Treatment of collagen gene related diseases by inhibition of natural antisense transcript to a collagen gene

Abstract

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

1 . A method of modulating a function of and/or the expression of a Collagen 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 consecutive nucleotides within nucleotides 1 to 387 of SEQ ID SEQ ID NO: 4, 1 to 561 of SEQ ID NO: 5, 1 to 335 of SEQ ID SEQ ID NO: 6, 1 to 613 of SEQ ID NO: 7, 1 to 177 of SEQ ID NO: 8, and 1 to 285 of SEQ ID NO: 9; thereby modulating a function of and/or the expression of the Collagen 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 Collagen 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 Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene 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 Collagen 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 oligonucleotide has at least 50% sequence identity to an antisense oligonucleotide to the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen 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 Collagen 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 a natural antisense oligonucleotide of the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen 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 Collagen gene 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 Collagen 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 Collagen gene polynucleotide. 
     
     
         8 . The method of  claim 4 , wherein the at least one antisense oligonucleotide targets overlapping and/or non-overlapping sequences of a Collagen 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′- 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: 10 to 29. 
     
     
         14 . A method of modulating a function of and/or the expression of a Collagen 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 Collagen gene 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 Collagen gene polynucleotide; and, modulating a function of and/or the expression of a Collagen 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 sequence of at least about five consecutive nucleic acids that is complementary to the antisense and/or sense nucleic acid molecule of the Collagen gene polynucleotide. 
     
     
         16 . A method of modulating a function of and/or the expression of a Collagen 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 Collagen 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 to 9; and, modulating the function and/or expression of the Collagen 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; wherein said oligonucleotide is an antisense compound which hybridizes to and modulates the function and/or expression of a Collagen 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 Collagen 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 Collagen 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 Collagen gene polynucleotide. 
     
     
         28 . The oligonucleotide of  claim 17 , wherein said oligonucleotide hybridizes to and modulates expression and/or function of at least one Collagen gene 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: 10 to 29. 
     
     
         30 . A composition comprising one or more oligonucleotides specific for one or more Collagen 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 29. 
     
     
         32 . The composition of  claim 30 , wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 10 to 29. 
     
     
         33 . The composition of  claim 32 , wherein the oligonucleotides set forth as SEQ ID NOS: 10 to 29 comprise one or more modifications or 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 Collagen 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 Collagen gene polynucleotide and modulates expression of said at least one Collagen gene polynucleotide; thereby preventing or treating the disease associated with the at least one Collagen 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 Collagen gene polynucleotide is selected from: a collagen disorder, age related collagen degradation, Osteogenesis imperfecta, Otosclerosis (OTSC), Osteoporosis, Osteoarthritis, Oesophageal squamous cell cancer, chondrodysplasia, atypical Marfan syndrome, Ehlers-Danlos Syndrome (EDS), Dystrophic epidermolysis bullosa (DEB), Caffey disease, aneurysms (eg. intracranial aneurysms), idiopathic pulmonary fibrosis, liver cirrhosis, kidney fibrosis, liver fibrosis, heart fibrosis, scleroderma, hypertrophic scars, keloids, cancer, inflammation, a genetic disease (e.g. Duchenne muscular dystrophy), a neurological disease or disorder (e.g. Parkinson's, Alzheimer's, Huntington's, Gaucher disease), a metabolic disease (e.g. type I diabetes), an autoimmune disease or disorder, trauma (e.g. spinal cord injury, burns, etc), ischemia, and other blood vessel, heart, a skin disease or disorder, skin aging, a skin disease or disorder or condition requiring skin engineering, a liver or kidney disease requiring transplantation; tendon, bone or tissue regeneration; skeletal repair, cartilage and bone repair. 
     
     
         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 the 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.   
     
     
         38 . A method of preventing or treating a skin condition associated with at least one Collagen gene 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 dose of at least one antisense oligonucleotide that binds to a natural antisense sequence of said at least one Collagen gene polynucleotide and modulates expression of said at least one Collagen gene polynucleotide;   thereby preventing or treating the disease skin condition associated with the at least one Collagen gene polynucleotide and/or at least one encoded product thereof.   
     
     
         39 . The method of  claim 38 , wherein the skin condition is caused by caused by inflammation, light damage or aging. 
     
     
         40 . The method of  claim 39 , 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. 
     
     
         41 . A Collagen gene based matrix composition, said composition comprising a three dimensional purified collagen matrix comprised of Collagen fibrils, wherein function of and/or the expression of a Collagen gene polynucleotide in the cells of collagen fibrils is modulated in vivo or in vitro by contacting collagen fibril cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide. 
     
     
         42 . The composition of  claim 41 , 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 29. 
     
     
         43 . The composition of  claim 41 , wherein the oligonucleotides comprise nucleotide sequences set forth as SEQ ID NOS: 10 to 29. 
     
     
         44 . The composition of  claim 41 , wherein the composition supports stem cells. 
     
     
         45 . A method of in vitro, ex vivo or in vivo wound healing or tissue regeneration or tissue engineering which comprises application of an extracellular matrix composition as defined in  claim 41  to a wound. 
     
     
         46 . A method of preparing a Collagen gene based matrix composition, comprising contacting collagen fibril cells or tissues with at least one antisense oligonucleotide that targets a region of a natural antisense oligonucleotide of the Collagen gene polynucleotide; thereby modulating a function of and/or the expression of the Collagen gene polynucleotide in patient cells or tissues in vivo or in vitro.