestive disease, pain associated with Crohn's disease, pain associated with autoimmune disease, pain associated with endocrine disease, pain associated with diabetic neuropathy, phantom limb pain, spontaneous pain, chronic post-surgical pain, chronic temporomandibular pain, causalgia, post-herpetic neuralgia, AIDS-related pain, complex regional pain syndromes type I and II, trigeminal neuralgia, chronic back pain, pain associated with spinal cord injury, pain associated with drug intake and recurrent acute pain, neuropathic pain), inappropriate neuronal activity resulting in neurodysthesias in a disease such as diabetes, an MS and a motor neuron disease, ataxias, muscular rigidity (spasticity), temporomandibular joint dysfunction, Reward deficiency syndrome (RDS), neurotoxicity caused by alcohol or substance abuse (e.g., ecstasy, methamphetamine etc.), mental retardation or cognitive impairment (e.g., nonsyndromic X-linked mental retardation, fragile X syndrome, Down's syndrome, autism), aphasia, Bell's palsy, Creutzfeldt-jacob disease, encephalitis, age related macular degeneration, ondine syndrome, WAGR syndrome, hearing loss, Rett syndrome, epilepsy, spinal cord injury, stroke, hypoxia, ischemia, brain injury, diabetic neuropathy, peripheral neuropathy, nerve transplantation complications, motor neuron disease, peripheral nerve injury, obesity, a metabolic syndrome, cancer, asthma, an atopic disease, inflammation, allergy, eczema, a neuro-oncological disease or disorder, neuro-immunological disease or disorder and neuro-otological disease or disorder; and a disease or disorder associated with aging and senescence. 
     
     
         38 . Use of an oligonucleotide selected from the group consisting of SEQ ID NOS 50-55 to target a natural antisense transcript (“NAT”) of a BDNF polynucleotide to modulate the expression of the BDNF polynucleotide wherein said NATs are selected from the group consisting of SEQ ID NOS. 3 to 11. 
     
     
         39 . Use of an oligonucleotide selected from the group consisting of SEQ ID NOS 50-55 to target a natural antisense transcript (NAT) of a BDNF polynucleotide to modulate the expression of the BDNF polynucleotide wherein said NAT is selected from the group consisting of SEQ ID NOS. 3, 4, 5, 7, 8, 9, 10 and 11.