DNA enzyme to inhibit plasminogen activator inhibitor-1

Abstract

The present invention provides DNAzymes and ribozymes that specifically cleave PAI-1-encoding mRNA. The present invention also provides antisense oligonucleotides that specifically inhibit translation of PAI-1-encoding mRNA. The invention also provides various methods of inhibiting the expression of PAI-1. Finally the invention provides pharmaceutical compositions containing the instant DNAzymes, ribozymes and antisense oligonucleotides as active ingredients.

What is claimed is:  
     
         1 . A catalytic nucleic acid that specifically cleaves an mRNA encoding a Plasminogen Activator Inhibitor-1 (PAI-1) comprising, in 5′ to 3′ order: 
 (a) consecutive nucleotides defining a first binding domain of at least 4 nucleotides;  
 (b) consecutive nucleotides defining a catalytic domain located contiguous with the 3′ end of the first binding domain, and capable of cleaving the PAI-1-encoding mRNA at a predetermined phosphodiester bond; and  
 (c) consecutive nucleotides defining a second binding domain of at least 4 nucleotides located contiguous with the 3′ end of the catalytic domain,  
 wherein the sequence of the nucleotides in each binding domain is complementary to a sequence of ribonucleotides in the PAI-1-encoding mRNA and wherein the catalytic nucleic acid hybridizes to and specifically cleaves the PAI-1-encoding mRNA.  
 
     
     
         2 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the first binding domain comprise at least one deoxyribonucleotide.  
     
     
         3 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the second binding domain comprise at least one deoxyribonucleotide.  
     
     
         4 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the first binding domain comprise at least one deoxyribonucleotide derivative.  
     
     
         5 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the second binding domain comprise at least one deoxyribonucleotide derivative.  
     
     
         6 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the first binding domain comprise at least one ribonucleotide.  
     
     
         7 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the second binding domain comprise at least one ribonucleotide.  
     
     
         8 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the first binding domain comprise at least one ribonucleotide derivative.  
     
     
         9 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the second binding domain comprise at least one ribonucleotide derivative.  
     
     
         10 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the first binding domain comprise at least one modified base.  
     
     
         11 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the second binding domain comprise at least one modified base.  
     
     
         12 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the first binding domain comprise at least one modified internucleoside bond.  
     
     
         13 . The catalytic nucleic acid of  claim 1 , wherein the nucleotides of the second binding domain comprise at least one modified internucleoside bond.  
     
     
         14 . The catalytic nucleic acid of  claim 12  or  13 , wherein the modified internucleoside bond is a phosphorothioate bond.  
     
     
         15 . The catalytic nucleic acid of  claim 1 , wherein the PAI-1-encoding mRNA encodes human PAI-1.  
     
     
         16 . The catalytic nucleic acid of  claim 15 , wherein the human PAI-1-encoding mRNA has the sequence set forth in SEQ ID NO:5.  
     
     
         17 . A pharmaceutical composition comprising the catalytic nucleic acid of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         18 . A method of specifically inhibiting the expression of PAI-1 in a cell that would otherwise express PAI-1, comprising contacting the cell with the catalytic nucleic acid of  claim 1  so as to specifically inhibit the expression of PAI-1 in the cell.  
     
     
         19 . A method of specifically inhibiting the expression of PAI-1 in a subject's cells comprising administering to the subject an amount of the catalytic nucleic acid of  claim 1  effective to specifically inhibit the expression of PAI-1 in the subject's cells.  
     
     
         20 . A method of specifically inhibiting the expression of PAI-1 in a subject's cells comprising administering to the subject an amount of the pharmaceutical composition of  claim 17  effective to specifically inhibit the expression of PAI-1 in the subject's cells.  
     
     
         21 . A method of treating a cardiovascular disease in a subject involving apoptosis of a cardiomyocyte in the subject which comprises administering to the subject an amount of the pharmaceutical composition of  claim 17  effective to inhibit apoptosis of the cardiomyocyte in the subject so as to thereby treat the cardiovascular disease.  
     
     
         22 . A method of treating a fibrotic disease in a subject involving fibrogenesis which comprises administering to the subject an amount of the pharmaceutical composition of  claim 17  effective to inhibit fibrogenesis in the subject so as to thereby treat the fibrotic disease.  
     
     
         23 . The method of  claim 22 , wherein the fibrotic disease is a renal disease.  
     
     
         24 . The method of  claim 22 , wherein the fibrotic disease is a hepatic disease.  
     
     
         25 . The method of  claim 22 , wherein the fibrotic disease is a disease of the lung.  
     
     
         26 . The method of  claim 22 , wherein the fibrotic disease is a disease of the skin.  
     
     
         27 . The method of  claim 22 , wherein the fibrotic disease is a disease of the eye.  
     
     
         28 . An oligonucleotide comprising consecutive nucleotides that hybridizes with a PAI-1-encoding mRNA under conditions of high stringency and is between 8 and 40 nucleotides in length.  
     
     
         29 . The oligonucleotide of  claim 28 , wherein at least one internucleoside linkage within the oligonucleotide comprises a phosphorothioate linkage.  
     
     
         30 . The oligonucleotide of  claim 28 , wherein the nucleotides comprise at least one deoxyribonucleotide.  
     
     
         31 . The oligonucleotide of  claim 28 , wherein the nucleotides comprise at least one ribonucleotide.  
     
     
         32 . The oligonucleotide of  claim 28 , wherein the PAI-1-encoding mRNA encodes human PAI-1.  
     
     
         33 . The oligonucleotide of  claim 32 , wherein the human PAI-1-encoding mRNA comprises consecutive nucleotides, the sequence of which is set forth in SEQ ID NO:5.  
     
     
         34 . A method of treating a subject which comprises administering to the subject an amount of the oligonucleotide of  claim 28  effective to inhibit expression of a PAI-1 in the subject so as to thereby treat the subject.  
     
     
         35 . A method of treating a cardiovascular disease in a subject involving apoptosis of a cardiomyocyte in the subject which comprises administering to the subject an amount of the oligonucleotide of  claim 28  effective to inhibit apoptosis of the cardiomyocyte in the subject so as to thereby treat the cardiovascular disease.  
     
     
         36 . A method of treating a fibrotic disease in a subject involving fibrogenesis in the subject which comprises administering to the subject an amount of the oligonucleotide of  claim 28  effective to inhibit fibrogenesis in the subject so as to thereby treat the fibrotic disease.  
     
     
         37 . The method of any of  claim 19 ,  20 ,  21 ,  22 ,  34 ,  35 , or  36 , wherein the subject is a mammal.  
     
     
         38 . The method of  claim 37 , wherein the mammal is a human being.