Antisense oligonucleotides directed against connective tissue growth factor and uses thereof

Abstract

This invention provides compounds which comprise modified oligonucleotides capable of inhibitory expression of connective tissue factor and composition containing same as well as methods of treating hyperprolific disorders and fibrotic diseases, and of reducing scarring resulting from wound healing using such compounds.

1 . A compound which comprises a modified oligonucleotide consisting of 12-30 linked nucleosides, at least a 12 nucleobase sequence portion of which is present within the region selected from nucleotides 718-751, 1388-1423, 1457-1689, 2040-2069, 2120-2147, 2728-2797, 2267-2301, 553-611, 1394-1423, 1469-1508, 1559-1605, 1659-1689, 2100-2129, 1399-1423, 380-399, 475-494, 501-520, 839-858, 1003-1022, 1273-1292, 1520-1539, 1965-1984, 1976-1995, 2007-2026, 2018-2037, 2088-2107, and 2219-2238 of SEQ ID NO: 9. 
     
     
         2 .- 3 . (canceled) 
     
     
         4 . A compound which comprises a modified oligonucleotide comprising at least 12 linked nucleosides, the nucleobase sequence of which is present within the nucleobase sequences set forth in SEQ ID NOs: 28, 30, 39, 40, 43, 44, 45, 50, 51, 52, 56, 78, and 166. 
     
     
         5 . The compound of  claim 4 , wherein the modified oligonucleotide comprises at least 14 linked nucleosides. 
     
     
         6 . The compound of  claim 1 , wherein:
 the modified oligonucleotide is a single-stranded oligonucleotide;   the modified oligonucleotide is a double-stranded oligonucleotide;   the modified oligonucleotide comprises at least one modified internucleoside linkage;   the modified oligonucleotide comprises at least one modified internucleoside linkage, wherein the at least one modified internucleoside linkage is a phosphothioate internucleoside linkage;   the modified oligonucleotide comprises at least one modified internucleoside linkage, wherein all of the internucleoside linkages are phosphothioate internucleoside linkages;   at least one nucleoside comprises a modified sugar;   at least one nucleoside comprises a modified sugar, wherein the modified sugar is a bicyclic sugar; or   at least one nucleoside comprises a modified sugar, wherein at least one of the modified sugar comprises a 2′-O-methoxyethyl.   
     
     
         7 .- 14 . (canceled) 
     
     
         15 . The compound of  claim 1 , comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring. 
     
     
         16 . The compound of  claim 15 , wherein each of the at least one tetrahydropyran modified nucleoside has the structure: 
       
         
           
           
               
               
           
         
         wherein Bx is an optionally protected heterocyclic base moiety. 
       
     
     
         17 . The compound of  claim 1 , wherein at least one nucleoside comprises a modified nucleobase. 
     
     
         18 . The compound of  claim 17 , wherein the modified nucleobase is a 5′-methylcytosine. 
     
     
         19 . The compound of  claim 1 , wherein the modified oligonucleotide comprises:
 (a) a gap segment consisting of linked deoxynucleosides;   (b) a 5′ wing segment consisting of linked modified nucleosides; and   (c) a 3′ wing segment consisting of linked modified nucleosides;   wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each modified nucleoside within each wing segment comprises a modified sugar.   
     
     
         20 . The compound of  claim 19 , wherein the modified oligonucleotide comprises:
 (a) a gap segment consisting of thirteen linked deoxynucleosides;   (b) a 5′ wing segment consisting of two linked modified nucleosides; and   (c) a 3′ wing segment consisting of five linked modified nucleosides;   wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each modified nucleoside within each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphothioate linkage.   
     
     
         21 . The compound of  claim 1 , wherein:
 the modified oligonucleotide consists of 20 linked nucleosides;   the nucleobase sequence is the sequence set forth in SEQ ID NO:39;   the nucleobase sequence is the sequence set forth in SEQ ID NO:40;   the nucleobase sequence is the sequence set forth in SEQ ID NO:45;   the nucleobase sequence is the sequence set forth in SEQ ID NO: 52; or   the nucleobase sequence is the sequence set forth in SEQ ID NO:166.   
     
     
         22 .- 26 . (canceled) 
     
     
         27 . A composition which comprises a modified oligonucleotide comprising linked nucleosides, the nucleobase sequence of which is a sequence set forth in one of SEQ ID NOs: 28, 30, 39, 40, 43, 44, 45, 50, 51, 52, 56, 78, 125 and 166, or a salt thereof, and a pharmaceutically acceptable carrier or diluent. 
     
     
         28 . The compound of  claim 1 , wherein the nucleobase sequence is 100% complimentary within the range of nucleotides (e.g. 1388-1423, A-B, C-D, and E-F) of SEQ ID NO:9 so long as a previously designed oligonucleotide does not fall within the range. 
     
     
         29 . The compound of  claim 28 , wherein:
 the modified oligonucleotide is a single-stranded oligonucleotide;   the modified oligonucleotide is a double-stranded oligonucleotide; or   the modified oligonucleotide consists of 20 linked nucleosides.   
     
     
         30 .- 31 . (canceled) 
     
     
         32 . A method for inhibiting expression of connective tissue growth factor in a cell or a tissue which comprises contacting the cell or tissue with the compound of  claim 1  under conditions such that expression of connective tissue growth factor is inhibited. 
     
     
         33 . A method of treating an animal having a disease or condition associated with expression of connective tissue growth factor which comprises administering to the animal an amount of the compound of  claim 1  effective to inhibit expression of connective tissue growth factor so as to thereby treat the animal. 
     
     
         34 . The method of  claim 33 , wherein the disease or condition is:
 a hyperproliferative disorder;   a cancer;   a fibrotic disease;   hypertrophic scarring, keloids, skin scarring, liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, or restenosis; or   joint fibrosis (including frozen shoulder syndrome, tendon and peripheral nerve damage), spinal cord damage, coronary bypass, abdominal and peritoneal adhesions (including endometriosis, uterine leiomyomata and fibroids), radial keratotomy and photorefractive keratectomy, retinal reattachment surgery, device mediated fibrosis (including for example diabetes), tendon adhesions, Dupuytren contracture, or scleroderma.   
     
     
         35 .- 38 . (canceled) 
     
     
         39 . A method for reducing scarring resulting from wound healing in a subject in need thereof which comprises administering to the subject an amount of the compound of  claim 1  effective to inhibit expression of connective tissue growth factor in the subject so as to thereby reduce scarring resulting from wound healing in the subject. 
     
     
         40 . (canceled) 
     
     
         41 . A compound which comprises a modified oligonucleotide consisting of 12-30 linked nucleosides, at least a 12 nucleobase sequence portion of which is present within the region selected from nucleotides 380-399, 475-494, 501-520, 839-858, 1003-1022, 1273-1292, 1520-1539, 1965-1984, 1976-1995, 2007-2026, 2018-2037, 2088-2107, and 2219-2238 of SEQ ID NO: 9. 
     
     
         42 . The compound of  claim 41 , which comprises a modified oligonucleotide comprising at least 12 linked nucleosides, the nucleobase sequence of which is present within the nucleobase sequences set forth in SEQ ID NOs: 161, 20, 24, 33, 12, 34, 48, 61, 63, 67, 69, 76, and 86. 
     
     
         43 . A compound which comprises a modified oligonucleotide comprising at least 12 linked nucleosides, the nucleobase sequence of which is present within the nucleobase sequences set forth in SEQ ID NOs: 161, 20, 24, 33, 12, 34, 48, 61, 63, 67, 69, 76, and 86. 
     
     
         44 . The compound of  claim 41 , wherein:
 the modified oligonucleotide is a single-stranded oligonucleotide;   the modified oligonucleotide is a double-stranded oligonucleotide;   the modified oligonucleotide comprises at least one modified internucleoside linkage;   the modified oligonucleotide comprises at least one modified internucleoside linkage, wherein the at least one modified internucleoside linkage is a phosphothioate internucleoside linkage;   the modified oligonucleotide comprises at least one modified internucleoside linkage, wherein all of the internucleoside linkages are phosphothioate internucleoside linkages;   at least one nucleoside comprises a modified sugar;   at least one nucleoside comprises a modified sugar, wherein the modified sugar is a bicyclic sugar; or   
       at least one nucleoside comprises a modified sugar, wherein at least one of the modified sugar comprises a 2′-O-methoxyethyl. 
     
     
         45 . The compound of  claim 41 , wherein the modified oligonucleotide comprises:
 (a) a gap segment consisting of thirteen linked deoxynucleosides;   (b) a 5′ wing segment consisting of two linked modified nucleosides; and   (c) a 3′ wing segment consisting of five linked modified nucleosides;   wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each modified nucleoside within each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphothioate linkage.   
     
     
         46 . A method of treating an animal having a disease or condition associated with expression of connective tissue growth factor which comprises administering to the animal an amount of the compound of  claim 41  effective to inhibit expression of connective tissue growth factor so as to thereby treat the animal, wherein the disease or condition is:
 a hyperproliferative disorder; 
 a cancer; 
 a fibrotic disease; 
 hypertrophic scarring, keloids, skin scarring, liver fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, or restenosis; or 
 joint fibrosis (including frozen shoulder syndrome, tendon and peripheral nerve damage), spinal cord damage, coronary bypass, abdominal and peritoneal adhesions (including endometriosis, uterine leiomyomata and fibroids), radial keratotomy and photorefractive keratectomy, retinal reattachment surgery, device mediated fibrosis (including for example diabetes), tendon adhesions, Dupuytren contracture, or scleroderma.