RNAi construct for inhibiting ASGR1 expression and method for its use

Abstract

To provide additional therapeutic agents for treatment of cardiovascular disease.SOLUTION: The present invention provides compositions and methods for modulating liver expression of asialoglycoprotein receptor 1 (ASGR1). Specifically, the present invention provides nucleic acid-based therapeutics for reducing ASGR1 expression via RNA interference, and methods of using such nucleic acid-based therapeutics to treat or prevent cardiovascular disease.SELECTED DRAWING: None

Claims

[Claim 1] An RNAi construct comprising a sense strand and an antisense strand, wherein the RNAi construct reduces the expression of human ASGR1 in liver cells, the sense strand and the antisense strand are each 19 to 27 nucleotides long, the sense strand and the antisense strand hybridize to form a double-stranded region of 15 to 26 base pairs long, and the antisense strand includes a region having a sequence complementary to the sequence of at least 15 consecutive nucleotides from nucleotides 886 to 910 of the ASGR1 mRNA sequence of SEQ ID NO:

1. [Claim 2] The RNAi construct according to claim 1, wherein the sense strand and the antisense strand are each 21 to 25 nucleotides long. [Claim 3] The RNAi construct according to claim 1, wherein the RNAi construct comprises at least one blunt end. [Claim 4] The RNAi construct according to claim 1, wherein the RNAi construct comprises at least one nucleotide overhang of 1 to 4 unpaired nucleotides. [Claim 5] The RNAi construct according to claim 4, wherein the RNAi construct includes a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or at the 3' ends of both the sense strand and the antisense strand. [Claim 6] The RNAi construct according to claim 1, wherein the RNAi construct comprises at least one modified nucleotide. [Claim 7] The RNAi construct according to claim 6, wherein the modified nucleotide is a 2'-modified nucleotide. [Claim 8] The RNAi construct according to claim 6, wherein the modified nucleotide is a 2'-fluoromodified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), or a combination thereof. [Claim 9] The RNAi construct according to claim 6, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides. [Claim 10] The RNAi construct according to claim 9, wherein the modified nucleotide is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a combination thereof. [Claim 11] The RNAi construct according to claim 6, wherein the RNAi construct comprises at least one phosphorothioate nucleotide bond. [Claim 12] The RNAi construct according to claim 11, wherein the RNAi construct includes two consecutive phosphorothioate nucleotide bonds at the 3' end of the antisense strand. [Claim 13] The RNAi construct according to claim 11, wherein the RNAi construct includes two consecutive phosphorothioate nucleotide bonds at both the 3' and 5' ends of the antisense strand, and includes two consecutive phosphorothioate nucleotide bonds at the 5' end of the sense strand. [Claim 14] The RNAi construct according to claim 1, wherein the antisense strand comprises a sequence selected from any one of sequence numbers 2867-2871 and 2873. [Claim 15] The RNAi construct according to claim 14, wherein the sense strand comprises a sequence selected from any one of sequence numbers 1364-1368 and 1370. [Claim 16] (a) The sense strand includes the sequence of sequence number 1364, and the antisense strand includes the sequence of sequence number 2867, (b) The sense strand includes the sequence of sequence number 1365, and the antisense strand includes the sequence of sequence number 2868, (c) The sense strand includes the sequence of sequence number 1366, and the antisense strand includes the sequence of sequence number 2869, (d) The sense strand includes the sequence of sequence number 1367, and the antisense strand includes the sequence of sequence number 2870, (e) The sense strand includes the sequence of sequence number 1368, and the antisense strand includes the sequence of sequence number 2871, or (f) The RNAi construct according to claim 14, wherein the sense strand comprises the sequence of SEQ ID NO: 1370, and the antisense strand comprises the sequence of SEQ ID NO: 2873. [Claim 17] (a) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4331, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4520, (b) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4381, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4565, (c) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4392, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4576, (d) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4413, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4520, (e) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4426, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4523, (f) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4441, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4523, (g) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4426, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4616, (h) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4463, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4638, (i) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4464, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4639, (j) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4466, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4641, (k) The sense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4467, and the antisense strand includes a sequence of modified nucleotides according to SEQ ID NO: 4642, or (l) The RNAi construct according to claim 16, wherein the sense strand comprises a sequence of modified nucleotides according to SEQ ID NO: 4468, and the antisense strand comprises a sequence of modified nucleotides according to SEQ ID NO: 4643. [Claim 18] The RNAi construct according to claim 1, wherein the RNAi construct further comprises a ligand. [Claim 19] The RNAi construct according to claim 18, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folic acid moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or an antigen-binding fragment thereof. [Claim 20] The RNAi construct according to claim 18, wherein the ligand targets the delivery of the RNAi construct to hepatocytes. [Claim 21] The RNAi construct according to claim 20, wherein the ligand comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to human ASGR1. [Claim 22] The RNAi construct according to claim 21, wherein the monoclonal antibody or its antigen-binding fragment comprises the substitution of at least one amino acid by a cysteine ​​amino acid, and the sense chain is covalently bound to the monoclonal antibody or its antigen-binding fragment via the side chain of the cysteine ​​amino acid. [Claim 23] The RNAi construct according to claim 18, wherein the ligand comprises galactose, galactosamine, N-acetyl-galactosamine, a polyvalent galactose moiety, or a polyvalent N-acetyl-galactosamine moiety. [Claim 24] The RNAi construct according to claim 23, wherein the polyvalent galactose moiety or the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. [Claim 25] The RNAi construct according to claim 18, wherein the ligand is covalently bound to the sense strand via a linker as needed. [Claim 26] The RNAi construct according to claim 25, wherein the ligand is covalently bound to the 3' or 5' end of the sense strand. [Claim 27] A pharmaceutical composition comprising an RNAi construct according to any one of claims 1 to 26 and a pharmaceutically acceptable carrier, excipient, or diluent. [Claim 28] A composition for reducing ASGR1 expression in a patient in need thereof, comprising an RNAi construct according to any one of claims 1 to 26, wherein the composition is administered to the patient. [Claim 29] The composition according to claim 28, wherein the patient is diagnosed with coronary artery disease, is at risk of coronary artery disease, or has a history of myocardial infarction. [Claim 30] The composition according to claim 28, wherein the patient has elevated levels of non-HDL cholesterol. [Claim 31] The composition according to claim 28, characterized in that the composition is administered to the patient via intravenous or subcutaneous administration. [Claim 32] A composition for reducing non-HDL cholesterol in a patient in need thereof, comprising an RNAi construct according to any one of claims 1 to 26, wherein the composition is administered to the patient. [Claim 33] The composition according to claim 32, characterized in that the composition is administered to the patient via intravenous or subcutaneous administration. [Claim 34] A composition for treating or preventing cardiovascular disease in a patient in need thereof, comprising an RNAi construct according to any one of claims 1 to 26, wherein the composition is administered to the patient. [Claim 35] The composition according to claim 34, wherein the cardiovascular disease is coronary artery disease or myocardial infarction. [Claim 36] The composition according to claim 34, characterized in that the composition is administered to the patient via intravenous or subcutaneous administration. [Claim 37] A composition for reducing the risk of myocardial infarction in a patient in need thereof, comprising an RNAi construct according to any one of claims 1 to 26, wherein the composition is administered to the patient. [Claim 38] The composition according to claim 37, characterized in that the composition is administered to the patient via intravenous or subcutaneous administration.

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