Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Tumor Suppressor genes, in particular, by targeting natural antisense polynucleotides of Tumor Suppressor genes. The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of Tumor Suppressor genes.

1 . A method of modulating a function of and/or the expression of a Tumor Suppressor gene 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 at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 nucleotides within nucleotides 1 to 1675 of SEQ ID NO: 4 or nucleotides 1 to 518 of SEQ ID NO: 5 or nucleotides 1 to 759 of SEQ ID NO: 6 or nucleotides 1 to 25892 of SEQ ID NO: 6a or nucleotides 1 to 279 of SEQ ID NO: 6b, or nucleotides 1 to 1982 of SEQ ID NO: 7, or nucleotides 1 to 789 of SEQ ID NO: 8, or nucleotides 1 to 467 of SEQ ID NO: 9 ( FIG. 5 ); thereby modulating a function of and/or the expression of the Tumor Suppressor gene polynucleotide in patient cells or tissues in vivo or in vitro.   
     
     
         2 . A method of modulating a function of and/or the expression of a Tumor Suppressor gene 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 at least one oligonucleotide has at least 50% sequence identity to a reverse complement of a natural antisense of a Tumor Suppressor gene polynucleotide; thereby modulating a function of and/or the expression of the Tumor Suppressor gene polynucleotide in patient cells or tissues in vivo or in vitro.   
     
     
         3 . A method of modulating a function of and/or expression of a Tumor Suppressor gene polynucleotide in patient cells or tissues in vivo or in vitro comprising:
 contacting said cells or tissues with an antisense oligonucleotide 5 to 30 nucleotides in length wherein said oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the Tumor Suppressor gene polynucleotide; thereby modulating a function of and/or the expression of the Tumor Suppressor gene polynucleotide in patient cells or tissues in vivo or in vitro.   
     
     
         4 . A method of modulating a function of and/or the expression of a Tumor Suppressor gene polynucleotide in patient cells or tissues in vivo or in vitro comprising:
 contacting said cells or tissues with at least one antisense oligonucleotide that targets a region of the natural antisense of a Tumor Suppressor gene polynucleotide; thereby modulating a function of and/or the expression of the Tumor Suppressor gene polynucleotide in patient cells or tissues in vivo or in vitro.   
     
     
         5 . The method of  claim 4 , wherein a function of and/or the expression of the Tumor Suppressor gene polynucleotide is increased in vivo or in vitro with respect to a control. 
     
     
         6 . The method of  claim 4 , wherein the at least one antisense oligonucleotide targets a natural antisense sequence of a Tumor Suppressor gene polynucleotide. 
     
     
         7 . The method of  claim 4 , wherein the at least one antisense oligonucleotide targets a nucleic acid sequence comprising coding and/or non-coding nucleic acid sequences of a Tumor Suppressor gene polynucleotide. 
     
     
         8 . The method of  claim 4 , wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Tumor Suppressor gene polynucleotide. 
     
     
         9 . The method of  claim 4 , 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. 
     
     
         10 . The method of  claim 9 , 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. 
     
     
         11 . The method of  claim 9 , wherein the one or more modifications comprise at least one modified internucleoside linkage selected from: a phosphorothioate, 2′-O-methoxyethyl (MOE), 2′-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, a carboxymethyl ester, and combinations thereof. 
     
     
         12 . The method of  claim 9 , 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. 
     
     
         13 . The method of  claim 1 , wherein the at least one oligonucleotide comprises at least one of the oligonucleotide sequences set forth as SEQ ID NOS: 10 to 30. 
     
     
         14 . A method of modulating a function of and/or the expression of a Tumor Suppressor 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 specific for an antisense polynucleotide of a Tumor Suppressor gene polynucleotide wherein said 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 a Tumor Suppressor gene polynucleotide; and, modulating a function of and/or the expression of the Tumor Suppressor gene in mammalian cells or tissues in vivo or in vitro.   
     
     
         15 . The method of  claim 14 , wherein said oligonucleotide has at least 80% 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 Tumor Suppressor gene polynucleotide. 
     
     
         16 . A method of modulating a function of and/or the expression of a Tumor Suppressor gene in mammalian cells or tissues in vivo or in vitro comprising:
 contacting said cells or tissues with at least one antisense oligonucleotide of about 5 to 30 nucleotides in length specific for noncoding and/or coding sequences of a sense and/or natural antisense strand of a Tumor Suppressor gene polynucleotide wherein said at least one antisense oligonucleotide has at least 50% sequence identity to at least one nucleic acid sequence set forth as SEQ ID NOS: 1, 1a, 1b, 2, 2a, 2b, 3, 3a, 4, 5, 6, 6a, 6b, 7, 8 and 9; and, modulating the function and/or expression of the Tumor Suppressor gene in mammalian cells or tissues in vivo or in vitro.   
     
     
         17 . A synthetic, modified oligonucleotide 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; and further wherein said oligonucleotide is an antisense compound which hybridizes to and modulates expression and/or function of a Tumor Suppressor gene polynucleotide in vivo or in vitro as compared to a normal control. 
     
     
         18 . The oligonucleotide of  claim 17 , 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. 
     
     
         19 . The oligonucleotide of  claim 17 , wherein said oligonucleotide comprises at least one phosphorothioate internucleotide linkage. 
     
     
         20 . The oligonucleotide of  claim 17 , wherein said oligonucleotide comprises a backbone of phosphorothioate internucleotide linkages. 
     
     
         21 . The oligonucleotide of  claim 17 , 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. 
     
     
         22 . The oligonucleotide of  claim 17 , wherein the oligonucleotide comprises a plurality of modifications, wherein said modifications comprise internucleotide linkages selected from: phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and a combination thereof. 
     
     
         23 . The oligonucleotide of  claim 17 , 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. 
     
     
         24 . The oligonucleotide of  claim 17 , 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. 
     
     
         25 . The oligonucleotide of  claim 17 , 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. 
     
     
         26 . The oligonucleotide of  claim 17 , 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 Tumor Suppressor gene polynucleotide wherein said oligonucleotide has at least about 20% 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 Tumor Suppressor gene polynucleotide. 
     
     
         27 . The oligonucleotide of  claim 17 , 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 Tumor Suppressor gene polynucleotide. 
     
     
         28 . The oligonucleotide of  claim 17 , wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Tumor Suppressor gene polynucleotide in vivo or in vitro, as compared to a normal control. 
     
     
         29 . The oligonucleotide of  claim 17 , wherein the oligonucleotide comprises one of the sequences set forth as SEQ ID NOS: 10 to 30. 
     
     
         30 . A composition comprising one or more oligonucleotides specific for one or more Tumor Suppressor gene polynucleotides, said polynucleotides comprising antisense sequences, complementary sequences, alleles, homologs, isoforms, variants, derivatives, mutants, fragments, or combinations thereof. 
     
     
         31 . The composition of  claim 30 , 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: 10 to 30. 
     
     
         32 . The composition of  claim 30 , wherein the one or more oligonucleotides comprise any of the nucleotide sequences set forth as SEQ ID NOS: 10 to 30. 
     
     
         33 . The composition of  claim 32 , wherein the oligonucleotides set forth as SEQ ID NOS: 10 to 30 comprise one or more modifications or nucleotide substitutions. 
     
     
         34 . The composition of  claim 33 , wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof. 
     
     
         35 . A method of preventing or treating a disease associated with at least one Tumor Suppressor gene polynucleotide and/or at least one encoded product thereof, comprising:
 administering to a patient a therapeutically effective dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Tumor Suppressor gene polynucleotide and modulates expression of said at least one Tumor Suppressor gene polynucleotide; thereby preventing or treating the disease associated with the at least one Tumor Suppressor gene polynucleotide and/or at least one encoded product thereof.   
     
     
         36 . The method of  claim 35 , wherein a disease associated with the at least one Tumor Suppressor gene polynucleotide is selected from: a disease associated with decreased or increased apoptosis, tissue/cell aging, a cancer (including those mentioned in Table 1), an autoimmune disease, an immunodeficiency disease including AIDS, senescence, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, ataxia telangiectasia, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease etc.), a hyperplastic disease (e.g., cheloid), rheumatoid arthritis, coronary heart disease ischemic cell death, a lymphoproliferative disorder, atherosclerosis, osteoporosis, a myelodysplastic syndrome, a toxin-induced disease, a viral infection, wound-healing, Cowden disease (CD), Lhermitte-Duclos disease (LDD), Bannayan-Zonana syndrome (BZS, also known as Bannayan-Riley-Ruvalcaba syndrome, Ruvalcaba-Myhre-Smith syndrome and Riley-Smith syndrome), transplantation, an apotosis-related disease or disorder, a metabolic disease or condition (e.g., diabetes), a kidney diseases or disorder, myocardial infarction/heart failure, ischemia, sepsis, an inflammatory disease where particular haematopoeitic inflammatory cells are in excess, a proliferative disease, or a disease or disorder wherein there is a therapeutic paradigm for treatment of inflammatory disease through increasing apoptosis. 
     
     
         37 . A method of identifying and selecting at least one oligonucleotide for in vivo administration comprising: selecting a target polynucleotide associated with a disease state; identifying at least one oligonucleotide comprising at least five consecutive nucleotides which are complementary to, or in an antisense orientation to the selected target polynucleotide; measuring the thermal melting point of an hybrid of an antisense oligonucleotide and the target polynucleotide under stringent hybridization conditions; and selecting at least one oligonucleotide for in vivo administration based on the information obtained. 
     
     
         38 . The method of  claim 2 , wherein the Tumor Suppressor gene encodes one of the Tumor Suppressor proteins listed in Table 1. 
     
     
         39 . The method of  claim 38 , wherein the Tumor Suppressor gene encodes P53, P73 or PTEN. 
     
     
         40 . The method of  claim 4 , wherein the Tumor Suppressor gene encodes one of the Tumor Suppressor proteins listed in Table 1. 
     
     
         41 . The method of  claim 40 , wherein the Tumor Suppressor gene encodes P53, P73 or PTEN. 
     
     
         42 . The method of  claim 35 , wherein the Tumor Suppressor gene encodes one of the Tumor Suppressor proteins listed in Table 1. 
     
     
         43 . The method of  claim 42 , wherein the Tumor Suppressor gene encodes P53, P73 or PTEN.