Pcsk9 targeting oligonucleotides for treating hypercholesterolemia and related conditions

Abstract

This disclosure relates to oligonucleotides, compositions and methods useful for reducing PCSK9 expression, particularly in hepatocytes. Disclosed oligonucleotides for the reduction of PCSK9 expression may be double-stranded or single-stranded, and may be modified for improved characteristics such as stronger resistance to nucleases and lower immunogenicity. Disclosed oligonucleotides for the reduction of PCSK9 expression may also include targeting ligands to target a particular cell or organ, such as the hepatocytes of the liver, and may be used to treat hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

1 - 50 . (canceled) 
     
     
         51 . A method of treating a subject having or at risk of having hypercholesterolemia or atherosclerosis, the method comprising administering to the subject an oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 1219-1222, 1231-1232, and 1269-1271, and a sense strand comprising a sequence as set forth in any one of SEQ ID NOs: 1179-1182, 1191-1192, and 1266-1268, wherein the sense strand forms a duplex region with the antisense strand. 
     
     
         52 . The method of  claim 51 , wherein the antisense strand is up to 27 nucleotides in length. 
     
     
         53 . A method of treating a subject having or at risk of having hypercholesterolemia or atherosclerosis, the method comprising administering to the subject an oligonucleotide for reducing expression of PCSK9, wherein the oligonucleotide comprises a pair of antisense strand and sense strand,
 wherein the antisense strand is 21 to 27 nucleotides in length comprising a sequence as set forth in any one of SEQ ID NOs: 1219-1222, 1231-1232, and 1269-1271, and has a region of complementarity to PCSK9,   wherein the sense strand comprises at its 3′-end a stem-loop set forth as: S 1 -L-S 2 , wherein S 1  is complementary to S 2 , and wherein L forms a loop between S 1  and S 2  of 3 to 5 nucleotides in length, and wherein the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length but are not covalently linked.   
     
     
         54 . The method of  claim 53 , wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1179-1182, 1191-1192, and 1266-1268. 
     
     
         55 . The method of  claim 51 , wherein the oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand. 
     
     
         56 . A method of treating a subject having or at risk of having hypercholesterolemia or atherosclerosis, the method comprising administering to the subject an oligonucleotide for reducing expression of PCSK9, wherein the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1219-1222, 1231-1232, and 1269-1271, and wherein the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1179-1182, 1191-1192, and 1266-1268; and
 wherein the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid.   
     
     
         57 . The method of  claim 56 , wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. 
     
     
         58 . The method of  claim 56 , wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. 
     
     
         59 . The method of  claim 56 , wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and wherein each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. 
     
     
         60 . The method of  claim 53 , wherein the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. 
     
     
         61 . The method of  claim 53 , wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. 
     
     
         62 . The method of  claim 53 , wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. 
     
     
         63 . The method of  claim 53 , wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety. 
     
     
         64 . The method of  claim 51 , wherein the oligonucleotide comprises at least one modified nucleotide, wherein the modified nucleotide comprises a 2′-modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. 
     
     
         65 . The method of  claim 51 , wherein the oligonucleotide comprises at least one modified internucleotide linkage, and wherein the at least one modified internucleotide linkage is a phosphorothioate linkage. 
     
     
         66 . The method of  claim 51 , wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog, and wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. 
     
     
         67 . The method of  claim 51 , wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety. 
     
     
         68 . The method of  claim 51 , wherein the subject has one or more symptoms or complications selected from the group consisting of coronary heart disease, angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke, feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and kidney problems. 
     
     
         69 . The method of  claim 53 , wherein the subject has one or more symptoms or complications selected from the group consisting of coronary heart disease, angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke, feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and kidney problems. 
     
     
         70 . The method of  claim 56 , wherein the subject has one or more symptoms or complications selected from the group consisting of coronary heart disease, angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke, feelings of weakness, confusion, difficulty speaking, dizziness, difficulty in walking or standing up straight, blurred vision, numbness of the face, arms, and legs, severe headaches, loss of consciousness, peripheral artery disease, and kidney problems.