Treatment of filaggrin (flg) related diseases by modulation of flg expression and activity

Abstract

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

1 . A method of modulating a function of and/or the expression of a Filaggrin (FLG) 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 consecutive nucleotides within nucleotides 1 to 4629 of SEQ ID NO: 2; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; thereby modulating a function of and/or the expression of the Filaggrin (FLG) polynucleotide in patient, cells or tissues in vivo or in vitro.   
     
     
         3 . A method of modulating a function of and/or the expression of a Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 a natural antisense oligonucleotide of the Filaggrin (FLG) polynucleotide; thereby modulating a function of and/or the expression of the Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide. 
     
     
         8 . The method of  claim 4 , wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Filaggrin (FLG) 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′-Omethoxyethyl (MOE),2′-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, 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 oligonucleotide sequences set forth as SEQ ID NOS: 3 to 13. 
     
     
         14 . A method of modulating a function of and/or the expression of a Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide; and, modulating a function of and/or the expression of Filaggrin (FLG) 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 sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the Filaggrin (FLG) polynucleotide. 
     
     
         16 . A method of modulating a function of and/or the expression of Filaggrin (FLG) 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 Filaggrin (FLG) 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 and 2; and, modulating the function and/or expression of the Filaggrin (FLG) 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, wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a Filaggrin (FLG) gene 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 modified nucleotides 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 Filaggrin (FLG) 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 Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide. 
     
     
         28 . The oligonucleotide of  claim 17 , wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Filaggrin (FLG) polynucleotide in vivo or in vitro, as compared to a normal control. 
     
     
         29 . The oligonucleotide of  claim 17 , wherein the oligonucleotide comprises the sequences set forth as SEQ ID NOS: 3 to 13. 
     
     
         30 . A composition comprising one or more oligonucleotides specific for one or more Filaggrin (FLG) 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 composition further comprises one or more FLG modulating molecules and a pharmaceutically acceptable carrier. 
     
     
         32 . The composition of  claim 31 , wherein the molecule is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine. 
     
     
         33 . 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: 3 to 1.3. 
     
     
         34 . The composition of  claim 30 , wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 3 to 13. 
     
     
         35 . The composition of  claim 34 , wherein the oligonucleotides set forth as SEQ ID NOS: 3 to 1.3 comprise one or more modifications or substitutions. 
     
     
         36 . The composition of  claim 35 , wherein the one or more modifications are selected from: phosphorothioate, methylphosphonate, peptide nucleic acid, locked nucleic acid (LNA) molecules, and combinations thereof. 
     
     
         37 . A composition for use in treatment of a dermatological disease or disorder, the composition comprising one or more FLG modulating molecules and a pharmaceutically acceptable earner. 
     
     
         38 . The composition of  claim 37 , wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine. 
     
     
         39 . The composition of  claim 37 , wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity. 
     
     
         40 . The composition of  claim 37 , wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject. 
     
     
         41 . The composition of  claim 40 , wherein the oligonucleotide comprises nucleotide sequences set forth as SEQ ID NOS: 3 to 13. 
     
     
         42 . The composition of  claim 40 , wherein the oligonucleotide set forth as SEQ ID NOS: 3 to 13 comprise one or more modifications or substitutions. 
     
     
         43 . A method of preventing or treating a disease associated with at least one Filaggrin (FLG) 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 Filaggrin (FLG) polynucleotide and modulates expression of said at least one Filaggrin (FLG) polynucleotide; thereby preventing or treating the disease associated with the at least one Filaggrin (FLG) polynucleotide and/or at least one encoded product thereof.   
     
     
         44 . The method of  claim 43 , wherein a disease associated with the at least one Filaggrin (FLG) polynucleotide is selected from: a disease or disorder associated with abnormal function and/or expression of FLG, a dermatological disease or disorder, sign of cutaneous aging, a skin condition caused due to external aggression, a allergy, psoriasis, asthma, eczema, hay lever, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immune-mediated disease or disorder, a hyper-immunity or hypoimmunity disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact type allergy, food allergy etc), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, Parkinson's disease, ALS etc.), AIDS wasting, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin. 
     
     
         45 . 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 rise selected target polynucleotide or to a polynucleotide that is antisense to the selected target polynucleotide; measuring the thermal melting point of a hybrid of an antisense oligonucleotide and the target polynucleotide or the polynucleotide that is antisense to the selected target polynucleotide under stringent hybridization conditions; and selecting at least one oligonucleotide for in vivo administration based on the information obtained.   
     
     
         46 . A method of treating an FLG associated disease or disorder in a subject, the method comprises administering to the subject a composition comprising one or more FLG modulating molecules and a pharmaceutically acceptable carrier. 
     
     
         47 . The method of  claim 46 , wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine. 
     
     
         48 . The method of  claim 46 , wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity. 
     
     
         49 . The method of  claim 46 , wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject. 
     
     
         50 . The method of  claim 46 , wherein a disease associated with the at least one Filaggrin polynucleotide is selected from: a dermatological disease or disorder, sign, of cutaneous aging, a skin condition caused due to external aggression, a allergy, psoriasis, asthma, eczema, hay fever, ichthyosis vulgaris, atopic dermatitis (AD), eczema herpeticum, rheumatoid arthritis, a cardiovascular disease or disorder, cancer, an inflammatory disease, an immune-mediated disease or disorder, a hyper-immunity or hypoimmunity disease or disorder, an autoimmune disease or disorder, asthma, psoriasis, an allergy (e.g., allergic rhinitis, contact type allergy, food allergy etc.), celiac disease, a neurological disease or disorder, a neurodegenerative disease or disorder (e.g. Alzheimer's disease, Parkinson's disease, ALS etc.), AIDS wasting, a disease or disorder associated with skin barrier function, a chronic inflammatory skin disease, clinical dry skin. 
     
     
         53 . A method of preventing or treating a skin condition associated with at least one Filaggrin (FLG) polynucleotide and/or at least one encoded product thereof, comprising: administering to a patient having a skin condition or at risk of developing a skin condition a therapeutically effective close, of a Filaggrin up-regulating compound, at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Filaggrin (FLG) polynucleotide and modulates expression of said at least one Filaggrin (FLG) polynucleotide and a pharmaceutically acceptable carrier; thereby preventing or treating the disease skin condition associated with the at least one Filaggrin (FLG) polynucleotide and/or at least one encoded product thereof. 
     
     
         52 . The method of claim  51 , wherein the compound, is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine. 
     
     
         53 . The method of claim  51 , wherein the skin condition is caused by caused by inflammation, light damage or aging. 
     
     
         54 . The method of claim  51 , wherein the skin condition is the development of wrinkles, contact dermatitis, atopic dermatitis, actinic keratosis, keratinization disorders, an epidermolysis bullosa disease, exfoliative dermatitis, seborrheic dermatitis, an erythema, discoid lupus erythematosus, dermatomyositis, skin cancer, or an effect of natural aging. 
     
     
         55 . A use of the composition of  claim 30 , in the manufacture of a medicament for the treatment of a dermatological disease or disorder. 
     
     
         56 . The use of  claim 55 , wherein the composition further comprises an antisense oligonucleotide that modulates FLG expression or activity. 
     
     
         57 . The use of  claim 55 , wherein the composition further comprises one antisense oligonucleotide to a Filaggrin natural antisense sequence, wherein the antisense oligonucleotide modulates the FLG expression in a subject. 
     
     
         58 . The use of  claim 55 , wherein the compound is selected from the group of Pioglitazone, Lomerizine, Bupropion, Phenprobamate, Benidipine, Piroxicam, Topiramate, Isradipine, Nicorandil, Piribedil, Oxaprozin, Glycopyrrolate, Granisetron, Memantine, Nimodipine and Amlodipine. 
     
     
         59 . The use of  claim 55 , wherein the dermatological disease or disorder is: development of wrinkles, contact dermatitis, atopic dermatitis, actinic keratosis, keratinization disorders, an epidermolysis bullosa disease, exfoliative dermatitis, seborrheic dermatitis, an erythema, discoid lupus erythematosus, dermatomyositis, skin cancer, or an effect of natural aging.