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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 17/048,846, filed Oct. 19, 2020, which is a National Stage application, filed under 35 U.S.C. § 371, of International Application No. PCT/US2019/025253, filed Apr. 1, 2019, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/659,693, filed Apr. 18, 2018, and U.S. Provisional Application No. 62/820,558, filed Mar. 19, 2019, the entire contents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present application relates to oligonucleotides and uses thereof, particularly uses relating to the treatment of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 400930-020USD1-195261.txt created on Nov. 30, 2022 which is 257 kilobytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cholesterol is one of three major classes of lipids manufactured by animal cells and used to construct cell membranes. Cholesterol is water insoluble and transported in the blood plasma within protein particles (lipoproteins). Any lipoprotein (e.g., very low density lipoprotein (VLDL), low density lipoprotein (LDL), intermediate density lipoprotein (IDL) and high density lipoprotein (HDL)) may carry cholesterol, but elevated levels of non-HDL cholesterol (most particularly LDL-cholesterol) are associated with an increased risk of atherosclerosis and coronary heart disease (e.g., coronary artery disease). This type of elevated cholesterol is known as hypercholesterolemia. Hypercholesterolemia can lead to the deposition of plaques on artery walls, known as atherosclerosis. Proprotein convertase subtilisin/kexin-9 (also known as PCSK9) is a serine protease that indirectly regulates plasma LDL cholesterol levels by controlling both hepatic and extrahepatic LDL receptor (LDLR) expression at the plasma membrane. Decreased expression of the PCSK9 protein increases expression of the LDLR receptor, thereby decreasing plasma LDL cholesterol and the resultant hypercholesterolemia and/or atherosclerosis as well as complications arising from the same.

BRIEF SUMMARY OF THE INVENTION

Aspects of the disclosure relate to oligonucleotides and related methods for treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject. In some embodiments, potent RNAi oligonucleotides have been developed for selectively inhibiting PCSK9 expression in a subject. In some embodiments, the RNAi oligonucleotides are useful for reducing PCSK9 activity, and thereby decreasing or preventing hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, and/or one or more symptoms or complications thereof. In some embodiments, key regions of PCSK9 mRNA (referred to as hotspots) have been identified herein that are particularly amenable to targeting using such oligonucleotide-based approaches (See Example 1).

One aspect of the present disclosure provides oligonucleotides for reducing expression of PCSK9. In some embodiments, the oligonucleotides comprise an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, or 1269-1271. In some embodiments, the oligonucleotides further comprise a sense strand that comprises a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, or 1266-1268. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, or 1269-1271. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, or 1266-1268. One aspect of the present disclosure provides oligonucleotides for reducing expression of PCSK9, in which the oligonucleotides comprise an antisense strand of 15 to 30 nucleotides in length. In some embodiments, the antisense strand has a region of complementarity to a target sequence of PCSK9 as set forth in any one of SEQ ID NOs: 1233-1244. In some embodiments, the region of complementarity is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides in length. In some embodiments, the region of complementarity is fully complementary to the target sequence of PCSK9. In some embodiments, the region of complementarity is at least 19 contiguous nucleotides in length.

In some embodiments, the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, or 1153-1192. In some embodiments, the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, or 1153-1192. In some embodiments, the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232. In some embodiments, the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232.

In some embodiments, the antisense strand is 19 to 27 nucleotides in length. In some embodiments, the antisense strand is 21 to 27 nucleotides in length. In some embodiments, the oligonucleotide further comprises a sense strand of 15 to 40 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand. In some embodiments, the sense strand is 19 to 40 nucleotides in length. In some embodiments, the duplex region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides in length. In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length. In some embodiments, the antisense strand and sense strand form a duplex region of 25 nucleotides in length.

In some embodiments, an oligonucleotide comprises an antisense strand and a sense strand that are each in a range of 21 to 23 nucleotides in length. In some embodiments, an oligonucleotide comprises a duplex structure in a range of 19 to 21 nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand. In some embodiments, an oligonucleotide further comprises a 3′-overhang sequence on the antisense strand of two nucleotides in length. In some embodiments, an oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, in which the 3′-overhang sequence is present on the antisense strand, and in which the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and antisense strand form a duplex of 21 nucleotides in length.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising an antisense strand and a sense strand, in which the antisense strand is 21 to 27 nucleotides in length and has a region of complementarity to PCSK9, in which the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of 3 to 5 nucleotides in length, and in which the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length but are not covalently linked. In some embodiments, the sense strand comprises at its 3′-end a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of 3 to 5 nucleotides in length. In some embodiments, the region of complementarity is fully complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides of PCSK9 mRNA. In some embodiments, L is a tetraloop. In some embodiments, L is 4 nucleotides in length. In some embodiments, L comprises a sequence set forth as GAAA.

In some embodiments, an oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2′-modification. In some embodiments, the 2′-modification is a modification selected from: 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. In some embodiments, all of the nucleotides of an oligonucleotide are modified.

In some embodiments, an oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.

In some embodiments, at least one nucleotide of an oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid. In some embodiments, each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety. In other embodiments, a bi-valent, tri-valent, or tetravalent GalNac moiety is conjugated to a single nucleotide, e.g., of the nucleotides of L of a stem loop. In some embodiments, the targeting ligand comprises an aptamer.

Another aspect of the present disclosure provides a composition comprising an oligonucleotide of the present disclosure and an excipient. Another aspect of the present disclosure provides a method comprising administering a composition of the present disclosure to a subject. In some embodiments, the method results in a decrease in level or severity of, or results in prevention of, hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), 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/or kidney problems (e.g., chronic kidney disease). Another aspect of the present disclosure provides a method for treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

Another aspect of the present disclosure provides an oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising a sense strand of 15 to 40 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, in which the sense strand forms a duplex region with the antisense strand, in which the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, or 1266-1268 and the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, or 1269-1271.

In some embodiments, the oligonucleotide comprises a pair of sense and antisense strands selected from a row of the table set forth in Table 4.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

FIGS. 1A and 1B are graphs showing the percentage of PCSK9 mRNA remaining after a screen of 576 PCSK9 oligonucleotides in Huh-7 cells. The nucleotide position in NM_174936.3 that corresponds to the 3′ end of the sense strand of each siRNA is indicated on the x axis.

FIGS. 2A-2D are a set of graphs showing the percentage of mRNA remaining after PCSK9 oligonucleotide screening of 96 PCSK9 oligonucleotides at two different concentrations (0.1 nM and 1 nM) in Huh-7 cells. The H number on the X-axis indicates the position in the PCSK9 mRNA mapping to the 5′ end of the antisense strand of the oligonucleotides.

FIG. 3 is a schematic showing a non-limiting example of a double-stranded oligonucleotide with a nicked tetraloop structure that has been conjugated to four GalNAc moieties (diamond shapes).

FIG. 4 is a graph showing the results of screening in a mouse hydrodynamic injection (HDI) model using PCSK9 tetraloop conjugates of 12 different base sequences with a single modification pattern. PBS, shown on the far left, was used as a control.

FIGS. 5A-5C are graphs showing the results of screening in Huh-7 cells (FIG. 5A) and in a mouse HDI model (FIGS. 5B and 5C) using PCSK9 oligonucleotides of different base sequences. FIG. 5A is a graph showing the percentage of PCSK9 mRNA remaining after screening of 40 nicked-tetraloop structures. The same modification pattern was used, and the oligonucleotides were tested at two different concentrations (0.03 nM and 0.1 nM; labeled as “Phase T2” in FIG. 5A). FIG. 5B shows a human-specific PCSK9 tetraloop conjugate screen in the mouse HDI model at a 2 mg/kg subcutaneous dose using three different modification patterns. FIG. 5C shows the same test as described in FIG. 5B, except at a 1 mg/kg subcutaneous dose (except for the control, which was dosed at both 1 and 2 mg/kg). Two different modification patterns were used. PBS was used as a control and is shown to the left.

FIGS. 6A and 6B are graphs showing the results of screening in a mouse hydrodynamic injection (HDI) model using three different PCSK9 tetraloop conjugates with varied modification patterns at three different concentrations. PBS, shown on the far left, was used as a control.

FIGS. 7A-7D are graphs showing an in vivo activity evaluation of PCSK9 oligonucleotides in a tetraloop conjugate in non-human primates. Candidate sequences were tested with different modifications. FIG. 7A shows the analysis of PCSK9 remaining and LDL-C lowering using a candidate PCSK9 tetraloop conjugate with two different modification patterns. The ability of the oligonucleotide to lower plasma PCSK9 through Day 30 (FIG. 7B) and through Day 90 (FIG. 7C) was measured using a PCSK9 ELISA. Serum levels of LDL were also measured, as shown in FIG. 7D.

DETAILED DESCRIPTION OF THE INVENTION

According to some aspects, the disclosure provides oligonucleotides targeting PCSK9 mRNA that are effective for reducing PCSK9 expression in cells, particularly liver cells (e.g., hepatocytes) for the treatment of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof. Accordingly, in related aspects, the disclosure provides methods of treating hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof that involve selectively reducing PCSK9 gene expression in liver. In certain embodiments, PCSK9 targeting oligonucleotides provided herein are designed for delivery to selected cells of target tissues (e.g., liver hepatocytes) to treat hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject.

Further aspects of the disclosure, including a description of defined terms, are provided below.

I. Definitions

Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

Administering: As used herein, the terms “administering” or “administration” means to provide a substance (e.g., an oligonucleotide) to a subject in a manner that is pharmacologically useful (e.g., to treat a condition in the subject).

Asialoglycoprotein receptor (ASGPR): As used herein, the term “Asialoglycoprotein receptor” or “ASGPR” refers to a bipartite C-type lectin formed by a major 48 kDa (ASGPR-1) and minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins).

Atherosclerosis: As used herein, the term “atherosclerosis” refers to a disease involving a narrowing of arteries (e.g., coronary, carotid, peripheral, and/or renal arteries) typically due to the buildup of plaques (made from fat, cholesterol, calcium, and other substances). In some embodiments, narrowing of the coronary arteries may produce symptoms such as angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, and/or palpitations. In some embodiments, narrowing of the carotid arteries may result in a stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain) and/or may produce symptoms such as 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, and/or loss of consciousness. In some embodiments, narrowing of the peripheral arteries may result in numbness or pain within the arms or legs. In some embodiments, narrowing of the renal arteries (resulting in decreased kidney blood flow) may result in chronic kidney disease. Complications of atherosclerosis may include coronary artery disease, stroke, peripheral artery disease, and kidney problems (e.g., chronic kidney disease).

Complementary: As used herein, the term “complementary” refers to a structural relationship between nucleotides (e.g., on two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. In some embodiments, two nucleic acids may have nucleotide sequences that are complementary to each other so as to form regions of complementarity, as described herein.

Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to a nucleotide having a hydrogen at the 2′ position of its pentose sugar as compared with a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the sugar, phosphate group or base.

Double-stranded oligonucleotide: As used herein, the term “double-stranded oligonucleotide” refers to an oligonucleotide that is substantially in a duplex form. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with one another. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

Duplex: As used herein, the term “duplex,” in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base-pairing of two antiparallel sequences of nucleotides.

Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

Hepatocyte: As used herein, the term “hepatocyte” or “hepatocytes” refers to cells of the parenchymal tissues of the liver. These cells make up approximately 70-85% of the liver's mass and manufacture serum albumin, fibrinogen, and the prothrombin group of clotting factors (except for Factors 3 and 4). Markers for hepatocyte lineage cells may include, but are not limited to: transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnfla), and hepatocyte nuclear factor 4a (Hnf4a). Markers for mature hepatocytes may include, but are not limited to: cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, e.g., Huch et al., (2013), Nature, 494(7436): 247-250, the contents of which relating to hepatocyte markers is incorporated herein by reference.

Hypercholesterolemia: As used herein, the term “hypercholesterolemia” refers to the presence of high levels of cholesterol (e.g., low density lipoprotein (LDL)-cholesterol) in the blood. Cholesterol is one of three major classes of lipids manufactured by animal cells and used to construct cell membranes. Cholesterol is water insoluble and transported in the blood plasma within protein particles (lipoproteins). Any lipoprotein (e.g., very low density lipoprotein (VLDL), low density lipoprotein (LDL), intermediate density lipoprotein (IDL) and high density lipoprotein (HDL)) may carry cholesterol, but elevated levels of non-HDL cholesterol (most particularly LDL-cholesterol) are associated with an increased risk of atherosclerosis and coronary heart disease (e.g., coronary artery disease).

Loop: As used herein, the term “loop” refers to an unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).

Modified Internucleotide Linkage: As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non-naturally occurring linkage. Typically, a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

Modified Nucleotide: As used herein, the term “modified nucleotide” refers to a nucleotide having one or more chemical modifications compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, a modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, a modified nucleotide has one or more chemical modifications in its sugar, nucleobase and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc. In certain embodiments, a modified nucleotide comprises a 2′-O-methyl or a 2′-F substitution at the 2′ position of the ribose ring.

Nicked Tetraloop Structure: A “nicked tetraloop structure” is a structure of a RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity to the antisense strand such that the two strands form a duplex, and in which at least one of the strands, generally the sense strand, extends from the duplex in which the extension contains a tetraloop and two self-complementary sequences forming a stem region adjacent to the tetraloop, in which the tetraloop is configured to stabilize the adjacent stem region formed by the self-complementary sequences of the at least one strand.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to a short nucleic acid, e.g., of less than 100 nucleotides in length. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and/or modified nucleotides including, for example, modified ribonucleotides. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA, or single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

Overhang: As used herein, the term “overhang” refers to terminal non-base-pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex. In some embodiments, an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5′ terminus or 3′ terminus of a double-stranded oligonucleotide. In certain embodiments, the overhang is a 3′ or 5′ overhang on the antisense strand or sense strand of a double-stranded oligonucleotide.

Phosphate analog: As used herein, the term “phosphate analog” refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5′ terminal nucleotide of an oligonucleotide in place of a 5′-phosphate, which is often susceptible to enzymatic removal. In some embodiments, a 5′ phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include 5′ phosphonates, such as 5′ methylenephosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP). In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”) at a 5′-terminal nucleotide. An example of a 4′-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, the contents of each of which relating to phosphate analogs are incorporated herein by reference. Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871; U.S. Pat. No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011, the contents of each of which relating to phosphate analogs are incorporated herein by reference).

Proprotein convertase subtilisin/kexin-9 (PCSK9): As used herein, the term “proprotein convertase subtilisin/kexin-9” (also known as PCSK9, NARC-1, neural apoptosis regulated convertase 1, HCHOLA3, and hypercholesterolemia, autosomal dominant 3) refers to the gene encoding PCSK9 protein.

Reduced expression: As used herein, the term “reduced expression” of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and/or a decrease in the amount of activity of the gene in a cell or subject, as compared to an appropriate reference cell or subject. For example, the act of treating a cell with a double-stranded oligonucleotide (e.g., one having an antisense strand that is complementary to PCSK9 mRNA sequence) may result in a decrease in the amount of RNA transcript, protein and/or enzymatic activity (e.g., encoded by the PCSK9 gene) compared to a cell that is not treated with the double-stranded oligonucleotide. Similarly, “reducing expression” as used herein refers to an act that results in reduced expression of a gene (e.g., PCSK9).

Region of Complementarity: As used herein, the term “region of complementarity” refers to a sequence of nucleotides of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides (e.g., a target nucleotide sequence within an mRNA) to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions, e.g., in a phosphate buffer, in a cell, etc. A region of complementarity may be fully complementary to a nucleotide sequence (e.g., a target nucleotide sequence present within an mRNA or portion thereof). For example, a region of complementary that is fully complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary, without any mismatches or gaps, to a corresponding sequence in the mRNA. Alternatively, a region of complementarity may be partially complementary to a nucleotide sequence (e.g., a nucleotide sequence present in an mRNA or portion thereof). For example, a region of complementary that is partially complementary to a nucleotide sequence present in an mRNA has a contiguous sequence of nucleotides that is complementary to a corresponding sequence in the mRNA but that contains one or more mismatches or gaps (e.g., 1, 2, 3, or more mismatches or gaps) compared with the corresponding sequence in the mRNA, provided that the region of complementarity remains capable of hybridizing with the mRNA under appropriate hybridization conditions.

Ribonucleotide: As used herein, the term “ribonucleotide” refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2′ position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the ribose, phosphate group or base.

RNAi Oligonucleotide: As used herein, the term “RNAi oligonucleotide” refers to either (a) a double stranded oligonucleotide having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a single stranded oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

Strand: As used herein, the term “strand” refers to a single contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends, e.g., a 5′-end and a 3′-end.

Subject: As used herein, the term “subject” means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or non-human primate. The terms “individual” or “patient” may be used interchangeably with “subject.”

Synthetic: As used herein, the term “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

Targeting ligand: As used herein, the term “targeting ligand” refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand, and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.

Tetraloop: As used herein, the term “tetraloop” refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable as an increase in melting temperature (T_m) of an adjacent stem duplex that is higher than the T_m of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides. For example, a tetraloop can confer a melting temperature of at least 50° C., at least 55° C., at least 56° C., at least 58° C., at least 60° C., at least 65° C., or at least 75° C. in 10 mM NaHPO₄ to a hairpin comprising a duplex of at least 2 base pairs in length. In some embodiments, a tetraloop may stabilize a base pair in an adjacent stem duplex by stacking interactions. In addition, interactions among the nucleotides in a tetraloop include, but are not limited to non-Watson-Crick base-pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). In some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and is typically 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety). In one embodiment, a tetraloop consists of four nucleotides. Any nucleotide may be used in the tetraloop and standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucl. Acids Res. 13: 3021-3030. For example, the letter “N” may be used to mean that any base may be in that position, the letter “R” may be used to show that A (adenine) or G (guanine) may be in that position, and “B” may be used to show that C (cytosine), G (guanine), or T (thymine) may be in that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA)), the d(GNRA) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example: Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000), which are incorporated by reference herein for their relevant disclosures. In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

Treat: As used herein, the term “treat” refers to the act of providing care to a subject in need thereof, e.g., through the administration a therapeutic agent (e.g., an oligonucleotide) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.

II. Oligonucleotide-Based Inhibitors

i. PCSK9 Targeting Oligonucleotides

Potent oligonucleotides have been identified herein through examination of the PCSK9 mRNA, including mRNAs of different species (human and Rhesus macaque, (see, e.g., Example 1)) and in vitro and in vivo testing. Such oligonucleotides can be used to achieve therapeutic benefit for subjects with a hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof by reducing PCSK9 activity, and consequently, by decreasing or preventing hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), 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/or kidney problems (e.g., chronic kidney disease). For example, potent RNAi oligonucleotides are provided herein that have a sense strand comprising, or consisting of, a sequence as set forth in any one of SEQ ID NO: 1-453, 907-1029, 1153-1192, 1248-1256, and 1266-1268 and an antisense strand comprising, or consisting of, a complementary sequence selected from SEQ ID NO: 454-906, 1030-1152, 1193-1232, 1257-1265, and 1269-1271, as is also arranged the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 454). The sequences can be put into multiple different structures (or formats), as described herein.

In some embodiments, it has been discovered that certain regions of PCSK9 mRNA are hotspots for targeting because they are more amenable than other regions to oligonucleotide-based inhibition. In some embodiments, a hotspot region of PCSK9 consists of a sequence as forth in any one of SEQ ID NOs: 1233-1244. These regions of PCSK9 mRNA may be targeted using oligonucleotides as discussed herein for purposes of inhibiting PCSK9 mRNA expression.

Accordingly, in some embodiments, oligonucleotides provided herein are designed so as to have regions of complementarity to PCSK9 mRNA (e.g., within a hotspot of PCSK9 mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression. The region of complementarity is generally of a suitable length and base content to enable annealing of the oligonucleotide (or a strand thereof) to PCSK9 mRNA for purposes of inhibiting its expression.

In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially complementary to a sequence as set forth in any of SEQ ID NOs: 1-453 or 907-1029, which include certain sequences mapping to within hotspot regions of PCSK9 mRNA. In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is fully complementary to a sequence as set forth in any of SEQ ID NOs: 1-453 or 907-1029. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any of SEQ ID NOs: 1-453 or 907-1029 spans the entire length of an antisense strand. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any one of any of SEQ ID NOs: 1-453 or 907-1029 spans a portion of the entire length of an antisense strand (e.g., all but two nucleotides at the 3′ end of the antisense strand). In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-19 of a sequence as set forth in SEQ ID NOs: 1153-1192.

In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to PCSK9 mRNA that is in the range of 12 to 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to PCSK9 mRNA that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, a region of complementarity to PCSK9 mRNA may have one or more mismatches compared with a corresponding sequence of PCSK9 mRNA. A region of complementarity on an oligonucleotide may have up to 1, up to 2, up to 3, up to 4, etc. mismatches provided that it maintains the ability to form complementary base pairs with PCSK9 mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an oligonucleotide may have no more than 1, no more than 2, no more than 3, or no more than 4 mismatches provided that it maintains the ability to form complementary base pairs with PCSK9 mRNA under appropriate hybridization conditions. In some embodiments, if there are more than one mismatches in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the oligonucleotide maintains the ability to form complementary base pairs with PCSK9 mRNA under appropriate hybridization conditions.

Still, in some embodiments, double-stranded oligonucleotides provided herein comprise, of consist of, a sense strand having a sequence as set forth in any one of SEQ ID NOs: 1-453, 907-1029, 1153-1192, 1248-1256, and 1266-1268 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265, and 1269-1271, as is arranged in the table provided in Table 4 (e.g., a sense strand comprising a sequence as set forth in SEQ ID NO: 1 and an antisense strand comprising a sequence as set forth in SEQ ID NO: 454).

ii. Oligonucleotide Structures

There are a variety of structures of oligonucleotides that are useful for targeting PCSK9 mRNA in the methods of the present disclosure, including RNAi, miRNA, etc. Any of the structures described herein or elsewhere may be used as a framework to incorporate or target a sequence described herein (e.g., a hotpot sequence of PCSK9 such as those illustrated in SEQ ID NOs: 1233-1244 or a sense or antisense strand that comprises or consists of a sequence as set forth as any of SEQ ID NOs: 1 to 453, 907-1029, and 1153-1192 or as set forth as any of SEQ ID NOs: 454-906, 1030-1152, and 1193-1232). Double-stranded oligonucleotides for targeting PCSK9 expression (e.g., via the RNAi pathway) generally have a sense strand and an antisense strand that form a duplex with one another. In some embodiments, the sense and antisense strands are not covalently linked. However, in some embodiments, the sense and antisense strands are covalently linked.

In some embodiments, double-stranded oligonucleotides for reducing PCSK9 expression engage RNA interference (RNAi). For example, RNAi oligonucleotides have been developed with each strand having sizes of 19-25 nucleotides with at least one 3′ overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by the Dicer enzyme to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, as well as WO2010033225, which are incorporated by reference herein for their disclosure of these oligonucleotides). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

In some embodiments, sequences described herein can be incorporated into, or targeted using, oligonucleotides that comprise separate sense and antisense strands that are both in the range of 17 to 40 nucleotides in length. In some embodiments, oligonucleotides incorporating such sequences are provided that have a tetraloop structure within a 3′ extension of their sense strand, and two terminal overhang nucleotides at the 3′ end of the separate antisense strand. In some embodiments, the two terminal overhang nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand is or are not complementary to the target.

In some embodiments, oligonucleotides incorporating such sequences are provided that have sense and antisense strands that are both in the range of 21 to 23 nucleotides in length. In some embodiments, a 3′ overhang is provided on the sense, antisense, or both sense and antisense strands that is 1 or 2 nucleotides in length. In some embodiments, an oligonucleotide has a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, in which the 3′-end of passenger strand and 5′-end of guide strand form a blunt end and where the guide strand has a two nucleotide 3′ overhang.

In some embodiments, oligonucleotides may be in the range of 21 to 23 nucleotides in length. In some embodiments, oligonucleotides may have an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3′ end of the sense and/or antisense strands. In some embodiments, oligonucleotides (e.g., siRNAs) may comprise a 21 nucleotide guide strand that is antisense to a target RNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3′ ends. See, for example, U.S. Pat. Nos. 9,012,138, 9,012,621, and 9,193,753, the contents of each of which are incorporated herein for their relevant disclosures.

In some embodiments, an oligonucleotide of the invention has a 36 nucleotide sense strand that comprises a region extending beyond the antisense-sense duplex, where the extension region has a stem-tetraloop structure where the stem is a six base pair duplex and where the tetraloop has four nucleotides. In certain of those embodiments, three or four of the tetraloop nucleotides are each conjugated to a monovalent GalNac ligand.

In some embodiments, an oligonucleotide of the invention comprises a 25 nucleotide sense strand and a 27 nucleotide antisense strand that when acted upon by a dicer enzyme results in an antisense strand that is incorporated into the mature RISC.

Other oligonucleotides designs for use with the compositions and methods disclosed herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629:141-158), blunt siRNAs (e.g., of 19 bps in length; see: e.g., Kraynack and Baker, RNA Vol. 12, p 163-176 (2006)), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric shorter-duplex siRNA (see, e.g., Chang et al., Mol Ther. 2009 April; 17(4): 725-32), fork siRNAs (see, e.g., Hohjoh, FEBS Letters, Vol 557, issues 1-3; January 2004, p 193-198), single-stranded siRNAs (Elsner; Nature Biotechnology 30, 1063 (2012)), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. J Am Chem Soc 129: 15108-15109 (2007)), and small internally segmented interfering RNA (sisiRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 September; 35(17): 5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the related disclosures therein. Further non-limiting examples of an oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of PCSK9 are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al., Embo J., 2002, 21(17): 4671-4679; see also U.S. Application No. 20090099115).

a. Antisense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting PCSK9 comprises an antisense strand comprising or consisting of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232. In some embodiments, an oligonucleotide comprises an antisense strand comprising or consisting of at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 454-906, 1030-1152, or 1193-1232.

In some embodiments, a double-stranded oligonucleotide may have an antisense strand of up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have an antisense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have an antisense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, an antisense strand of an oligonucleotide may be referred to as a “guide strand.” For example, if an antisense strand can engage with RNA-induced silencing complex (RISC) and bind to an Argonaut protein, or engage with or bind to one or more similar factors, and direct silencing of a target gene, it may be referred to as a guide strand. In some embodiments, a sense strand complementary to a guide strand may be referred to as a “passenger strand.”

b. Sense Strands

In some embodiments, an oligonucleotide disclosed herein for targeting PCSK9 comprises or consists of a sense strand sequence as set forth in in any one of SEQ ID NOs: 1 to 453, 907-1029, and 1153-1192. In some embodiments, an oligonucleotide has a sense strand that comprises or consists of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1 to 453, 907-1029, and 1153-1192.

In some embodiments, an oligonucleotide may have a sense strand (or passenger strand) of up to 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand in a range of 12 to 40 (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

In some embodiments, a sense strand comprises a stem-loop structure at its 3′-end. In some embodiments, a sense strand comprises a stem-loop structure at its 5′-end. In some embodiments, a stem is a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 base pairs in length. In some embodiments, a stem-loop provides the molecule better protection against degradation (e.g., enzymatic degradation) and facilitates targeting characteristics for delivery to a target cell. For example, in some embodiments, a loop provides added nucleotides on which modification can be made without substantially affecting the gene expression inhibition activity of an oligonucleotide. In certain embodiments, an oligonucleotide is provided herein in which the sense strand comprises (e.g., at its 3′-end) a stem-loop set forth as: S₁-L-S₂, in which S₁ is complementary to S₂, and in which L forms a loop between S₁ and S₂ of up to 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). FIG. 3 depicts a non-limiting example of such an oligonucleotide.

In some embodiments, a loop (L) of a stem-loop is a tetraloop (e.g., within a nicked tetraloop structure). A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides.

c. Duplex Length

In some embodiments, a duplex formed between a sense and antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30, or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In certain embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.

d. Oligonucleotide Ends

In some embodiments, an oligonucleotide provided herein comprises sense and antisense strands, such that there is a 3′-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, oligonucleotides provided herein have one 5′ end that is thermodynamically less stable compared to the other 5′ end. In some embodiments, an asymmetric oligonucleotide is provided that includes a blunt end at the 3′ end of a sense strand and an overhang at the 3′ end of an antisense strand. In some embodiments, a 3′ overhang on an antisense strand is 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length).

Typically, an oligonucleotide for RNAi has a two nucleotide overhang on the 3′ end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, an overhang is a 3′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. However, in some embodiments, the overhang is a 5′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.

In some embodiments, one or more (e.g., 2, 3, 4) terminal nucleotides of the 3′ end or 5′ end of a sense and/or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides of the 3′ end of an antisense strand are modified. In some embodiments, the last nucleotide at the 3′ end of an antisense strand is modified, e.g., comprises 2′-modification, e.g., a 2′-O-methoxyethyl. In some embodiments, the last one or two terminal nucleotides at the 3′ end of an antisense strand are complementary to the target. In some embodiments, the last one or two nucleotides at the 3′ end of the antisense strand are not complementary to the target. In some embodiments, the 5′ end and/or the 3′ end of a sense or antisense strand has an inverted cap nucleotide.

e. Mismatches

In some embodiments, there is one or more (e.g., 1, 2, 3, or 4) mismatches between a sense and antisense strand. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3′-terminus of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3′ terminus of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3′-end of the sense strand of the oligonucleotide improved the potency of synthetic duplexes in RNAi, possibly through facilitating processing by Dicer.

iii. Single-Stranded Oligonucleotides

In some embodiments, an oligonucleotide for reducing PCSK9 expression as described herein is single-stranded. Such structures may include, but are not limited to single-stranded RNAi oligonucleotides. Recent efforts have demonstrated the activity of single-stranded RNAi oligonucleotides (see, e.g., Matsui et al. (May 2016), Molecular Therapy, Vol. 24(5), 946-955). However, in some embodiments, oligonucleotides provided herein are antisense oligonucleotides (ASOs). An antisense oligonucleotide is a single-stranded oligonucleotide that has a nucleobase sequence which, when written in the 5′ to 3′ direction, comprises the reverse complement of a targeted segment of a particular nucleic acid and is suitably modified (e.g., as a gapmer) so as to induce RNaseH mediated cleavage of its target RNA in cells or (e.g., as a mixmer) so as to inhibit translation of the target mRNA in cells. Antisense oligonucleotides for use in the instant disclosure may be modified in any suitable manner known in the art including, for example, as shown in U.S. Pat. No. 9,567,587, which is incorporated by reference herein for its disclosure regarding modification of antisense oligonucleotides (including, e.g., length, sugar moieties of the nucleobase (pyrimidine, purine), and alterations of the heterocyclic portion of the nucleobase). Further, antisense molecules have been used for decades to reduce expression of specific target genes (see, e.g., Bennett et al.; Pharmacology of Antisense Drugs, Annual Review of Pharmacology and Toxicology, Vol. 57: 81-105).

iv. Oligonucleotide Modifications

Oligonucleotides may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-paring properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3 (2012): 1-22). Accordingly, in some embodiments, oligonucleotides of the present disclosure may include one or more suitable modifications. In some embodiments, a modified nucleotide has a modification in its base (or nucleobase), the sugar (e.g., ribose, deoxyribose), or the phosphate group.

The number of modifications on an oligonucleotide and the positions of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an oligonucleotide is not protected by an LNP or similar carrier (e.g., “naked delivery”), it may be advantageous for at least some of its nucleotides to be modified. Accordingly, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all of the nucleotides of an oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, with naked delivery, every nucleotide is modified at the 2′-position of the sugar group of that nucleotide. These modifications may be reversible or irreversible. Typically, the 2′ position modification is a 2′-fluoro, 2′-O-methyl, etc. In some embodiments, an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristic (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).

a. Sugar Modifications

In some embodiments, a modified sugar (also referred to herein as a sugar analog) includes a modified deoxyribose or ribose moiety, e.g., in which one or more modifications occur at the 2′, 3′, 4′, and/or 5′ carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy—Nucleic Acids, 2, e103), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated by reference herein for their disclosures relating to sugar modifications.

In some embodiments, a nucleotide modification in a sugar comprises a 2′-modification. In some embodiments, the 2′-modification may be 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, or 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2′-fluoro, 2′-O-methyl, or 2′-O-methoxyethyl. However, a large variety of 2′ position modifications that have been developed for use in oligonucleotides can be employed in oligonucleotides disclosed herein. See, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881. In some embodiments, a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a linkage between the 2′-carbon and a 1′-carbon or 4′-carbon of the sugar. For example, the linkage may comprise an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2′-carbon to 3′-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4′ position of the sugar.

In some embodiments, the terminal 3′-end group (e.g., a 3′-hydroxyl) is a phosphate group or other group, which can be used, for example, to attach linkers, adapters or labels or for the direct ligation of an oligonucleotide to another nucleic acid.

b. 5′ Terminal Phosphates

5′-terminal phosphate groups of oligonucleotides may or in some circumstances enhance the interaction with Argonaut 2. However, oligonucleotides comprising a 5′-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5′ phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5′ end of an oligonucleotide strand is attached to a chemical moiety that mimics the electrostatic and steric properties of a natural 5′-phosphate group (“phosphate mimic”) (see, e.g., Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar. 31; 43(6): 2993-3011, the contents of which relating to phosphate analogs are incorporated herein by reference). Many phosphate mimics have been developed that can be attached to the 5′ end (see, e.g., U.S. Pat. No. 8,927,513, the contents of which relating to phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871, the contents of which relating to phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5′ end of the oligonucleotide.

In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”). See, for example, International Patent Application PCT/US2017/049909, filed on Sep. 1, 2017, U.S. Provisional Application Nos. 62/383,207, entitled 4′ Phosphate Analogs and Oligonucleotides Comprising the Same, filed on Sep. 2, 2016, and 62/393,401, filed on Sep. 12, 2016, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, the contents of each of which relating to phosphate analogs are incorporated herein by reference. In some embodiments, an oligonucleotide provided herein comprises a 4′-phosphate analog at a 5′-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. In other embodiments, a 4′-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4′-carbon of the sugar moiety or analog thereof. In certain embodiments, a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula —O—CH₂—PO(OH)₂ or —O—CH₂—PO(OR)₂, in which R is independently selected from H, CH₃, an alkyl group, CH₂CH₂CN, CH₂OCOC(CH₃)₃, CH₂OCH₂CH₂Si(CH₃)₃, or a protecting group. In certain embodiments, the alkyl group is CH₂CH₃. More typically, R is independently selected from H, CH₃, or CH₂CH₃.

c. Modified Internucleoside Linkages

In some embodiments, the oligonucleotide may comprise a modified internucleoside linkage. In some embodiments, phosphate modifications or substitutions may result in an oligonucleotide that comprises at least one (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage

d. Base Modifications

In some embodiments, oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1′ position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain a nitrogen atom. See e.g., U.S. Published Patent Application No. 20080274462. In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic).

In some embodiments, a universal base is a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering the structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower T_m than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher T_m than a duplex formed with the nucleic acid comprising the mismatched base.

Non-limiting examples of universal-binding nucleotides include inosine, ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct. 11; 22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications).

e. Reversible Modifications

While certain modifications to protect an oligonucleotide from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione).

In some embodiments, a reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance. See U.S. Published Application No. 2011/0294869 originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015/188197 to Solstice Biologics, Ltd. (“Solstice”), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 (“Meade”), PCT Publication No. WO 2014/088920 to Merck Sharp & Dohme Corp, each of which are incorporated by reference for their disclosures of such modifications. This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g. glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and/or lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed and the result is a cleaved oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2′-carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5′-carbon of a sugar, particularly when the modified nucleotide is the 5′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3′-carbon of a sugar, particularly when the modified nucleotide is the 3′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, e.g., International Patent Application PCT/US2017/048239, which published on Mar. 1, 2018 as International Patent Publication WO2018/039364, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof, which was filed on Aug. 23, 2016, the contents of which are incorporated by reference herein for its relevant disclosures.

v. Targeting Ligands

In some embodiments, it may be desirable to target the oligonucleotides of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs, or may avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit for the oligonucleotide. Accordingly, in some embodiments, oligonucleotides disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, oligonucleotides disclosed herein may be modified to facilitate delivery of the oligonucleotide to the hepatocytes of the liver. In some embodiments, an oligonucleotide comprises a nucleotide that is conjugated to one or more targeting ligands.

A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer. For example, a targeting ligand may be an RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferrin, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand, as described, for example, in International Patent Application Publication WO 2016/100401, which was published on Jun. 23, 2016, the relevant contents of which are incorporated herein by reference.

In some embodiments, it is desirable to target an oligonucleotide that reduces the expression of PCSK9 to the hepatocytes of the liver of a subject. Any suitable hepatocyte targeting moiety may be used for this purpose.

GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotides of the instant disclosure may be used to target these oligonucleotides to the ASGPR expressed on these hepatocyte cells.

In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide of the instant disclosure is conjugated to one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3, or 4 nucleotides of the loop of the stem may be individually conjugated to a GalNAc moiety. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand, where each GalNAc moiety is conjugated to one nucleotide.

Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication Number WO2016100401 A1, which published on Jun. 23, 2016, and the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is fairly stable. In some embodiments, a duplex extension (up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide.

III. Formulations

Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce the expression of PCSK9. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enters the cell to reduce PCSK9 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of PCSK9 as disclosed herein. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate-buffered saline solutions, liposomes, micellar structures, and capsids. In some embodiments, naked oligonucleotides or conjugates thereof are formulated in water or in an aqueous solution (e.g., water with pH adjustments). In some embodiments, naked oligonucleotides or conjugates thereof are formulated in basic buffered aqueous solutions (e.g., PBS)

Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotides into cells. For example, cationic lipids, such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche) all of which can be used according to the manufacturer's instructions.

Accordingly, in some embodiments, a formulation comprises a lipid nanoparticle. In some embodiments, an excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013).

In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent (e.g., an oligonucleotide for reducing PCSK9 expression) or more, although the percentage of the active ingredient(s) may be between about 1% and about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

Even though a number of embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated.

IV. Methods of Use

i. Reducing PCSK9 Expression in Cells

In some embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of PCSK9 in the cell. Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses PCSK9 (e.g., liver, lung, kidney, spleen, testis, adipose, and intestinal cells). In some embodiments, the cell is a primary cell that has been obtained from a subject and that may have undergone a limited number of a passages, such that the cell substantially maintains its natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides). In specific embodiments, methods are provided for delivering to a cell an effective amount any one of the oligonucleotides disclosed herein for purposes of reducing expression of PCSK9 solely or primarily in hepatocytes.

In some embodiments, oligonucleotides disclosed herein can be introduced using appropriate nucleic acid delivery methods including injection of a solution containing the oligonucleotides, bombardment by particles covered by the oligonucleotides, exposing the cell or organism to a solution containing the oligonucleotides, or electroporation of cell membranes in the presence of the oligonucleotides. Other appropriate methods for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

The consequences of inhibition can be confirmed by an appropriate assay to evaluate one or more properties of a cell or subject, or by biochemical techniques that evaluate molecules indicative of PCSK9 expression (e.g., RNA, protein). In some embodiments, the extent to which an oligonucleotide provided herein reduces levels of expression of PCSK9 is evaluated by comparing expression levels (e.g., mRNA or protein levels of PCSK9 to an appropriate control (e.g., a level of PCSK9 expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of PCSK9 expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

In some embodiments, administration of an oligonucleotide as described herein results in a reduction in the level of PCSK9 expression in a cell. In some embodiments, the reduction in levels of PCSK9 expression may be a reduction to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower compared with an appropriate control level of PCSK9. The appropriate control level may be a level of PCSK9 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell according to a method disclosed herein is assessed after a finite period of time. For example, levels of PCSK9 may be analyzed in a cell at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introduction of the oligonucleotide into the cell.

In some embodiments, an oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotides disclosed herein (e.g., in the form of an shRNA). In some embodiments, an oligonucleotide is delivered using a transgene that is engineered to express any oligonucleotide disclosed herein. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject.

ii. Treatment Methods

Aspects of the disclosure relate to methods for reducing PCSK9 expression for the treatment of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject. In some embodiments, the methods may comprise administering to a subject in need thereof an effective amount of any one of the oligonucleotides disclosed herein. In some embodiments, such treatments may be used, for example, to decrease or prevent hypercholesterolemia (high levels of low density lipoprotein (LDL)-cholesterol), atherosclerosis, coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), 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/or kidney problems (e.g., chronic kidney disease). In some embodiments, such treatments may be used, for example, to treat or prevent one or more symptoms associated with hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof.

Accordingly, in some embodiments, the present disclosure provides methods of treating a subject at risk of (or susceptible to) hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof including coronary heart disease (e.g., coronary artery disease), angina, shortness of breath, sweating, nausea, dizziness, shortness of breath, arrhythmias, heart palpitations, stroke (i.e., death of brain cells resulting from insufficient blood and oxygen flow to the brain), 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/or kidney problems (e.g., chronic kidney disease).

In certain aspects, the disclosure provides a method for preventing in a subject, a disease, disorder, symptom, or condition as described herein by administering to the subject a therapeutic agent (e.g., an oligonucleotide or vector or transgene encoding same). In some embodiments, the subject to be treated is a subject who will benefit therapeutically from a reduction in the amount of PCSK9 protein, e.g., in the liver.

Methods described herein typically involve administering to a subject an effective amount of an oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that is capable of treating a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

In some embodiments, a subject is administered any one of the compositions disclosed herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides disclosed herein are administered intravenously or subcutaneously.

In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 25 mg/kg (e.g., 1 mg/kg to 5 mg/kg). In some embodiments, oligonucleotides are administered at a dose in a range of 0.1 mg/kg to 5 mg/kg or in a range of 0.5 mg/kg to 5 mg/kg.

As a non-limiting set of examples, the oligonucleotides of the instant disclosure would typically be administered once per year, twice per year, quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly.

In some embodiments, the subject to be treated is a human (e.g., a human patient) or non-human primate or other mammalian subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

EXAMPLES

Example 1: Development of PCSK9 Oligonucleotide Inhibitors Using Human and Mouse Cell-Based Assays

Human and mouse-based assays were used to develop candidate oligonucleotides for inhibition of PCSK9 expression. First, a computer-based algorithm was used to generate candidate oligonucleotide sequences (25-27-mer) for PCSK9 inhibition. Cell-based assays and PCR assays were then employed for evaluation of candidate oligonucleotides for their ability to reduce PCSK9 expression.

The computer-based algorithm provided oligonucleotides that were complementary to human PCSK9 mRNA (SEQ ID NO: 1245, Table 1), of which certain sequences were also complementary to Rhesus monkey PCSK9 mRNA (SEQ ID NO: 1246, Table 1).

TABLE 1
Sequences of human and Rhesus monkey PCSK9 mRNA
	Species	GenBank RefSeq #	SEQ ID NO.
	Human	NM_174936.3	1245
	Rhesus monkey	NM_001112660.1	1246

Of the oligonucleotides that the algorithm provided, 576 oligonucleotides were selected as candidates for experimental evaluation in a Huh-7 cell-based assay. In this assay, Huh-7 human liver cells stably expressing PCSK9 were transfected with the oligonucleotides. Cells were maintained for a period of time following transfection and then levels of remaining PCSK9 mRNA were interrogated using TAQMAN®-based qPCR assays. Two qPCR assays, a 3′ assay and a 5′ assay, were used to determine mRNA levels as measured by HEX (housekeeping gene—SFRS9) and FAM probes, respectively. The results of the cell-based assay with the 576 oligonucleotides are shown in FIGS. 1A and 1B. The percent mRNA remaining is shown for each of the 5′ assay (circle shapes) and the 3′ assay (diamond shapes) in FIG. 1B. Oligonucleotides with the lowest percentage of mRNA remaining compared to mock transfection controls were considered hits. Oligonucleotides with low complementarity to the human genome were used as negative controls.

Based on the activity and locations of these oligonucleotides, hotspots on the human PCSK9 mRNA were defined. A hotspot was identified as a stretch on the human PCSK9 mRNA sequence associated with at least one oligonucleotide resulting in mRNA levels that were less than or equal to 35% in either assay compared with controls. Accordingly, the following hotspots within the human PCSK9 mRNA sequence (NM_174936.3) were identified: 746-783, 2602-2639, 2737-2792, 2880-2923, 2956-2996, 3015-3075, 3099-3178, 3190-3244, 3297-3359, 3649-3446, 3457-3499, and 3532-3715.

The sequences of the hotspots are outlined in Table 2.

TABLE 2
Sequences of Hotspots
Hotspot
Position
In Human
PCSK9		SEQ
mRNA	Sequence	ID NO.
746-783	CGACCTGCTGGAGCTGGCCTTGAAGTTGCC	1233
	CCATGTCG
2602-2639	AGCCTCCTTGCCTGGAACTCACTCACTCTG	1234
	GGTGCCTC
2737-2792	CAATGTGCCGATGTCCGTGGGCAGAATGAC	1235
	TTTTATTGAGCTCTTGTTCCGTGCCA
2880-2923	CGTTGGGGGGTGAGTGTGAAAGGTGCTGAT	1236
	GGCCCTCATCTCCA
2956-2996	GATTAATGGAGGCTTAGCTTTCTGGATGGC	1237
	ATCTAGCCAGA
3015-3075	CCCTGGTGGTCACAGGCTGTGCCTTGGTTT	1238
	CCTGAGCCACCTTTACTCTGCTCTATGCCA
	G
3099-3178	TGGCCTGCGGGGAGCCATCACCTAGGACTG	1239
	ACTCGGCAGTGTGCAGTGGTGCATGCACTG
	TCTCAGCCAACCCGCTCCAC
3190-3244	GTACACATTCGCACCCCTACTTCACAGAGG	1240
	AAGAAACCTGGAACCAGAGGGGGCG
3297-3359	GCTCTGAAGCCAAGCCTCTTCTTACTTCAC	1241
	CCGGCTGGGCTCCTCATTTTTACGGGTAAC
	AGT
3469-3446	AACGATGCCTGCAGGCATGGAACTTTTTCC	1242
	GTTATCACCCAGGCCT
3457-3499	TTCACTGGCCTGGCGGAGATGCTTCTAAGG	1243
	CATGGTCGGGGGA
3532-3715	GCCCCACCCAAGCAAGCAGACATTTATCTT	1244
	TTGGGTCTGTCCTCTCTGTTGCCTTTTTAC
	AGCCAACTTTTCTAGACCTGTTTTGCTTTT
	GTAACTTGAAGATATTTATTCTGGGTTTTG
	TAGCATTTTTATTAATATGGTGACTTTTTA
	AAATAAAAACAAACAAACGTTGTCCTAACA
	AAAA

Dose Response Analysis

Of the 576 oligonucleotides evaluated in the initial Huh-7 cell-based assay, 96 particularly active oligonucleotides were selected as hits based on their ability to knock down PCSK9 levels and were subjected to a secondary screen (FIGS. 2A and 2B).

In this secondary screen, the candidate oligonucleotides were tested using the same assay as in the primary screen, but at two different concentrations 0.1 nM and 1 nM (FIGS. 2A and 2B). The target mRNA levels were generally normalized based on splicing factor, arginine/serine-rich 9 (SFRS9), a housekeeping gene that provides a stable expression reference across samples, to generate the percent mRNA shown in FIGS. 2A and 2B. The tested oligonucleotides in each of FIGS. 2A and 2B are shown compared to mock transfection control. All 96 oligonucleotides had the same modification pattern, designated M1, which contains a combination of ribonucleotides, deoxyribonucleotides and 2′-O-methyl modified nucleotides. The sequences of the 96 oligonucleotides tested are provided in Table 3.

TABLE 3
Candidate oligonucleotide Sequences for Huh-7 Cell-Based
Assay
Sense                           Corresponding Antisense
SEQ ID NO.                      SEQ ID NO.
35, 41, 51, 53, 56-58, 66, 177- 488, 494, 504, 506, 509-511,
180, 187, 192, 196, 201-204,    519, 630-633, 640, 645, 649,
219-225, 227, 237-241, 243,     654-657, 672-678, 680, 690-
248, 249, 257, 261, 262, 264,   694, 696, 701, 702, 710, 714,
266, 268, 274, 280, 281, 288-   715, 717, 719, 721, 727, 733,
292, 297, 304-306, 315, 316,    734, 741-745, 750, 757-759,
320-322, 328-330, 333, 334,     768, 769, 773-775, 781-783,
344, 345, 347, 349, 351, 352,   786, 787, 797, 798, 800, 802,
374, 375, 385-395, 400-402,     804, 805, 827, 828, 838-848,
405, 408-411, 418, 433, 434,    853-855, 858, 861-864, 871,
440-442                         886, 887, 893-895
Sense and antisense SEQ ID NO. columns provide the sense strand and respective antisense strand, in relative order, that are hybridized to make each oligonucleotide. For example, sense strand of SEQ ID NO: 35 hybridizes with antisense strand of SEQ ID NO: 488; each of the oligonucleotides tested had the same modification pattern.

At this stage, the most potent sequences from the testing were selected for further analysis. The selected sequences were converted to a nicked tetraloop conjugate structure format (a 36-mer passenger strand with a 22-mer guide strand). See FIG. 3 for a generic tetraloop conjugate structure. Four GalNAc moieties were conjugated to nucleotides in the tetraloop of the sense strand. Conjugation was performed using a click linker. The GalNAc used was as shown below:

These oligonucleotides were then tested as before, and each oligonucleotide was evaluated at two concentrations for its ability to reduce PCSK9 mRNA expression in vitro, using Huh-7 cells, as well as in vivo, using a mouse HDI model.

In Vivo Murine Screening and In Vitro Human Cell Line Screening

Data from the above in vitro experiments were assessed to identify tetraloops and modification patterns that would improve delivery properties while maintaining activity for reduction of PCSK9 expression in the mouse hepatocytes. As shown in FIG. 4, 12 human PCSK9 tetraloop conjugates with a range of modifications were dosed subcutaneously into mice at a concentration of 3 mg/kg. Animals were administered 2 ml of human PCSK9 plasmid (pcDNA3.1-hPCSK9, total 16 μg) suspended in PBS per animal by tail vein (intravenous) injection on day 3 after the subcutaneous dosing of tetraloop conjugates. Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate PCSK9 mRNA levels by RT-qPCR. The percent PCSK9 mRNA as compared to PBS control mRNA was determined based on these measurements.

Further tetraloop sequences were tested in human Huh-7 cells at two different concentrations (0.03 nM and 0.1 nM in tetraloop formation; labeled as “Phase T2”) (FIG. 5A). From the 40 tetraloop oligonucleotides tested (shown in FIG. 5A), 21 different base sequences were selected to be scaled up as 5′-MOP/GalNAc conjugates for further in vivo testing (FIGS. 5B and 5C). The PCSK9 oligonucleotides were subcutaneously administered to CD-1 mice transiently expressing human PCSK9 mRNA by hydrodynamic injection (HDI) of a human PCSK9 expression plasmid (pcDNA3.1-hPCSK9, total 16 μg). Mice were euthanized on day 4 following administration. Liver samples were obtained and RNA was extracted to evaluate PCSK9 mRNA levels by RT-qPCR. The percent PCSK9 mRNA as compared to PBS control mRNA was determined based on these measurements. As shown in FIGS. 5B-5C, different concentrations (1 mg/kg and 2 mg/kg) were used for the candidate molecules. A candidate of sense sequence SEQ ID NO: 1182 and antisense sequence SEQ ID NO: 1222 may be seen in both FIG. 5B and FIG. 5C.

Additional testing of certain PCSK9 oligonucleotides in the mouse HDI model described above was performed using three different PCSK9 tetraloop conjugates with varied modification patterns at three different concentrations (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg). Results are shown in FIGS. 6A and 6B.

In Vivo Non-Human Primate Screening

An additional study was performed to evaluate PCSK9 mRNA KD with tetraloop conjugates in non-human primates. Cynomolgus monkeys (n=4 per group) were administered 3 or 6 mg/kg subcutaneously in a single dose. Clinical observations were recorded daily, and blood samples were taken three times prior to the dosing and twice a week until day 36 and weekly through day 90. Serum samples were analyzed for a standard LFT panel (ALT, AST, ALP, and GGT), as well as LDL-c, HDL-c, total cholesterol, and TG. Three sets of sequences (sense and antisense) were tested: S1266-AS1269, S1267-AS1270, and S1268-AS1271 and results are shown in FIGS. 7A-7C. All three sets of sequences were able to reduce plasma levels of PCSK9 relative to the pre-dose levels.

Materials and Methods

Transfection

For the first screen, Lipofectamine RNAiMAX™ was used to complex the oligonucleotides for efficient transfection. Oligonucleotides, RNAiMAX and Opti-MEM incubated together at room temperature for 20 minutes and then 50 μL of this mix was added per well to plates prior to transfection. Media was aspirated from a flask of actively passaging cells and the cells were incubated at 37° C. in the presence of trypsin for 3-5 minutes. After cells no longer adhered to the flask, cell growth media (lacking penicillin and streptomycin) was added to neutralize the trypsin and to suspend the cells. A 10 μL aliquot was removed and cells were counted with a hemocytometer to quantify the cells on a per milliliter basis. A diluted cell suspension was added to the 96-well transfection plates, which already contained the oligonucleotides in Opti-MEM. The transfection plates were then incubated for 24 hours at 37° C. After 24 hours of incubation, media was aspirated from each well.

For subsequent screens and experiments, e.g., the secondary screen, Lipofectamine RNAiMAX was used to complex the oligonucleotides for reverse transfection. The complexes were made by mixing RNAiMAX and siRNAs in OptiMEM medium for 15 minutes. The transfection mixture was transferred to multi-well plates and cell suspension was added to the wells. After 24 hours incubation the cells were washed once with PBS and then processed described above.

Hydrodynamic Injection (HDI)

CD-1 female mice were obtained from Charles River Laboratories. All mice were maintained in an AALAC and IACUC approved animal facility at the Dicerna Pharmaceuticals. Animals were divided into appropriate number of study groups and dosed with the test article assigned to that group. Animals were dosed subcutaneously with the PCSK tetraloop conjugates. Animals were administered with 2 ml hPCSK9 plasmid suspended in PBS per animal by tail vein intravenous injection on day 3 after the subcutaneous dosing of tetraloop conjugate. Mice were sacrificed on days 4 via CO₂ asphyxiation and liver tissue was collected. Liver tissue was collected by taking two 4 mm punch biopsies and processed to RNA isolation, cDNA synthesis, q-RT PCR, according the manufacturer's protocol. pcDNA3.1-hPCSK9 plasmid encoding the human PCSK9 (NM 174936.3) gene (hPCSK9) was synthesized by Genewiz.

cDNA Synthesis

Cells were lysed for 5 minutes using the iScript RT-qPCR sample preparation buffer from Bio-Rad. The supernatants containing total RNA were then stored at −80° C. or used for reverse transcription using the High Capacity Reverse Transcription kit (Life Technologies) in a 10 microliter reaction. The cDNA was then diluted to 50 μL with nuclease free water and used for quantitative PCR with multiplexed 5′-endonuclease assays and SSoFast qPCR mastermix (Bio-Rad laboratories).

qPCR Assays

For each target, mRNA levels were quantified by two 5′ nuclease assays. In general, several assays are screened for each target. The two assays selected displayed a combination of good efficiency, low limit of detection, and broad 5′43′ coverage of the gene of interest (GOI). Both assays against one GOI could be combined in one reaction when different fluorophores were used on the respective probes. Thus, the final step in assay validation was to determine the efficiency of the selected assays when they were combined in the same qPCR or “multi-plexed.”

Linearized plasmids for both assays in 10-fold dilutions were combined and qPCR was performed. The efficiency of each assay was determined as described above. The accepted efficiency rate was 90-110%.

While validating multi-plexed reactions using linearized plasmid standards, Cq values for the target of interest were also assessed using cDNA as the template. The cDNA, in this case, was derived from RNA isolated on the Corbett (˜5 ng/μl in water) from untransfected cells. In this way, the observed Cq values from this sample cDNA were representative of the expected Cq values from a 96-well plate transfection. In cases where Cq values were greater than 30, other cell lines were sought that exhibit higher expression levels of the gene of interest. A library of total RNA isolated from via high-throughput methods on the Corbett from each human and mouse line was generated and used to screen for acceptable levels of target expression.

Description of Oligonucleotide Nomenclature

All oligonucleotides described herein are designated either SN₁-ASN₂-MN₃. The following designations apply:

• • N₁: sequence identifier number of the sense strand sequence
  • N₂: sequence identifier number of the antisense strand sequence
  • N₃: reference number of modification pattern, in which each number represents a pattern of modified nucleotides in the oligonucleotide.
    For example, S1-AS454-M1 represents an oligonucleotide with a sense sequence that is set forth by SEQ ID NO: 1, an antisense sequence that is set forth by SEQ ID NO: 454, and which is adapted to a modification pattern identified as M1.

TABLE 4
		S SEQ		AS SEQ
App Name	Sense Sequence/mRNA seq	ID NO	Antisense Sequence	ID NO
S1-AS454-	AAGCACCCACACCCUAGAAUGUUTC	1	GAAACAUUCUAGGGUGUGG	454
M1			GUGCUUGA
S2-AS455-	AGCACCCACACCCUAGAAGUUUUCC	2	GGAAAACUUCUAGGGUGUG	455
M1			GGUGCUUG
S3-AS456-	GCACCCACACCCUAGAAGGUUUCCG	3	CGGAAACCUUCUAGGGUGU	456
M1			GGGUGCUU
S4-AS457-	ACCCACACCCUAGAAGGUUUCCGCA	4	UGCGGAAACCUUCUAGGGU	457
M1			GUGGGUGC
S5-AS458-	CCCACACCCUAGAAGGUUUUCGCAG	5	CUGCGAAAACCUUCUAGGG	458
M1			UGUGGGUG
S6-AS459-	AGUUCAGGGUCUGAGCCUGUAGGAG	6	CUCCUACAGGCUCAGACCC	459
M1			UGAACUGA
S7-AS460-	GUUCAGGGUCUGAGCCUGGAGGAGT	7	ACUCCUCCAGGCUCAGACC	460
M1			CUGAACUG
S8-AS461-	UUCAGGGUCUGAGCCUGGAUGAGTG	8	CACUCAUCCAGGCUCAGAC	461
M1			CCUGAACU
S9-AS462-	UCAGGGUCUGAGCCUGGAGUAGUGA	9	UCACUACUCCAGGCUCAGA	462
M1			CCCUGAAC
S10-AS463-	AGGGUCUGAGCCUGGAGGAUUGAGC	10	GCUCAAUCCUCCAGGCUCA	463
M1			GACCCUGA
S11-AS464-	GGUCUGAGCCUGGAGGAGUUAGCCA	11	UGGCUAACUCCUCCAGGCU	464
M1			CAGACCCU
S12-AS465-	AGGAUUCCGCGCGCCCCUUUACGCG	12	CGCGUAAAGGGGCGCGCGG	465
M1			AAUCCUGG
S13-AS466-	GGAUUCCGCGCGCCCCUUCACGCGC	13	GCGCGUGAAGGGGCGCGCG	466
M1			GAAUCCUG
S14-AS467-	UCACGCGCCCUGCUCCUGAACUUCA	14	UGAAGUUCAGGAGCAGGGC	467
M1			GCGUGAAG
S15-AS468-	CACGCGCCCUGCUCCUGAAUUUCAG	15	CUGAAAUUCAGGAGCAGGG	468
M1			CGCGUGAA
S16-AS469-	CCCUGCUCCUGAACUUCAGUUCCTG	16	CAGGAACUGAAGUUCAGGA	469
M1			GCAGGGCG
S17-AS470-	CUGCUCCUGAACUUCAGCUUCUGCA	17	UGCAGAAGCUGAAGUUCAG	470
M1			GAGCAGGG
S18-AS471-	UGCUCCUGAACUUCAGCUCUUGCAC	18	GUGCAAGAGCUGAAGUUCA	471
M1			GGAGCAGG
S19-AS472-	GCUCCUGAACUUCAGCUCCUGCACA	19	UGUGCAGGAGCUGAAGUUC	472
M1			AGGAGCAG
S20-AS473-	CUCCUGAACUUCAGCUCCUUCACAG	20	CUGUGAAGGAGCUGAAGUU	473
M1			CAGGAGCA
S21-AS474-	UCCUGAACUUCAGCUCCUGUACAGT	21	ACUGUACAGGAGCUGAAGU	474
M1			UCAGGAGC
S22-AS475-	CCUGAACUUCAGCUCCUGCACAGTC	22	GACUGUGCAGGAGCUGAAG	475
M1			UUCAGGAG
S23-AS476-	CUGAACUUCAGCUCCUGCAUAGUCC	23	GGACUAUGCAGGAGCUGAA	476
M1			GUUCAGGA
S24-AS477-	UGAACUUCAGCUCCUGCACAGUCCT	24	AGGACUGUGCAGGAGCUGA	477
M1			AGUUCAGG
S25-AS478-	GAACUUCAGCUCCUGCACAUUCCTC	25	GAGGAAUGUGCAGGAGCUG	478
M1			AAGUUCAG
S26-AS479-	AACUUCAGCUCCUGCACAGUCCUCC	26	GGAGGACUGUGCAGGAGCU	479
M1			GAAGUUCA
S27-AS480-	ACUUCAGCUCCUGCACAGUUCUCCC	27	GGGAGAACUGUGCAGGAGC	480
M1			UGAAGUUC
S28-AS481-	CUUCAGCUCCUGCACAGUCUUCCCC	28	GGGGAAGACUGUGCAGGAG	481
M1			CUGAAGUU
S29-AS482-	ACAGUCCUCCCCACCGCAAUGCUCA	29	UGAGCAUUGCGGUGGGGAG	482
M1			GACUGUGC
S30-AS483-	CAGUCCUCCCCACCGCAAGUCUCAA	30	UUGAGACUUGCGGUGGGGA	483
M1			GGACUGUG
S31-AS484-	GCCUCUAGGUCUCCUCGCCAGGACA	31	UGUCCUGGCGAGGAGACCU	484
M1			AGAGGCCG
S32-AS485-	GCCAGGACAGCAACCUCUCUCCUGG	32	CCAGGAGAGAGGUUGCUGU	485
M1			CCUGGCGA
S33-AS486-	GGACAGCAACCUCUCCCCUUGCCCT	33	AGGGCAAGGGGAGAGGUUG	486
M1			CUGUCCUG
S34-AS487-	CCCCUGGCCCUCAUGGGCAUCGUCA	34	UGACGAUGCCCAUGAGGGC	487
M1			CAGGGGAG
S35-AS488-	UGGCCCUCAUGGGCACCGUUAGCTC	35	GAGCUAACGGUGCCCAUGA	488
M1			GGGCCAGG
S36-AS489-	GGCCCUCAUGGGCACCGUCAGCUCC	36	GGAGCUGACGGUGCCCAUG	489
M1			AGGGCCAG
S37-AS490-	GCCCUCAUGGGCACCGUCAUCUCCA	37	UGGAGAUGACGGUGCCCAU	490
M1			GAGGGCCA
S38-AS491-	GCGGUCCUGGUGGCCGCUGUCACTG	38	CAGUGACAGCGGCCACCAG	491
M1			GACCGCCU
S39-AS492-	GGCCUGGCCGAAGCACCCGAGCACG	39	CGUGCUCGGGUGCUUCGGC	492
M1			CAGGCCGU
S40-AS493-	ACCCGAGCACGGAACCACAUCCACC	40	GGUGGAUGUGGUUCCGUGC	493
M1			UCGGGUGC
S41-AS494-	AGCACGGAACCACAGCCACUUUCCA	41	UGGAAAGUGGCUGUGGUUC	494
M1			CGUGCUCG
S42-AS495-	CACGGAACCACAGCCACCUUCCACC	42	GGUGGAAGGUGGCUGUGGU	495
M1			UCCGUGCU
S43-AS496-	ACGGAACCACAGCCACCUUUCACCG	43	CGGUGAAAGGUGGCUGUGG	496
M1			UUCCGUGC
S44-AS497-	GCCAAGGAUCCGUGGAGGUUGCCTG	44	CAGGCAACCUCCACGGAUC	497
M1			CUUGGCGC
S45-AS498-	CCAAGGAUCCGUGGAGGUUUCCUGG	45	CCAGGAAACCUCCACGGAU	498
M1			CCUUGGCG
S46-AS499-	AAGGAUCCGUGGAGGUUGCUUGGCA	46	UGCCAAGCAACCUCCACGG	499
M1			AUCCUUGG
S47-AS500-	GGAUCCGUGGAGGUUGCCUUGCACC	47	GGUGCAAGGCAACCUCCAC	500
M1			GGAUCCUU
S48-AS501-	UGGAGGUUGCCUGGCACCUACGUGG	48	CCACGUAGGUGCCAGGCAA	501
M1			CCUCCACG
S49-AS502-	UGCCUGGCACCUACGUGGUUGUGCT	49	AGCACAACCACGUAGGUGC	502
M1			CAGGCAAC
S50-AS503-	GCCUGGCACCUACGUGGUGUUGCTG	50	CAGCAACACCACGUAGGUG	503
M1			CCAGGCAA
S51-AS504-	AGGAGGAGACCCACCUCUCUCAGTC	51	GACUGAGAGAGGUGGGUCU	504
M1			CCUCCUUC
S52-AS505-	CCUGCAUGUCUUCCAUGGCUUUCTT	52	AAGAAAGCCAUGGAAGACA	505
M1			UGCAGGAU
S53-AS506-	UGCAUGUCUUCCAUGGCCUUCUUCC	53	GGAAGAAGGCCAUGGAAGA	506
M1			CAUGCAGG
S54-AS507-	ACCUGCUGGAGCUGGCCUUUAAGTT	54	AACUUAAAGGCCAGCUCCA	507
M1			GCAGGUCG
S55-AS508-	CUGCUGGAGCUGGCCUUGAAGUUGC	55	GCAACUUCAAGGCCAGCUC	508
M1			CAGCAGGU
S56-AS509-	UGCUGGAGCUGGCCUUGAAUUUGCC	56	GGCAAAUUCAAGGCCAGCU	509
M1			CCAGCAGG
S57-AS510-	UGGAGCUGGCCUUGAAGUUUCCCCA	57	UGGGGAAACUUCAAGGCCA	510
M1			GCUCCAGC
S58-AS511-	GGCCUUGAAGUUGCCCCAUUUCGAC	58	GUCGAAAUGGGGCAACUUC	511
M1			AAGGCCAG
S59-AS512-	GCCUUGAAGUUGCCCCAUGUCGACT	59	AGUCGACAUGGGGCAACUU	512
M1			CAAGGCCA
S60-AS513-	CCUUGAAGUUGCCCCAUGUUGACTA	60	UAGUCAACAUGGGGCAACU	513
M1			UCAAGGCC
S61-AS514-	CUUGAAGUUGCCCCAUGUCUACUAC	61	GUAGUAGACAUGGGGCAAC	514
M1			UUCAAGGC
S62-AS515-	ACUCCUCUGUCUUUGCCCAUAGCAT	62	AUGCUAUGGGCAAAGACAG	515
M1			AGGAGUCC
S63-AS516-	CUCCUCUGUCUUUGCCCAGAGCATC	63	GAUGCUCUGGGCAAAGACA	516
M1			GAGGAGUC
S64-AS517-	UCCUCUGUCUUUGCCCAGAUCAUCC	64	GGAUGAUCUGGGCAAAGAC	517
M1			AGAGGAGU
S65-AS518-	CCUCUGUCUUUGCCCAGAGUAUCCC	65	GGGAUACUCUGGGCAAAGA	518
M1			CAGAGGAG
S66-AS519-	UCUGUCUUUGCCCAGAGCAUCCCGT	66	ACGGGAUGCUCUGGGCAAA	519
M1			GACAGAGG
S67-AS520-	CUGUCUUUGCCCAGAGCAUUCCGTG	67	CACGGAAUGCUCUGGGCAA	520
M1			AGACAGAG
S68-AS521-	GUCUUUGCCCAGAGCAUCCUGUGGA	68	UCCACAGGAUGCUCUGGGC	521
M1			AAAGACAG
S69-AS522-	UCUUUGCCCAGAGCAUCCCUUGGAA	69	UUCCAAGGGAUGCUCUGGG	522
M1			CAAAGACA
S70-AS523-	UUUGCCCAGAGCAUCCCGUUGAACC	70	GGUUCAACGGGAUGCUCUG	523
M1			GGCAAAGA
S71-AS524-	AGAGCAUCCCGUGGAACCUUGAGCG	71	CGCUCAAGGUUCCACGGGA	524
M1			UGCUCUGG
S72-AS525-	GAGCAUCCCGUGGAACCUGUAGCGG	72	CCGCUACAGGUUCCACGGG	525
M1			AUGCUCUG
S73-AS526-	AGCAUCCCGUGGAACCUGGAGCGGA	73	UCCGCUCCAGGUUCCACGG	526
M1			GAUGCUCU
S74-AS527-	GCAUCCCGUGGAACCUGGAUCGGAT	74	AUCCGAUCCAGGUUCCACG	527
M1			GGAUGCUC
S75-AS528-	CAUCCCGUGGAACCUGGAGUGGATT	75	AAUCCACUCCAGGUUCCAC	528
M1			GGGAUGCU
S76-AS529-	AUCCCGUGGAACCUGGAGCUGAUTA	76	UAAUCAGCUCCAGGUUCCA	529
M1			CGGGAUGC
S77-AS530-	UCCCGUGGAACCUGGAGCGUAUUAC	77	GUAAUACGCUCCAGGUUCC	530
M1			ACGGGAUG
S78-AS531-	CCCGUGGAACCUGGAGCGGAUUACC	78	GGUAAUCCGCUCCAGGUUC	531
M1			CACGGGAU
S79-AS532-	CCGUGGAACCUGGAGCGGAUUACCC	79	GGGUAAUCCGCUCCAGGUU	532
M1			CCACGGGA
S80-AS533-	CUGGAGCGGAUUACCCCUCUACGGT	80	ACCGUAGAGGGGUAAUCCG	533
M1			CUCCAGGU
S81-AS534-	UGGAGCGGAUUACCCCUCCACGGTA	81	UACCGUGGAGGGGUAAUCC	534
M1			GCUCCAGG
S82-AS535-	GGAGCGGAUUACCCCUCCAUGGUAC	82	GUACCAUGGAGGGGUAAUC	535
M1			CGCUCCAG
S83-AS536-	GAGCGGAUUACCCCUCCACUGUACC	83	GGUACAGUGGAGGGGUAAU	536
M1			CCGCUCCA
S84-AS537-	AGCGGAUUACCCCUCCACGUUACCG	84	CGGUAACGUGGAGGGGUAA	537
M1			UCCGCUCC
S85-AS538-	CGGAUUACCCCUCCACGGUACCGGG	85	CCCGGUACCGUGGAGGGGU	538
M1			AAUCCGCU
S86-AS539-	GGAUUACCCCUCCACGGUAUCGGGC	86	GCCCGAUACCGUGGAGGGG	539
M1			UAAUCCGC
S87-AS540-	UCCACGGUACCGGGCGGAUUAAUAC	87	GUAUUAAUCCGCCCGGUAC	540
M1			CGUGGAGG
S88-AS541-	CGGAGGCAGCCUGGUGGAGUUGUAT	88	AUACAACUCCACCAGGCUG	541
M1			CCUCCGUC
S89-AS542-	AGACACCAGCAUACAGAGUUACCAC	89	GUGGUAACUCUGUAUGCUG	542
M1			GUGUCUAG
S90-AS543-	GCAUACAGAGUGACCACCGUGAAAT	90	AUUUCACGGUGGUCACUCU	543
M1			GUAUGCUG
S91-AS544-	CGAGAAUGUGCCCGAGGAGUACGGG	91	CCCGUACUCCUCGGGCACA	544
M1			UUCUCGAA
S92-AS545-	GAGAAUGUGCCCGAGGAGGACGGGA	92	UCCCGUCCUCCUCGGGCAC	545
M1			AUUCUCGA
S93-AS546-	AGAAUGUGCCCGAGGAGGAUGGGAC	93	GUCCCAUCCUCCUCGGGCA	546
M1			CAUUCUCG
S94-AS547-	GCAAGUGUGACAGUCAUGGUACCCA	94	UGGGUACCAUGACUGUCAC	547
M1			ACUUGCUG
S95-AS548-	CAAGUGUGACAGUCAUGGCACCCAC	95	GUGGGUGCCAUGACUGUCA	548
M1			CACUUGCU
S96-AS549-	AAGUGUGACAGUCAUGGCAUCCACC	96	GGUGGAUGCCAUGACUGUC	549
M1			ACACUUGC
S97-AS550-	CGCAGCCUGCGCGUGCUCAACUGCC	97	GGCAGUUGAGCACGCGCAG	550
M1			GCUGCGCA
S98-AS551-	GCAGCCUGCGCGUGCUCAAUUGCCA	98	UGGCAAUUGAGCACGCGCA	551
M1			GGCUGCGC
S99-AS552-	AGCCUGUGGGGCCACUGGUUGUGCT	99	AGCACAACCAGUGGCCCCA	552
M1			CAGGCUGG
S100-	CCUCUACUCCCCAGCCUCAUCUCCC	100	GGGAGAUGAGGCUGGGGAG	553
AS553-M1			UAGAGGCA
S101-	CAGCCUCAGCUCCCGAGGUUAUCAC	101	GUGAUAACCUCGGGAGCUG	554
AS554-M1			AGGCUGGG
S102-	GCCACCAAUGCCCAAGACCAGCCGG	102	CCGGCUGGUCUUGGGCAUU	555
AS555-M1			GGUGGCCC
S103-	AUGCCCAAGACCAGCCGGUUACCCT	103	AGGGUAACCGGCUGGUCUU	556
AS556-M1			GGGCAUUG
S104-	UGCCCAAGACCAGCCGGUGACCCTG	104	CAGGGUCACCGGCUGGUCU	557
AS557-M1			UGGGCAUU
S105-	GUCACAGAGUGGGACAUCAUAGGCT	105	AGCCUAUGAUGUCCCACUC	558
AS558-M1			UGUGACAC
S106-	GAGUGGGACAUCACAGGCUUCUGCC	106	GGCAGAAGCCUGUGAUGUC	559
AS559-M1			CCACUCUG
S107-	UGGGACAUCACAGGCUGCUUCCCAC	107	GUGGGAAGCAGCCUGUGAU	560
AS560-M1			GUCCCACU
S108-	GGGACAUCACAGGCUGCUGUCCACG	108	CGUGGACAGCAGCCUGUGA	561
AS561-M1			UGUCCCAC
S109-	CUCACCCUGGCCGAGUUGAUGCAGA	109	UCUGCAUCAACUCGGCCAG	562
AS562-M1			GGUGAGCU
S110-	ACCCUGGCCGAGUUGAGGCAGAGAC	110	GUCUCUGCCUCAACUCGGC	563
AS563-M1			CAGGGUGA
S111-	ACUUCUCUGCCAAAGAUGUUAUCAA	111	UUGAUAACAUCUUUGGCAG	564
AS564-M1			AGAAGUGG
S112-	CCCAUGGGGCAGGUUGGCAUCUGTT	112	AACAGAUGCCAACCUGCCC	565
AS565-M1			CAUGGGUG
S113-	UGGGGCAGGUUGGCAGCUGUUUUGC	113	GCAAAACAGCUGCCAACCU	566
AS566-M1			GCCCCAUG
S114-	CUGUUUUGCAGGACUGUAUUGUCAG	114	CUGACAAUACAGUCCUGCA	567
AS567-M1			AAACAGCU
S115-	UUUUGCAGGACUGUAUGGUUAGCAC	115	GUGCUAACCAUACAGUCCU	568
AS568-M1			GCAAAACA
S116-	CAGGACUGUAUGGUCAGCAUACUCG	116	CGAGUAUGCUGACCAUACA	569
AS569-M1			GUCCUGCA
S117-	GGACUGUAUGGUCAGCACAUUCGGG	117	CCCGAAUGUGCUGACCAUA	570
AS570-M1			CAGUCCUG
S118-	CGCUGCGCCCCAGAUGAGGAGCUGC	118	GCAGCUCCUCAUCUGGGGC	571
AS571-M1			GCAGCGGG
S119-	GCGCCCCAGAUGAGGAGCUUCUGAG	119	CUCAGAAGCUCCUCAUCUG	572
AS572-M1			GGGCGCAG
S120-	CCCCAGAUGAGGAGCUGCUUAGCTG	120	CAGCUAAGCAGCUCCUCAU	573
AS573-M1			CUGGGGCG
S121-	CCCAGAUGAGGAGCUGCUGAGCUGC	121	GCAGCUCAGCAGCUCCUCA	574
AS574-M1			UCUGGGGC
S122-	CCAGAUGAGGAGCUGCUGAUCUGCT	122	AGCAGAUCAGCAGCUCCUC	575
AS575-M1			AUCUGGGG
S123-	CGGCGGGGCGAGCGCAUGGAGGCCC	123	GGGCCUCCAUGCGCUCGCC	576
AS576-M1			CCGCCGCU
S124-	GGCGGGGCGAGCGCAUGGAUGCCCA	124	UGGGCAUCCAUGCGCUCGC	577
AS577-M1			CCCGCCGC
S125-	GGCGAGCGCAUGGAGGCCCAAGGGG	125	CCCCUUGGGCCUCCAUGCG	578
AS578-M1			CUCGCCCC
S126-	CUGGUCUGCCGGGCCCACAACGCTT	126	AAGCGUUGUGGGCCCGGCA	579
AS579-M1			GACCAGCU
S127-	UGCCUGCUACCCCAGGCCAACUGCA	127	UGCAGUUGGCCUGGGGUAG	580
AS580-M1			CAGGCAGC
S128-	GCCUGCUACCCCAGGCCAAUUGCAG	128	CUGCAAUUGGCCUGGGGUA	581
AS581-M1			GCAGGCAG
S129-	CCCAGGCCAACUGCAGCGUUCACAC	129	GUGUGAACGCUGCAGUUGG	582
AS582-M1			CCUGGGGU
S130-	GGCCCCUCAGGAGCAGGUGACCGTG	130	CACGGUCACCUGCUCCUGA	583
AS583-M1			GGGGCCGG
S131-	UGACCGUGGCCUGCGAGGAUGGCTG	131	CAGCCAUCCUCGCAGGCCA	584
AS584-M1			CGGUCACC
S132-	GCGAGGAGGGCUGGACCCUUACUGG	132	CCAGUAAGGGUCCAGCCCU	585
AS585-M1			CCUCGCAG
S133-	CGAGGAGGGCUGGACCCUGACUGGC	133	GCCAGUCAGGGUCCAGCCC	586
AS586-M1			UCCUCGCA
S134-	GGGCUGGACCCUGACUGGCUGCAGT	134	ACUGCAGCCAGUCAGGGUC	587
AS587-M1			CAGCCCUC
S135-	GGCUGGACCCUGACUGGCUUCAGTG	135	CACUGAAGCCAGUCAGGGU	588
AS588-M1			CCAGCCCU
S136-	UGGACCCUGACUGGCUGCAUUGCCC	136	GGGCAAUGCAGCCAGUCAG	589
AS589-M1			GGUCCAGC
S137-	GGCUGCAGUGCCCUCCCUGUGACCT	137	AGGUCACAGGGAGGGCACU	590
AS590-M1			GCAGCCAG
S138-	UCCCUGGGACCUCCCACGUUCUGGG	138	CCCAGAACGUGGGAGGUCC	591
AS591-M1			CAGGGAGG
S139-	CCCUGGGACCUCCCACGUCUUGGGG	139	CCCCAAGACGUGGGAGGUC	592
AS592-M1			CCAGGGAG
S140-	GGGCCUACGCCGUAGACAAUACGTG	140	CACGUAUUGUCUACGGCGU	593
AS593-M1			AGGCCCCC
S141-	GACGUCAGCACUACAGGCAUCACCA	141	UGGUGAUGCCUGUAGUGCU	594
AS594-M1			GACGUCCC
S142-	CAGCACUACAGGCAGCACCAGCGAA	142	UUCGCUGGUGCUGCCUGUA	595
AS595-M1			GUGCUGAC
S143-	AGCACUACAGGCAGCACCAUCGAAG	143	CUUCGAUGGUGCUGCCUGU	596
AS596-M1			AGUGCUGA
S144-	GCACUACAGGCAGCACCAGUGAAGG	144	CCUUCACUGGUGCUGCCUG	597
AS597-M1			UAGUGCUG
S145-	GGGGCCGUGACAGCCGUUGUCAUCT	145	AGAUGACAACGGCUGUCAC	598
AS598-M1			GGCCCCUU
S146-	GGAGCUCCAGUGACAGCCCUAUCCC	146	GGGAUAGGGCUGUCACUGG	599
AS599-M1			AGCUCCUG
S147-	AGGAUGGGUGUCUGGGGAGUGUCAA	147	UUGACACUCCCCAGACACC	600
AS600-M1			CAUCCUGG
S148-	UGGGUGUCUGGGGAGGGUCAAGGGC	148	GCCCUUGACCCUCCCCAGA	601
AS601-M1			CACCCAUC
S149-	GGGUGUCUGGGGAGGGUCAAGGGCT	149	AGCCCUUGACCCUCCCCAG	602
AS602-M1			CAACCCAU
S150-	GGUGUCUGGGGAGGGUCAAUGGCTG	150	CAGCCAUUGACCCUCCCCA	603
AS603-M1			AGCACCCA
S151-	AGGGUCAAGGGCUGGGGCUUAGCTT	151	AAGCUAAGCCCCAGCCCUU	604
AS604-M1			AGCCCUCC
S152-	GGGUCAAGGGCUGGGGCUGAGCUTT	152	AAAGCUCAGCCCCAGCCCU	605
AS605-M1			UGACCCUC
S153-	GACUUGUCCCUCUCUCAGCUCUCCA	153	UGGAGAGCUGAGAGAGGGA	606
AS606-M1			CAAGUCGG
S154-	ACUUGUCCCUCUCUCAGCCUUCCAT	154	AUGGAAGGCUGAGAGAGGG	607
AS607-M1			ACAAGUCG
S155-	CUUGUCCCUCUCUCAGCCCUCCATG	155	CAUGGAGGGCUGAGAGAGG	608
AS608-M1			GACAAGUC
S156-	UUGUCCCUCUCUCAGCCCUUCAUGG	156	CCAUGAAGGGCUGAGAGAG	609
AS609-M1			GGACAAGU
S157-	UCCCUCUCUCAGCCCUCCAUGGCCT	157	AGGCCAUGGAGGGCUGAGA	610
AS610-M1			GAGGGACA
S158-	UGGCCUGGCACGAGGGGAUUGGGAT	158	AUCCCAAUCCCCUCGUGCC	611
AS611-M1			AGGCCAUG
S159-	UGGCACGAGGGGAUGGGGAUGCUTC	159	GAAGCAUCCCCAUCCCCUC	612
AS612-M1			GUGCCAGG
S160-	CGAGGGGAUGGGGAUGCUUUCGCCT	160	AGGCGAAAGCAUCCCCAUC	613
AS613-M1			CCCUCGUG
S161-	GAGGGGAUGGGGAUGCUUCUGCCTT	161	AAGGCAGAAGCAUCCCCAU	614
AS614-M1			CCCCUCGU
S162-	GGGAUGGGGAUGCUUCCGCUUUUCC	162	GGAAAAGCGGAAGCAUCCC	615
AS615-M1			CAUCCCCU
S163-	AUGGGGAUGCUUCCGCCUUUCCGGG	163	CCCGGAAAGGCGGAAGCAU	616
AS616-M1			CCCCAUCC
S164-	UGGGGAUGCUUCCGCCUUUUCGGGG	164	CCCCGAAAAGGCGGAAGCA	617
AS617-M1			UCCCCAUC
S165-	GGGGAUGCUUCCGCCUUUCUGGGGC	165	GCCCCAGAAAGGCGGAAGC	618
AS618-M1			AUCCCCAU
S166-	GGGAUGCUUCCGCCUUUCCUGGGCT	166	AGCCCAGGAAAGGCGGAAG	619
AS619-M1			CAUCCCCA
S167-	CCCUUGAGUGGGGCAGCCUUCUUGC	167	GCAAGAAGGCUGCCCCACU	620
AS620-M1			CAAGGGCC
S168-	UGAGUGGGGCAGCCUCCUUUCCUGG	168	CCAGGAAAGGAGGCUGCCC	621
AS621-M1			CACUCAAG
S169-	GGGGCAGCCUCCUUGCCUGUAACTC	169	GAGUUACAGGCAAGGAGGC	622
AS622-M1			UGCCCCAC
S170-	GGCAGCCUCCUUGCCUGGAACUCAC	170	GUGAGUUCCAGGCAAGGAG	623
AS623-M1			GCUGCCCC
S171-	GCAGCCUCCUUGCCUGGAAUUCACT	171	AGUGAAUUCCAGGCAAGGA	624
AS624-M1			GGCUGCCC
S172-	AGCCUCCUUGCCUGGAACUUACUCA	172	UGAGUAAGUUCCAGGCAAG	625
AS625-M1			GAGGCUGC
S173-	GCCUCCUUGCCUGGAACUCACUCAC	173	GUGAGUGAGUUCCAGGCAA	626
AS626-M1			GGAGGCUG
S174-	CCUCCUUGCCUGGAACUCAUUCACT	174	AGUGAAUGAGUUCCAGGCA	627
AS627-M1			AGGAGGCU
S175-	CUCCUUGCCUGGAACUCACUCACTC	175	GAGUGAGUGAGUUCCAGGC	628
AS628-M1			AAGGAGGC
S176-	UCCUUGCCUGGAACUCACUUACUCT	176	AGAGUAAGUGAGUUCCAGG	629
AS629-M1			CAAGGAGG
S177-	CCUUGCCUGGAACUCACUCACUCTG	177	CAGAGUGAGUGAGUUCCAG	630
AS630-M1			GCAAGGAG
S178-	CUUGCCUGGAACUCACUCAUUCUGG	178	CCAGAAUGAGUGAGUUCCA	631
AS631-M1			GGCAAGGA
S179-	UUGCCUGGAACUCACUCACUCUGGG	179	CCCAGAGUGAGUGAGUUCC	632
AS632-M1			AGGCAAGG
S180-	UGCCUGGAACUCACUCACUUUGGGT	180	ACCCAAAGUGAGUGAGUUC	633
AS633-M1			CAGGCAAG
S181-	UCUGGGUGCCUCCUCCCCAUGUGGA	181	UCCACAUGGGGAGGAGGCA	634
AS634-M1			CCCAGAGU
S182-	CCCAGGUGGAGGUGCCAGGAAGCTC	182	GAGCUUCCUGGCACCUCCA	635
AS635-M1			CCUGGGGA
S183-	CCAGGAAGCUCCCUCCCUCACUGTG	183	CACAGUGAGGGAGGGAGCU	636
AS636-M1			UCCUGGCA
S184-	GGAAGCUCCCUCCCUCACUUUGGGG	184	CCCCAAAGUGAGGGAGGGA	637
AS637-M1			GCUUCCUG
S185-	AGCUCCCUCCCUCACUGUGUGGCAT	185	AUGCCACACAGUGAGGGAG	638
AS638-M1			GGAGCUUC
S186-	GCUCCCUCCCUCACUGUGGUGCATT	186	AAUGCACCACAGUGAGGGA	639
AS639-M1			GGGAGCUU
S187-	GGGGCAUUUCACCAUUCAAACAGGT	187	ACCUGUUUGAAUGGUGAAA	640
AS640-M1			UGCCCCAC
S188-	GGGCAUUUCACCAUUCAAAUAGGTC	188	GACCUAUUUGAAUGGUGAA	641
AS641-M1			AUGCCCCA
S189-	CACCAUUCAAACAGGUCGAUCUGTG	189	CACAGAUCGACCUGUUUGA	642
AS642-M1			AUGGUGAA
S190-	ACCAUUCAAACAGGUCGAGUUGUGC	190	GCACAACUCGACCUGUUUG	643
AS643-M1			AAUGGUGA
S191-	UGCUCGGGUGCUGCCAGCUUCUCCC	191	GGGAGAAGCUGGCAGCACC	644
AS644-M1			CGAGCACA
S192-	CGGGUGCUGCCAGCUGCUCUCAATG	192	CAUUGAGAGCAGCUGGCAG	645
AS645-M1			CACCCGAG
S193-	GGGUGCUGCCAGCUGCUCCUAAUGT	193	ACAUUAGGAGCAGCUGGCA	646
AS646-M1			GCACCCGA
S194-	GCCAGCUGCUCCCAAUGUGUCGATG	194	CAUCGACACAUUGGGAGCA	647
AS647-M1			GCUGGCAG
S195-	CCAGCUGCUCCCAAUGUGCUGAUGT	195	ACAUCAGCACAUUGGGAGC	648
AS648-M1			AGCUGGCA
S196-	UGCCGAUGUCCGUGGGCAGAAUGAC	196	GUCAUUCUGCCCACGGACA	649
AS649-M1			UCGGCACA
S197-	GCAGAAUGACUUUUAUUGAUCUCTT	197	AAGAGAUCAAUAAAAGUCA	650
AS650-M1			UUCUGCCC
S198-	CAGAAUGACUUUUAUUGAGUUCUTG	198	CAAGAACUCAAUAAAAGUC	651
AS651-M1			AUUCUGCC
S199-	AGAAUGACUUUUAUUGAGCUCUUGT	199	ACAAGAGCUCAAUAAAAGU	652
AS652-M1			CAUUCUGC
S200-	GAAUGACUUUUAUUGAGCUUUUGTT	200	AACAAAAGCUCAAUAAAAG	653
AS653-M1			UCAUUCUG
S201-	AAUGACUUUUAUUGAGCUCUUGUTC	201	GAACAAGAGCUCAAUAAAA	654
AS654-M1			GUCAUUCU
S202-	AUGACUUUUAUUGAGCUCUUGUUCC	202	GGAACAAGAGCUCAAUAAA	655
AS655-M1			AGUCAUUC
S203-	UGACUUUUAUUGAGCUCUUUUUCCG	203	CGGAAAAAGAGCUCAAUAA	656
AS656-M1			AAGUCAUU
S204-	CUUGUUCCGUGCCAGGCAUUCAATC	204	GAUUGAAUGCCUGGCACGG	657
AS657-M1			AACAAGAG
S205-	CCAGGCAUUCAAUCCUCAGUUCUCC	205	GGAGAACUGAGGAUUGAAU	658
AS658-M1			GCCUGGCA
S206-	CAUUCAAUCCUCAGGUCUCUACCAA	206	UUGGUAGAGACCUGAGGAU	659
AS659-M1			UGAAUGCC
S207-	AUUCAAUCCUCAGGUCUCCACCAAG	207	CUUGGUGGAGACCUGAGGA	660
AS660-M1			UUGAAUGC
S208-	UUCAAUCCUCAGGUCUCCAUCAAGG	208	CCUUGAUGGAGACCUGAGG	661
AS661-M1			AUUGAAUG
S209-	CCUCAGGUCUCCACCAAGGAGGCAG	209	CUGCCUCCUUGGUGGAGAC	662
AS662-M1			CUGAGGAU
S210-	CUCAGGUCUCCACCAAGGAUGCAGG	210	CCUGCAUCCUUGGUGGAGA	663
AS663-M1			CCUGAGGA
S211-	GCGGUAGGGGCUGCAGGGAUAAACA	211	UGUUUAUCCCUGCAGCCCC	664
AS664-M1			UACCGCCC
S212-	CGGUAGGGGCUGCAGGGACAAACAT	212	AUGUUUGUCCCUGCAGCCC	665
AS665-M1			CUACCGCC
S213-	GGUAGGGGCUGCAGGGACAAACATC	213	GAUGUUUGUCCCUGCAGCC	666
AS666-M1			CCUACCGC
S214-	UAGGGGCUGCAGGGACAAAUAUCGT	214	ACGAUAUUUGUCCCUGCAG	667
AS667-M1			CCCCUACC
S215-	AGGGGCUGCAGGGACAAACAUCGTT	215	AACGAUGUUUGUCCCUGCA	668
AS668-M1			GCCCCUAC
S216-	GGGGCUGCAGGGACAAACAUCGUTG	216	CAACGAUGUUUGUCCCUGC	669
AS669-M1			AGCCCCUA
S217-	GGGCUGCAGGGACAAACAUUGUUGG	217	CCAACAAUGUUUGUCCCUG	670
AS670-M1			CAGCCCCU
S218-	GGCUGCAGGGACAAACAUCUUUGGG	218	CCCAAAGAUGUUUGUCCCU	671
AS671-M1			GCAGCCCC
S219-	GGGGUGAGUGUGAAAGGUGUUGATG	219	CAUCAACACCUUUCACACU	672
AS672-M1			CACCCCCC
S220-	GGGUGAGUGUGAAAGGUGCUGAUGG	220	CCAUCAGCACCUUUCACAC	673
AS673-M1			UCACCCCC
S221-	GGUGAGUGUGAAAGGUGCUUAUGGC	221	GCCAUAAGCACCUUUCACA	674
AS674-M1			CUCACCCC
S222-	GUGAGUGUGAAAGGUGCUGAUGGCC	222	GGCCAUCAGCACCUUUCAC	675
AS675-M1			ACUCACCC
S223-	UGAGUGUGAAAGGUGCUGAUGGCCC	223	GGGCCAUCAGCACCUUUCA	676
AS676-M1			CACUCACC
S224-	GAGUGUGAAAGGUGCUGAUUGCCCT	224	AGGGCAAUCAGCACCUUUC	677
AS677-M1			ACACUCAC
S225-	AGUGUGAAAGGUGCUGAUGUCCCTC	225	GAGGGACAUCAGCACCUUU	678
AS678-M1			CACACUCA
S226-	GUGUGAAAGGUGCUGAUGGUCCUCA	226	UGAGGACCAUCAGCACCUU	679
AS679-M1			UCACACUC
S227-	UGUGAAAGGUGCUGAUGGCUCUCAT	227	AUGAGAGCCAUCAGCACCU	680
AS680-M1			UUCACACU
S228-	GUGAAAGGUGCUGAUGGCCUUCATC	228	GAUGAAGGCCAUCAGCACC	681
AS681-M1			UUUCACAC
S229-	UGAAAGGUGCUGAUGGCCCUCAUCT	229	AGAUGAGGGCCAUCAGCAC	682
AS682-M1			CUUUCACA
S230-	GAAAGGUGCUGAUGGCCCUUAUCTC	230	GAGAUAAGGGCCAUCAGCA	683
AS683-M1			CCUUUCAC
S231-	CUCAUCUCCAGCUAACUGUUGAGAA	231	UUCUCAACAGUUAGCUGGA	684
AS684-M1			GAUGAGGG
S232-	CCAGCUAACUGUGGAGAAGUCCCTG	232	CAGGGACUUCUCCACAGUU	685
AS685-M1			AGCUGGAG
S233-	CAGCUAACUGUGGAGAAGCUCCUGG	233	CCAGGAGCUUCUCCACAGU	686
AS686-M1			UAGCUGGA
S234-	AGCUAACUGUGGAGAAGCCUCUGGG	234	CCCAGAGGCUUCUCCACAG	687
AS687-M1			UUAGCUGG
S235-	GCUAACUGUGGAGAAGCCCUUGGGG	235	CCCCAAGGGCUUCUCCACA	688
AS688-M1			GUUAGCUG
S236-	GGGCUCCCUGAUUAAUGGAUGCUTA	236	UAAGCAUCCAUUAAUCAGG	689
AS689-M1			GAGCCCCC
S237-	AUGGAGGCUUAGCUUUCUGUAUGGC	237	GCCAUACAGAAAGCUAAGC	690
AS690-M1			CUCCAUUA
S238-	UGGAGGCUUAGCUUUCUGGAUGGCA	238	UGCCAUCCAGAAAGCUAAG	691
AS691-M1			CCUCCAUU
S239-	GGAGGCUUAGCUUUCUGGAUGGCAT	239	AUGCCAUCCAGAAAGCUAA	692
AS692-M1			GCCUCCAU
S240-	GAGGCUUAGCUUUCUGGAUUGCATC	240	GAUGCAAUCCAGAAAGCUA	693
AS693-M1			AGCCUCCA
S241-	AGGCUUAGCUUUCUGGAUGUCAUCT	241	AGAUGACAUCCAGAAAGCU	694
AS694-M1			AAGCCUCC
S242-	GGCUUAGCUUUCUGGAUGGUAUCTA	242	UAGAUACCAUCCAGAAAGC	695
AS695-M1			UAAGCCUC
S243-	GCUUAGCUUUCUGGAUGGCAUCUAG	243	CUAGAUGCCAUCCAGAAAG	696
AS696-M1			CUAAGCCU
S244-	GACAGGUGCGCCCCUGGUGUUCACA	244	UGUGAACACCAGGGGCGCA	697
AS697-M1			CCUGUCUC
S245-	GCGCCCCUGGUGGUCACAGUCUGTG	245	CACAGACUGUGACCACCAG	698
AS698-M1			GGGCGCAC
S246-	CCCCUGGUGGUCACAGGCUUUGCCT	246	AGGCAAAGCCUGUGACCAC	699
AS699-M1			CAGGGGCG
S247-	CCCUGGUGGUCACAGGCUGUGCCTT	247	AAGGCACAGCCUGUGACCA	700
AS700-M1			CCAGGGGC
S248-	GUGGUCACAGGCUGUGCCUUGGUTT	248	AAACCAAGGCACAGCCUGU	701
AS701-M1			GACCACCA
S249-	UGGUCACAGGCUGUGCCUUUGUUTC	249	GAAACAAAGGCACAGCCUG	702
AS702-M1			UGACCACC
S250-	GGUCACAGGCUGUGCCUUGUUUUCC	250	GGAAAACAAGGCACAGCCU	703
AS703-M1			GUGACCAC
S251-	GUCACAGGCUGUGCCUUGGUUUCCT	251	AGGAAACCAAGGCACAGCC	704
AS704-M1			UGUGACCA
S252-	GGCUGUGCCUUGGUUUCCUUAGCCA	252	UGGCUAAGGAAACCAAGGC	705
AS705-M1			ACAGCCUG
S253-	GCUGUGCCUUGGUUUCCUGAGCCAC	253	GUGGCUCAGGAAACCAAGG	706
AS706-M1			CACAGCCU
S254-	CUGUGCCUUGGUUUCCUGAUCCACC	254	GGUGGAUCAGGAAACCAAG	707
AS707-M1			GCACAGCC
S255-	UGUGCCUUGGUUUCCUGAGUCACCT	255	AGGUGACUCAGGAAACCAA	708
AS708-M1			GGCACAGC
S256-	GUGCCUUGGUUUCCUGAGCUACCTT	256	AAGGUAGCUCAGGAAACCA	709
AS709-M1			AGGCACAG
S257-	UGCCUUGGUUUCCUGAGCCACCUTT	257	AAAGGUGGCUCAGGAAACC	710
AS710-M1			AAGGCACA
S258-	GCCUUGGUUUCCUGAGCCAUCUUTA	258	UAAAGAUGGCUCAGGAAAC	711
AS711-M1			CAAGGCAC
S259-	CCUUGGUUUCCUGAGCCACUUUUAC	259	GUAAAAGUGGCUCAGGAAA	712
AS712-M1			CCAAGGCA
S260-	CUUGGUUUCCUGAGCCACCUUUACT	260	AGUAAAGGUGGCUCAGGAA	713
AS713-M1			ACCAAGGC
S261-	UUGGUUUCCUGAGCCACCUUUACTC	261	GAGUAAAGGUGGCUCAGGA	714
AS714-M1			AACCAAGG
S262-	UGGUUUCCUGAGCCACCUUUACUCT	262	AGAGUAAAGGUGGCUCAGG	715
AS715-M1			AAACCAAG
S263-	GGUUUCCUGAGCCACCUUUACUCTG	263	CAGAGUAAAGGUGGCUCAG	716
AS716-M1			GAAACCAA
S264-	GUUUCCUGAGCCACCUUUAUUCUGC	264	GCAGAAUAAAGGUGGCUCA	717
AS717-M1			GGAAACCA
S265-	CUGAGCCACCUUUACUCUGUUCUAT	265	AUAGAACAGAGUAAAGGUG	718
AS718-M1			GCUCAGGA
S266-	CCAGGCUGUGCUAGCAACAUCCAAA	266	UUUGGAUGUUGCUAGCACA	719
AS719-M1			GCCUGGCA
S267-	CUGCGGGGAGCCAUCACCUAGGACT	267	AGUCCUAGGUGAUGGCUCC	720
AS720-M1			CCGCAGGC
S268-	UGCGGGGAGCCAUCACCUAUGACTG	268	CAGUCAUAGGUGAUGGCUC	721
AS721-M1			CCCGCAGG
S269-	GCGGGGAGCCAUCACCUAGUACUGA	269	UCAGUACUAGGUGAUGGCU	722
AS722-M1			CCCCGCAG
S270-	CGGGGAGCCAUCACCUAGGACUGAC	270	GUCAGUCCUAGGUGAUGGC	723
AS723-M1			UCCCCGCA
S271-	GGGGAGCCAUCACCUAGGAUUGACT	271	AGUCAAUCCUAGGUGAUGG	724
AS724-M1			CUCCCCGC
S272-	GCCAUCACCUAGGACUGACUCGGCA	272	UGCCGAGUCAGUCCUAGGU	725
AS725-M1			GAUGGCUC
S273-	CCAUCACCUAGGACUGACUUGGCAG	273	CUGCCAAGUCAGUCCUAGG	726
AS726-M1			UGAUGGCU
S274-	CAUCACCUAGGACUGACUCUGCAGT	274	ACUGCAGAGUCAGUCCUAG	727
AS727-M1			GUGAUGGC
S275-	CUAGGACUGACUCGGCAGUUUGCAG	275	CUGCAAACUGCCGAGUCAG	728
AS728-M1			UCCUAGGU
S276-	UGACUCGGCAGUGUGCAGUUGUGCA	276	UGCACAACUGCACACUGCC	729
AS729-M1			GAGUCAGU
S277-	GACUCGGCAGUGUGCAGUGUUGCAT	277	AUGCAACACUGCACACUGC	730
AS730-M1			CGAGUCAG
S278-	CUCGGCAGUGUGCAGUGGUUCAUGC	278	GCAUGAACCACUGCACACU	731
AS731-M1			GCCGAGUC
S279-	UCGGCAGUGUGCAGUGGUGUAUGCA	279	UGCAUACACCACUGCACAC	732
AS732-M1			UGCCGAGU
S280-	CGGCAGUGUGCAGUGGUGCAUGCAC	280	GUGCAUGCACCACUGCACA	733
AS733-M1			CUGCCGAG
S281-	GUGUGCAGUGGUGCAUGCAUUGUCT	281	AGACAAUGCAUGCACCACU	734
AS734-M1			GCACACUG
S282-	UGUGCAGUGGUGCAUGCACUGUCTC	282	GAGACAGUGCAUGCACCAC	735
AS735-M1			UGCACACU
S283-	GUGCAGUGGUGCAUGCACUUUCUCA	283	UGAGAAAGUGCAUGCACCA	736
AS736-M1			CUGCACAC
S284-	UGCAGUGGUGCAUGCACUGUCUCAG	284	CUGAGACAGUGCAUGCACC	737
AS737-M1			ACUGCACA
S285-	GCAGUGGUGCAUGCACUGUUUCAGC	285	GCUGAAACAGUGCAUGCAC	738
AS738-M1			CACUGCAC
S286-	CAGUGGUGCAUGCACUGUCUCAGCC	286	GGCUGAGACAGUGCAUGCA	739
AS739-M1			CCACUGCA
S287-	AGUGGUGCAUGCACUGUCUUAGCCA	287	UGGCUAAGACAGUGCAUGC	740
AS740-M1			ACCACUGC
S288-	UGCAUGCACUGUCUCAGCCAACCCG	288	CGGGUUGGCUGAGACAGUG	741
AS741-M1			CAUGCACC
S289-	GCAUGCACUGUCUCAGCCAACCCGC	289	GCGGGUUGGCUGAGACAGU	742
AS742-M1			GCAUGCAC
S290-	CAUUCGCACCCCUACUUCAUAGAGG	290	CCUCUAUGAAGUAGGGGUG	743
AS743-M1			CGAAUGUG
S291-	AUUCGCACCCCUACUUCACAGAGGA	291	UCCUCUGUGAAGUAGGGGU	744
AS744-M1			GCGAAUGU
S292-	UUCGCACCCCUACUUCACAUAGGAA	292	UUCCUAUGUGAAGUAGGGG	745
AS745-M1			UGCGAAUG
S293-	UCGCACCCCUACUUCACAGAGGAAG	293	CUUCCUCUGUGAAGUAGGG	746
AS746-M1			GUGCGAAU
S294-	CGCACCCCUACUUCACAGAUGAAGA	294	UCUUCAUCUGUGAAGUAGG	747
AS747-M1			GGUGCGAA
S295-	GCACCCCUACUUCACAGAGUAAGAA	295	UUCUUACUCUGUGAAGUAG	748
AS748-M1			GGGUGCGA
S296-	CACCCCUACUUCACAGAGGAAGAAA	296	UUUCUUCCUCUGUGAAGUA	749
AS749-M1			GGGGUGCG
S297-	ACCCCUACUUCACAGAGGAAGAAAC	297	GUUUCUUCCUCUGUGAAGU	750
AS750-M1			AGGGGUGC
S298-	CCCCUACUUCACAGAGGAAUAAACC	298	GGUUUAUUCCUCUGUGAAG	751
AS751-M1			UAGGGGUG
S299-	CCCUACUUCACAGAGGAAGAAACCT	299	AGGUUUCUUCCUCUGUGAA	752
AS752-M1			GUAGGGGU
S300-	CUUCACAGAGGAAGAAACCUGGAAC	300	GUUCCAGGUUUCUUCCUCU	753
AS753-M1			GUGAAGUA
S301-	UUCACAGAGGAAGAAACCUUGAACC	301	GGUUCAAGGUUUCUUCCUC	754
AS754-M1			UGUGAAGU
S302-	UCACAGAGGAAGAAACCUGUAACCA	302	UGGUUACAGGUUUCUUCCU	755
AS755-M1			CUGUGAAG
S303-	CACAGAGGAAGAAACCUGGAACCAG	303	CUGGUUCCAGGUUUCUUCC	756
AS756-M1			UCUGUGAA
S304-	ACAGAGGAAGAAACCUGGAACCAGA	304	UCUGGUUCCAGGUUUCUUC	757
AS757-M1			CUCUGUGA
S305-	CAGAGGAAGAAACCUGGAAUCAGAG	305	CUCUGAUUCCAGGUUUCUU	758
AS758-M1			CCUCUGUG
S306-	AGAGGAAGAAACCUGGAACUAGAGG	306	CCUCUAGUUCCAGGUUUCU	759
AS759-M1			UCCUCUGU
S307-	GAGGAAGAAACCUGGAACCAGAGGG	307	CCCUCUGGUUCCAGGUUUC	760
AS760-M1			UUCCUCUG
S308-	AGGAAGAAACCUGGAACCAUAGGGG	308	CCCCUAUGGUUCCAGGUUU	761
AS761-M1			CUUCCUCU
S309-	GCAGAUUGGGCUGGCUCUGAAGCCA	309	UGGCUUCAGAGCCAGCCCA	762
AS762-M1			AUCUGCGU
S310-	CAGAUUGGGCUGGCUCUGAAGCCAA	310	UUGGCUUCAGAGCCAGCCC	763
AS763-M1			AAUCUGCG
S311-	AGAUUGGGCUGGCUCUGAAUCCAAG	311	CUUGGAUUCAGAGCCAGCC	764
AS764-M1			CAAUCUGC
S312-	UGGGCUGGCUCUGAAGCCAAGCCTC	312	GAGGCUUGGCUUCAGAGCC	765
AS765-M1			AGCCCAAU
S313-	GGGCUGGCUCUGAAGCCAAUCCUCT	313	AGAGGAUUGGCUUCAGAGC	766
AS766-M1			CAGCCCAA
S314-	GAAGCCAAGCCUCUUCUUAUUUCAC	314	GUGAAAUAAGAAGAGGCUU	767
AS767-M1			GGCUUCAG
S315-	AAGCCUCUUCUUACUUCACUCGGCT	315	AGCCGAGUGAAGUAAGAAG	768
AS768-M1			AGGCUUGG
S316-	AGCCUCUUCUUACUUCACCUGGCTG	316	CAGCCAGGUGAAGUAAGAA	769
AS769-M1			GAGGCUUG
S317-	GCCUCUUCUUACUUCACCCUGCUGG	317	CCAGCAGGGUGAAGUAAGA	770
AS770-M1			AGAGGCUU
S318-	CCCGGCUGGGCUCCUCAUUUUUACG	318	CGUAAAAAUGAGGAGCCCA	771
AS771-M1			GCCGGGUG
S319-	CCGGCUGGGCUCCUCAUUUUUACGG	319	CCGUAAAAAUGAGGAGCCC	772
AS772-M1			AGCCGGGU
S320-	CGGCUGGGCUCCUCAUUUUUACGGG	320	CCCGUAAAAAUGAGGAGCC	773
AS773-M1			CAGCCGGG
S321-	GGCUGGGCUCCUCAUUUUUACGGGT	321	ACCCGUAAAAAUGAGGAGC	774
AS774-M1			CCAGCCGG
S322-	GCUGGGCUCCUCAUUUUUAUGGGTA	322	UACCCAUAAAAAUGAGGAG	775
AS775-M1			CCCAGCCG
S323-	ACGGGUAACAGUGAGGCUGUGAAGG	323	CCUUCACAGCCUCACUGUU	776
AS776-M1			ACCCGUAA
S324-	AGCUCGGUGAGUGAUGGCAUAACGA	324	UCGUUAUGCCAUCACUCAC	777
AS777-M1			CGAGCUUC
S325-	GCUCGGUGAGUGAUGGCAGAACGAT	325	AUCGUUCUGCCAUCACUCA	778
AS778-M1			CCGAGCUU
S326-	CUCGGUGAGUGAUGGCAGAACGATG	326	CAUCGUUCUGCCAUCACUC	779
AS779-M1			ACCGAGCU
S327-	UCGGUGAGUGAUGGCAGAAUGAUGC	327	GCAUCAUUCUGCCAUCACU	780
AS780-M1			CACCGAGC
S328-	CGGUGAGUGAUGGCAGAACUAUGCC	328	GGCAUAGUUCUGCCAUCAC	781
AS781-M1			UCACCGAG
S329-	AUGCCUGCAGGCAUGGAACUUUUTC	329	GAAAAAGUUCCAUGCCUGC	782
AS782-M1			AGGCAUCG
S330-	UGCCUGCAGGCAUGGAACUUUUUCC	330	GGAAAAAGUUCCAUGCCUG	783
AS783-M1			CAGGCAUC
S331-	GCCUGCAGGCAUGGAACUUUUUCCG	331	CGGAAAAAGUUCCAUGCCU	784
AS784-M1			GCAGGCAU
S332-	CCUGCAGGCAUGGAACUUUUUCCGT	332	ACGGAAAAAGUUCCAUGCC	785
AS785-M1			UGCAGGCA
S333-	CUGCAGGCAUGGAACUUUUUCCGTT	333	AACGGAAAAAGUUCCAUGC	786
AS786-M1			CUGCAGGC
S334-	AUGGAACUUUUUCCGUUAUUACCCA	334	UGGGUAAUAACGGAAAAAG	787
AS787-M1			UUCCAUGC
S335-	UUUUUCCGUUAUCACCCAGUCCUGA	335	UCAGGACUGGGUGAUAACG	788
AS788-M1			GAAAAAGU
S336-	UUUUCCGUUAUCACCCAGGUCUGAT	336	AUCAGACCUGGGUGAUAAC	789
AS789-M1			GGAAAAAG
S337-	UUUCCGUUAUCACCCAGGCUUGATT	337	AAUCAAGCCUGGGUGAUAA	790
AS790-M1			CGGAAAAA
S338-	UUCCGUUAUCACCCAGGCCUGAUTC	338	GAAUCAGGCCUGGGUGAUA	791
AS791-M1			ACGGAAAA
S339-	UCCGUUAUCACCCAGGCCUUAUUCA	339	UGAAUAAGGCCUGGGUGAU	792
AS792-M1			AACGGAAA
S340-	CCGUUAUCACCCAGGCCUGAUUCAC	340	GUGAAUCAGGCCUGGGUGA	793
AS793-M1			UAACGGAA
S341-	CGUUAUCACCCAGGCCUGAUUCACT	341	AGUGAAUCAGGCCUGGGUG	794
AS794-M1			AUAACGGA
S342-	CACCCAGGCCUGAUUCACUUGCCTG	342	CAGGCAAGUGAAUCAGGCC	795
AS795-M1			UGGGUGAU
S343-	ACCCAGGCCUGAUUCACUGUCCUGG	343	CCAGGACAGUGAAUCAGGC	796
AS796-M1			CUGGGUGA
S344-	UGGCCUGGCGGAGAUGCUUUUAAGG	344	CCUUAAAAGCAUCUCCGCC	797
AS797-M1			AGGCCAGU
S345-	GGCCUGGCGGAGAUGCUUCUAAGGC	345	GCCUUAGAAGCAUCUCCGC	798
AS798-M1			CAGGCCAG
S346-	GCCUGGCGGAGAUGCUUCUAAGGCA	346	UGCCUUAGAAGCAUCUCCG	799
AS799-M1			CCAGGCCA
S347-	CCUGGCGGAGAUGCUUCUAAGGCAT	347	AUGCCUUAGAAGCAUCUCC	800
AS800-M1			GCCAGGCC
S348-	CUGGCGGAGAUGCUUCUAAUGCATG	348	CAUGCAUUAGAAGCAUCUC	801
AS801-M1			CGCCAGGC
S349-	UGGCGGAGAUGCUUCUAAGUCAUGG	349	CCAUGACUUAGAAGCAUCU	802
AS802-M1			CCGCCAGG
S350-	GGCGGAGAUGCUUCUAAGGUAUGGT	350	ACCAUACCUUAGAAGCAUC	803
AS803-M1			UCCGCCAG
S351-	GCGGAGAUGCUUCUAAGGCAUGGTC	351	GACCAUGCCUUAGAAGCAU	804
AS804-M1			CUCCGCCA
S352-	CGGAGAUGCUUCUAAGGCAUGGUCG	352	CGACCAUGCCUUAGAAGCA	805
AS805-M1			UCUCCGCC
S353-	GGAGAUGCUUCUAAGGCAUUGUCGG	353	CCGACAAUGCCUUAGAAGC	806
AS806-M1			AUCUCCGC
S354-	GAGAUGCUUCUAAGGCAUGUUCGGG	354	CCCGAACAUGCCUUAGAAG	807
AS807-M1			CAUCUCCG
S355-	GGAGAGGGCCAACAACUGUUCCUCC	355	GGAGGAACAGUUGUUGGCC	808
AS808-M1			CUCUCCCC
S356-	GCCAACAACUGUCCCUCCUUGAGCA	356	UGCUCAAGGAGGGACAGUU	809
AS809-M1			GUUGGCCC
S357-	CCAACAACUGUCCCUCCUUUAGCAC	357	GUGCUAAAGGAGGGACAGU	810
AS810-M1			UGUUGGCC
S358-	UUGAGCACCAGCCCCACCCAAGCAA	358	UUGCUUGGGUGGGGCUGGU	811
AS811-M1			GCUCAAGG
S359-	UGAGCACCAGCCCCACCCAAGCAAG	359	CUUGCUUGGGUGGGGCUGG	812
AS812-M1			UGCUCAAG
S360-	GAGCACCAGCCCCACCCAAUCAAGC	360	GCUUGAUUGGGUGGGGCUG	813
AS813-M1			GUGCUCAA
S361-	AGCACCAGCCCCACCCAAGUAAGCA	361	UGCUUACUUGGGUGGGGCU	814
AS814-M1			GGUGCUCA
S362-	ACCCAAGCAAGCAGACAUUUAUCTT	362	AAGAUAAAUGUCUGCUUGC	815
AS815-M1			UUGGGUGG
S363-	CCCAAGCAAGCAGACAUUUAUCUTT	363	AAAGAUAAAUGUCUGCUUG	816
AS816-M1			CUUGGGUG
S364-	CCAAGCAAGCAGACAUUUAUCUUTT	364	AAAAGAUAAAUGUCUGCUU	817
AS817-M1			GCUUGGGU
S365-	CAAGCAAGCAGACAUUUAUUUUUTG	365	CAAAAAAUAAAUGUCUGCU	818
AS818-M1			UGCUUGGG
S366-	AAGCAAGCAGACAUUUAUCUUUUGG	366	CCAAAAGAUAAAUGUCUGC	819
AS819-M1			UUGCUUGG
S367-	AGCAAGCAGACAUUUAUCUUUUGGG	367	CCCAAAAGAUAAAUGUCUG	820
AS820-M1			CUUGCUUG
S368-	GCAAGCAGACAUUUAUCUUUUGGGT	368	ACCCAAAAGAUAAAUGUCU	821
AS821-M1			GCUUGCUU
S369-	AAGCAGACAUUUAUCUUUUUGGUCT	369	AGACCAAAAAGAUAAAUGU	822
AS822-M1			CUGCUUGC
S370-	AGCAGACAUUUAUCUUUUGUGUCTG	370	CAGACACAAAAGAUAAAUG	823
AS823-M1			UCUGCUUG
S371-	GCAGACAUUUAUCUUUUGGUUCUGT	371	ACAGAACCAAAAGAUAAAU	824
AS824-M1			GUCUGCUU
S372-	UGUUGCCUUUUUACAGCCAACUUTT	372	AAAAGUUGGCUGUAAAAAG	825
AS825-M1			GCAACAGA
S373-	GUUGCCUUUUUACAGCCAAUUUUTC	373	GAAAAAUUGGCUGUAAAAA	826
AS826-M1			GGCAACAG
S374-	UUUACAGCCAACUUUUCUAUACCTG	374	CAGGUAUAGAAAAGUUGGC	827
AS827-M1			UGUAAAAA
S375-	UUACAGCCAACUUUUCUAGACCUGT	375	ACAGGUCUAGAAAAGUUGG	828
AS828-M1			CUGUAAAA
S376-	UUUUCUAGACCUGUUUUGCUUUUGT	376	ACAAAAGCAAAACAGGUCU	829
AS829-M1			AGAAAAGU
S377-	UUUCUAGACCUGUUUUGCUUUUGTA	377	UACAAAAGCAAAACAGGUC	830
AS830-M1			UAGAAAAG
S378-	UUCUAGACCUGUUUUGCUUUUGUAA	378	UUACAAAAGCAAAACAGGU	831
AS831-M1			CUAGAAAA
S379-	UCUAGACCUGUUUUGCUUUUGUAAC	379	GUUACAAAAGCAAAACAGG	832
AS832-M1			UCUAGAAA
S380-	CUAGACCUGUUUUGCUUUUUUAACT	380	AGUUAAAAAAGCAAAACAG	833
AS833-M1			GUCUAGAA
S381-	UAGACCUGUUUUGCUUUUGUAACTT	381	AAGUUACAAAAGCAAAACA	834
AS834-M1			GGUCUAGA
S382-	AGACCUGUUUUGCUUUUGUAACUTG	382	CAAGUUACAAAAGCAAAAC	835
AS835-M1			AGGUCUAG
S383-	GACCUGUUUUGCUUUUGUAACUUGA	383	UCAAGUUACAAAAGCAAAA	836
AS836-M1			CAGGUCUA
S384-	ACCUGUUUUGCUUUUGUAAUUUGAA	384	UUCAAAUUACAAAAGCAAA	837
AS837-M1			ACAGGUCU
S385-	CCUGUUUUGCUUUUGUAACUUGAAG	385	CUUCAAGUUACAAAAGCAA	838
AS838-M1			AACAGGUC
5386-	CUGUUUUGCUUUUGUAACUUGAAGA	386	UCUUCAAGUUACAAAAGCA	839
AS839-M1			AAACAGGU
S387-	UGUUUUGCUUUUGUAACUUUAAGAT	387	AUCUUAAAGUUACAAAAGC	840
AS840-M1			AAAACAGG
S388-	GUUUUGCUUUUGUAACUUGAAGATA	388	UAUCUUCAAGUUACAAAAG	841
AS841-M1			CAAAACAG
S389-	UUUUGCUUUUGUAACUUGAAGAUAT	389	AUAUCUUCAAGUUACAAAA	842
AS842-M1			GCAAAACA
S390-	UUUGCUUUUGUAACUUGAAUAUATT	390	AAUAUAUUCAAGUUACAAA	843
AS843-M1			AGCAAAAC
S391-	UUGCUUUUGUAACUUGAAGAUAUTT	391	AAAUAUCUUCAAGUUACAA	844
AS844-M1			AAGCAAAA
S392-	UGCUUUUGUAACUUGAAGAUAUUTA	392	UAAAUAUCUUCAAGUUACA	845
AS845-M1			AAAGCAAA
S393-	GCUUUUGUAACUUGAAGAUAUUUAT	393	AUAAAUAUCUUCAAGUUAC	846
AS846-M1			AAAAGCAA
S394-	CUUUUGUAACUUGAAGAUAUUUATT	394	AAUAAAUAUCUUCAAGUUA	847
AS847-M1			CAAAAGCA
S395-	UUUUGUAACUUGAAGAUAUUUAUTC	395	GAAUAAAUAUCUUCAAGUU	848
AS848-M1			ACAAAAGC
S396-	UUUGUAACUUGAAGAUAUUUAUUCT	396	AGAAUAAAUAUCUUCAAGU	849
AS849-M1			UACAAAAG
S397-	UUGUAACUUGAAGAUAUUUAUUCTG	397	CAGAAUAAAUAUCUUCAAG	850
AS850-M1			UUACAAAA
S398-	UGUAACUUGAAGAUAUUUAUUCUGG	398	CCAGAAUAAAUAUCUUCAA	851
AS851-M1			GUUACAAA
S399-	GUAACUUGAAGAUAUUUAUUCUGGG	399	CCCAGAAUAAAUAUCUUCA	852
AS852-M1			AGUUACAA
S400-	UAACUUGAAGAUAUUUAUUUUGGGT	400	ACCCAAAAUAAAUAUCUUC	853
AS853-M1			AAGUUACA
S401-	ACUUGAAGAUAUUUAUUCUUGGUTT	401	AAACCAAGAAUAAAUAUCU	854
AS854-M1			UCAAGUUA
S402-	CUUGAAGAUAUUUAUUCUGUGUUTT	402	AAAACACAGAAUAAAUAUC	855
AS855-M1			UUCAAGUU
S403-	UUGAAGAUAUUUAUUCUGGUUUUTG	403	CAAAAACCAGAAUAAAUAU	856
AS856-M1			CUUCAAGU
S404-	GAAGAUAUUUAUUCUGGGUUUUGTA	404	UACAAAACCCAGAAUAAAU	857
AS857-M1			AUCUUCAA
S405-	AAGAUAUUUAUUCUGGGUUUUGUAG	405	CUACAAAACCCAGAAUAAA	858
AS858-M1			UAUCUUCA
S406-	AUAUUUAUUCUGGGUUUUGUAGCAT	406	AUGCUACAAAACCCAGAAU	859
AS859-M1			AAAUAUCU
S407-	UAUUUAUUCUGGGUUUUGUAGCATT	407	AAUGCUACAAAACCCAGAA	860
AS860-M1			UAAAUAUC
S408-	AUUUAUUCUGGGUUUUGUAUCAUTT	408	AAAUGAUACAAAACCCAGA	861
AS861-M1			AUAAAUAU
S409-	UUUAUUCUGGGUUUUGUAGUAUUTT	409	AAAAUACUACAAAACCCAG	862
AS862-M1			AAUAAAUA
S410-	AUUCUGGGUUUUGUAGCAUUUUUAT	410	AUAAAAAUGCUACAAAACC	863
AS863-M1			CAGAAUAA
S411-	UUCUGGGUUUUGUAGCAUUUUUATT	411	AAUAAAAAUGCUACAAAAC	864
AS864-M1			CCAGAAUA
S412-	UCUGGGUUUUGUAGCAUUUUUAUTA	412	UAAUAAAAAUGCUACAAAA	865
AS865-M1			CCCAGAAU
S413-	CUGGGUUUUGUAGCAUUUUUAUUAA	413	UUAAUAAAAAUGCUACAAA	866
AS866-M1			ACCCAGAA
S414-	UGGGUUUUGUAGCAUUUUUAUUAAT	414	AUUAAUAAAAAUGCUACAA	867
AS867-M1			AACCCAGA
S415-	GGGUUUUGUAGCAUUUUUAUUAATA	415	UAUUAAUAAAAAUGCUACA	868
AS868-M1			AAACCCAG
S416-	GGUUUUGUAGCAUUUUUAUUAAUAT	416	AUAUUAAUAAAAAUGCUAC	869
AS869-M1			AAAACCCA
S417-	GUUUUGUAGCAUUUUUAUUAAUATG	417	CAUAUUAAUAAAAAUGCUA	870
AS870-M1			CAAAACCC
S418-	UUUUGUAGCAUUUUUAUUAAUAUGG	418	CCAUAUUAAUAAAAAUGCU	871
AS871-M1			ACAAAACC
S419-	UUUGUAGCAUUUUUAUUAAUAUGGT	419	ACCAUAUUAAUAAAAAUGC	872
AS872-M1			UACAAAAC
S420-	UUGUAGCAUUUUUAUUAAUAUGGTG	420	CACCAUAUUAAUAAAAAUG	873
AS873-M1			CUACAAAA
S421-	UGUAGCAUUUUUAUUAAUAUGGUGA	421	UCACCAUAUUAAUAAAAAU	874
AS874-M1			GCUACAAA
S422-	GUAGCAUUUUUAUUAAUAUUGUGAC	422	GUCACAAUAUUAAUAAAAA	875
AS875-M1			UGCUACAA
S423-	UAGCAUUUUUAUUAAUAUGUUGACT	423	AGUCAACAUAUUAAUAAAA	876
AS876-M1			AUGCUACA
S424-	AGCAUUUUUAUUAAUAUGGUGACTT	424	AAGUCACCAUAUUAAUAAA	877
AS877-M1			AAUGCUAC
S425-	GCAUUUUUAUUAAUAUGGUUACUTT	425	AAAGUAACCAUAUUAAUAA	878
AS878-M1			AAAUGCUA
S426-	CAUUUUUAUUAAUAUGGUGACUUTT	426	AAAAGUCACCAUAUUAAUA	879
AS879-M1			AAAAUGCU
S427-	AUUUUUAUUAAUAUGGUGAUUUUTT	427	AAAAAAUCACCAUAUUAAU	880
AS880-M1			AAAAAUGC
S428-	UUUUUAUUAAUAUGGUGACUUUUTA	428	UAAAAAGUCACCAUAUUAA	881
AS881-M1			UAAAAAUG
S429-	UUUUAUUAAUAUGGUGACUUUUUAA	429	UUAAAAAGUCACCAUAUUA	882
AS882-M1			AUAAAAAU
S430-	UUUAUUAAUAUGGUGACUUUUUAAA	430	UUUAAAAAGUCACCAUAUU	883
AS883-M1			AAUAAAAA
S431-	UUAUUAAUAUGGUGACUUUUUAAAA	431	UUUUAAAAAGUCACCAUAU	884
AS884-M1			UAAUAAAA
S432-	UAUUAAUAUGGUGACUUUUUAAAAT	432	AUUUUAAAAAGUCACCAUA	885
AS885-M1			UUAAUAAA
S433-	AUUAAUAUGGUGACUUUUUAAAATA	433	UAUUUUAAAAAGUCACCAU	886
AS886-M1			AUUAAUAA
S434-	UUAAUAUGGUGACUUUUUAAAAUAA	434	UUAUUUUAAAAAGUCACCA	887
AS887-M1			UAUUAAUA
S435-	UAAUAUGGUGACUUUUUAAAAUAAA	435	UUUAUUUUAAAAAGUCACC	888
AS888-M1			AUAUUAAU
S436-	AAUAUGGUGACUUUUUAAAAUAAAA	436	UUUUAUUUUAAAAAGUCAC	889
AS889-M1			CAUAUUAA
S437-	AUAUGGUGACUUUUUAAAAUAAAAA	437	UUUUUAUUUUAAAAAGUCA	890
AS890-M1			CCAUAUUA
S438-	UAUGGUGACUUUUUAAAAUAAAAAC	438	GUUUUUAUUUUAAAAAGUC	891
AS891-M1			ACCAUAUU
S439-	AUGGUGACUUUUUAAAAUAAAAACA	439	UGUUUUUAUUUUAAAAAGU	892
AS892-M1			CACCAUAU
S440-	UGGUGACUUUUUAAAAUAAAAACAA	440	UUGUUUUUAUUUUAAAAAG	893
AS893-M1			UCACCAUA
S441-	GGUGACUUUUUAAAAUAAAAACAAA	441	UUUGUUUUUAUUUUAAAAA	894
AS894-M1			GUCACCAU
S442-	GUGACUUUUUAAAAUAAAAACAAAC	442	GUUUGUUUUUAUUUUAAAA	895
AS895-M1			AGUCACCA
S443-	UGACUUUUUAAAAUAAAAAUAAACA	443	UGUUUAUUUUUAUUUUAAA	896
AS896-M1			AAGUCACC
S444-	GACUUUUUAAAAUAAAAACAAACAA	444	UUGUUUGUUUUUAUUUUAA	897
AS897-M1			AAAGUCAC
S445-	ACUUUUUAAAAUAAAAACAAACAAA	445	UUUGUUUGUUUUUAUUUUA	898
AS898-M1			AAAAGUCA
S446-	UUUUAAAAUAAAAACAAACAAACGT	446	ACGUUUGUUUGUUUUUAUU	899
AS899-M1			UUAAAAAG
S447-	UUUAAAAUAAAAACAAACAAACGTT	447	AACGUUUGUUUGUUUUUAU	900
AS900-M1			UUUAAAAA
S448-	UUAAAAUAAAAACAAACAAACGUTG	448	CAACGUUUGUUUGUUUUUA	901
AS901-M1			UUUUAAAA
S449-	UAAAAUAAAAACAAACAAAUGUUGT	449	ACAACAUUUGUUUGUUUUU	902
AS902-M1			AUUUUAAA
S450-	AAAAACAAACAAACGUUGUUCUAAC	450	GUUAGAACAACGUUUGUUU	903
AS903-M1			GUUUUUAU
S451-	CAAACAAACGUUGUCCUAAUAAAAA	451	UUUUUAUUAGGACAACGUU	904
AS904-M1			UGUUUGUU
S452-	AAACAAACGUUGUCCUAACAAAAAA	452	UUUUUUGUUAGGACAACGU	905
AS905-M1			UUGUUUGU
S453-	AACAAACGUUGUCCUAACAAAAAAA	453	UUUUUUUGUUAGGACAACG	906
AS906-M1			UUUGUUUG
S907-	CUCCAGGCGGUCCUGGUGGUCGCTG	907	CAGCGACCACCAGGACCGC	1030
AS1030-M1			CUGGAGCU
S908-	UCCAGGCGGUCCUGGUGGCUGCUGC	908	GCAGCAGCCACCAGGACCG	1031
AS1031-M1			CCUGGAGC
S909-	GCCGCUGCCACUGCUGCUGUUGCTG	909	CAGCAACAGCAGCAGUGGC	1032
AS1032-M1			AGCGGCCA
S910-	CCGCUGCCACUGCUGCUGCUGCUGC	910	GCAGCAGCAGCAGCAGUGG	1033
AS1033-M1			CAGCGGCC
S911-	GCCCGUGCGCAGGAGGACGAGGACG	911	CGUCCUCGUCCUCCUGCGC	1034
AS1034-M1			ACGGGCGC
S912-	CCCGUGCGCAGGAGGACGAUGACGG	912	CCGUCAUCGUCCUCCUGCG	1035
AS1035-M1			CACGGGCG
S913-	CCGUGCGCAGGAGGACGAGUACGGC	913	GCCGUACUCGUCCUCCUGC	1036
AS1036-M1			GCACGGGC
S914-	CGUGCGCAGGAGGACGAGGACGGCG	914	CGCCGUCCUCGUCCUCCUG	1037
AS1037-M1			CGCACGGG
S915-	GUGCGCAGGAGGACGAGGAUGGCGA	915	UCGCCAUCCUCGUCCUCCU	1038
AS1038-M1			GCGCACGG
S916-	UGCGCAGGAGGACGAGGACUGCGAC	916	GUCGCAGUCCUCGUCCUCC	1039
AS1039-M1			UGCGCACG
S917-	GCGCAGGAGGACGAGGACGUCGACT	917	AGUCGACGUCCUCGUCCUC	1040
AS1040-M1			CUGCGCAC
S918-	GGAGGACGAGGACGGCGACUACGAG	918	CUCGUAGUCGCCGUCCUCG	1041
AS1041-M1			UCCUCCUG
S919-	GCGUUCCGAGGAGGACGGCUUGGCC	919	GGCCAAGCCGUCCUCCUCG	1042
AS1042-M1			GAACGCAA
S920-	CGUUCCGAGGAGGACGGCCUGGCCG	920	CGGCCAGGCCGUCCUCCUC	1043
AS1043-M1			GGAACGCA
S921-	GUUCCGAGGAGGACGGCCUUGCCGA	921	UCGGCAAGGCCGUCCUCCU	1044
AS1044-M1			CGGAACGC
S922-	UUCCGAGGAGGACGGCCUGUCCGAA	922	UUCGGACAGGCCGUCCUCC	1045
AS1045-M1			UCGGAACG
S923-	UCCGAGGAGGACGGCCUGGUCGAAG	923	CUUCGACCAGGCCGUCCUC	1046
AS1046-M1			CUCGGAAC
S924-	CCGAGGAGGACGGCCUGGCUGAAGC	924	GCUUCAGCCAGGCCGUCCU	1047
AS1047-M1			CCUCGGAA
S925-	CGAGGAGGACGGCCUGGCCUAAGCA	925	UGCUUAGGCCAGGCCGUCC	1048
AS1048-M1			UCCUCGGA
S926-	GAGGAGGACGGCCUGGCCGAAGCAC	926	GUGCUUCGGCCAGGCCGUC	1049
AS1049-M1			CUCCUCGG
S927-	GCCACCUUCCACCGCUGCGUCAAGG	927	CCUUGACGCAGCGGUGGAA	1050
AS1050-M1			GGUGGCUG
S928-	CCACCUUCCACCGCUGCGCUAAGGA	928	UCCUUAGCGCAGCGGUGGA	1051
AS1051-M1			AGGUGGCU
S929-	CACCUUCCACCGCUGCGCCAAGGAT	929	AUCCUUGGCGCAGCGGUGG	1052
AS1052-M1			AAGGUGGC
S930-	ACCUUCCACCGCUGCGCCAAGGATC	930	GAUCCUUGGCGCAGCGGUG	1053
AS1053-M1			GAAGGUGG
S931-	AGCGCACUGCCCGCCGCCUUCAGGC	931	GCCUGAAGGCGGCGGGCAG	1054
AS1054-M1			UGCGCUCU
S932-	GCGCACUGCCCGCCGCCUGUAGGCC	932	GGCCUACAGGCGGCGGGCA	1055
AS1055-M1			GUGCGCUC
S933-	CGCACUGCCCGCCGCCUGCAGGCCC	933	GGGCCUGCAGGCGGCGGGC	1056
AS1056-M1			AGUGCGCU
S934-	GCACUGCCCGCCGCCUGCAUGCCCA	934	UGGGCAUGCAGGCGGCGGG	1057
AS1057-M1			CAGUGCGC
S935-	CACUGCCCGCCGCCUGCAGUCCCAG	935	CUGGGACUGCAGGCGGCGG	1058
AS1058-M1			GCAGUGCG
S936-	ACUGCCCGCCGCCUGCAGGUCCAGG	936	CCUGGACCUGCAGGCGGCG	1059
AS1059-M1			GGCAGUGC
S937-	CUGCCCGCCGCCUGCAGGCUCAGGC	937	GCCUGAGCCUGCAGGCGGC	1060
AS1060-M1			GGGCAGUG
S938-	UGCCCGCCGCCUGCAGGCCUAGGCT	938	AGCCUAGGCCUGCAGGCGG	1061
AS1061-M1			CGGGCAGU
S939-	GCCCGCCGCCUGCAGGCCCAGGCTG	939	CAGCCUGGGCCUGCAGGCG	1062
AS1062-M1			GCGGGCAG
S940-	CCCGCCGCCUGCAGGCCCAUGCUGC	940	GCAGCAUGGGCCUGCAGGC	1063
AS1063-M1			GGCGGGCA
S941-	UGGCGACCUGCUGGAGCUGUCCUTG	941	CAAGGACAGCUCCAGCAGG	1064
AS1064-M1			UCGCCACU
S942-	GGCGACCUGCUGGAGCUGGUCUUGA	942	UCAAGACCAGCUCCAGCAG	1065
AS1065-M1			GUCGCCAC
S943-	GCGACCUGCUGGAGCUGGCUUUGAA	943	UUCAAAGCCAGCUCCAGCA	1066
AS1066-M1			GGUCGCCA
S944-	CGACCUGCUGGAGCUGGCCUUGAAG	944	CUUCAAGGCCAGCUCCAGC	1067
AS1067-M1			AGGUCGCC
S945-	GAGGCAGCCUGGUGGAGGUUUAUC	945	AGAUAAACCUCCACCAGGC	1068
AS1068-M1			UGCCUCCG
S946-	AGGCAGCCUGGUGGAGGUGUAUCTC	946	GAGAUACACCUCCACCAGG	1069
AS1069-M1			CUGCCUCC
S947-	UGUGCCCGAGGAGGACGGGACCCGC	947	GCGGGUCCCGUCCUCCUCG	1070
AS1070-M1			GGCACAUU
S948-	GUGCCCGAGGAGGACGGGAUCCGCT	948	AGCGGAUCCCGUCCUCCUC	1071
AS1071-M1			GGGCACAU
S949-	UGCCCGAGGAGGACGGGACUCGCTT	949	AAGCGAGUCCCGUCCUCCU	1072
AS1072-M1			CGGGCACA
S950-	GCCCGAGGAGGACGGGACCUGCUTC	950	GAAGCAGGUCCCGUCCUCC	1073
AS1073-M1			UCGGGCAC
S951-	CCCGAGGAGGACGGGACCCUCUUCC	951	GGAAGAGGGUCCCGUCCUC	1074
AS1074-M1			CUCGGGCA
S952-	CCGAGGAGGACGGGACCCGUUUCCA	952	UGGAAACGGGUCCCGUCCU	1075
AS1075-M1			CCUCGGGC
S953-	CGAGGAGGACGGGACCCGCUUCCAC	953	GUGGAAGCGGGUCCCGUCC	1076
AS1076-M1			UCCUCGGG
S954-	GGCAGGGGUGGUCAGCGGCUGGGAT	954	AUCCCAGCCGCUGACCACC	1077
AS1077-M1			CCUGCCAG
S955-	GCAGGGGUGGUCAGCGGCCUGGATG	955	CAUCCAGGCCGCUGACCAC	1078
AS1078-M1			CCCUGCCA
S956-	CAGGGGUGGUCAGCGGCCGUGAUGC	956	GCAUCACGGCCGCUGACCA	1079
AS1079-M1			CCCCUGCC
S957-	GUGCUGCUGCCCCUGGCGGUUGGGT	957	ACCCAACCGCCAGGGGCAG	1080
AS1080-M1			CAGCACCA
S958-	UGCUGCUGCCCCUGGCGGGUGGGTA	958	UACCCACCCGCCAGGGGCA	1081
AS1081-M1			GCAGCACC
S959-	GCUGCUGCCCCUGGCGGGUUGGUAC	959	GUACCAACCCGCCAGGGGC	1082
AS1082-M1			AGCAGCAC
S960-	CUGCUGCCCCUGGCGGGUGUGUACA	960	UGUACACACCCGCCAGGGG	1083
AS1083-M1			CAGCAGCA
S961-	UGCUGCCCCUGGCGGGUGGUUACAG	961	CUGUAACCACCCGCCAGGG	1084
AS1084-M1			GCAGCAGC
S962-	GCUGCCCCUGGCGGGUGGGUACAGC	962	GCUGUACCCACCCGCCAGG	1085
AS1085-M1			GGCAGCAG
S963-	CUGCCCCUGGCGGGUGGGUACAGCC	963	GGCUGUACCCACCCGCCAG	1086
AS1086-M1			GGGCAGCA
S964-	UGCCCCUGGCGGGUGGGUAUAGCCG	964	CGGCUAUACCCACCCGCCA	1087
AS1087-M1			GGGGCAGC
S965-	GCCCCUGGCGGGUGGGUACAGCCGC	965	GCGGCUGUACCCACCCGCC	1088
AS1088-M1			AGGGGCAG
S966-	UCAACGCCGCCUGCCAGCGUCUGGC	966	GCCAGACGCUGGCAGGCGG	1089
AS1089-M1			CGUUGAGG
S967-	CAACGCCGCCUGCCAGCGCUUGGCG	967	CGCCAAGCGCUGGCAGGCG	1090
AS1090-M1			GCGUUGAG
S968-	AACGCCGCCUGCCAGCGCCUGGCGA	968	UCGCCAGGCGCUGGCAGGC	1091
AS1091-M1			GGCGUUGA
S969-	ACGCCGCCUGCCAGCGCCUUGCGAG	969	CUCGCAAGGCGCUGGCAGG	1092
AS1092-M1			CGGCGUUG
S970-	CGCCGCCUGCCAGCGCCUGUCGAGG	970	CCUCGACAGGCGCUGGCAG	1093
AS1093-M1			GCGGCGUU
S971-	GCCGCCUGCCAGCGCCUGGUGAGGG	971	CCCUCACCAGGCGCUGGCA	1094
AS1094-M1			GGCGGCGU
S972-	CCGCCUGCCAGCGCCUGGCUAGGGC	972	GCCCUAGCCAGGCGCUGGC	1095
AS1095-M1			AGGCGGCG
S973-	CGCCUGCCAGCGCCUGGCGAGGGCT	973	AGCCCUCGCCAGGCGCUGG	1096
AS1096-M1			CAGGCGGC
S974-	GCCUGCCAGCGCCUGGCGAUGGCTG	974	CAGCCAUCGCCAGGCGCUG	1097
AS1097-M1			GCAGGCGG
S975-	CCAGCGCCUGGCGAGGGCUUGGGTC	975	GACCCAAGCCCUCGCCAGG	1098
AS1098-M1			CGCUGGCA
S976-	CAGCGCCUGGCGAGGGCUGUGGUCG	976	CGACCACAGCCCUCGCCAG	1099
AS1099-M1			GCGCUGGC
S977-	AGCGCCUGGCGAGGGCUGGUGUCGT	977	ACGACACCAGCCCUCGCCA	1100
AS1100-M1			GGCGCUGG
S978-	GCGCCUGGCGAGGGCUGGGUUCGTG	978	CACGAACCCAGCCCUCGCC	1101
AS1101-M1			AGGCGCUG
S979-	CGCCUGGCGAGGGCUGGGGUCGUGC	979	GCACGACCCCAGCCCUCGC	1102
AS1102-M1			CAGGCGCU
S980-	GCGAGGGCUGGGGUCGUGCUGGUCA	980	UGACCAGCACGACCCCAGC	1103
AS1103-M1			CCUCGCCA
S981-	AUGCCUGCCUCUACUCCCCAGCCTC	981	GAGGCUGGGGAGUAGAGGC	1104
AS1104-M1			AGGCAUCG
S982-	GCCUCUACUCCCCAGCCUCAGCUCC	982	GGAGCUGAGGCUGGGGAGU	1105
AS1105-M1			AGAGGCAG
S983-	GACCUCUUUGCCCCAGGGGAGGACA	983	UGUCCUCCCCUGGGGCAAA	1106
AS1106-M1			GAGGUCCA
S984-	CUUUGCCCCAGGGGAGGACAUCATT	984	AAUGAUGUCCUCCCCUGGG	1107
AS1107-M1			GCAAAGAG
S985-	UUUGCCCCAGGGGAGGACAUCAUTG	985	CAAUGAUGUCCUCCCCUGG	1108
AS1108-M1			GGCAAAGA
S986-	UUGCCCCAGGGGAGGACAUUAUUGG	986	CCAAUAAUGUCCUCCCCUG	1109
AS1109-M1			GGGCAAAG
S987-	UGCCCCAGGGGAGGACAUCAUUGGT	987	ACCAAUGAUGUCCUCCCCU	1110
AS1110-M1			GGGGCAAA
S988-	GCCCCAGGGGAGGACAUCAUUGGTG	988	CACCAAUGAUGUCCUCCCC	1111
AS1111-M1			UGGGGCAA
S989-	ACACGGAUGGCCACAGCCGUCGCCC	989	GGGCGACGGCUGUGGCCAU	1112
AS1112-M1			CCGUGUAG
S990-	CUCCAGGAGUGGGAAGCGGUGGGGC	990	GCCCCACCGCUUCCCACUC	1113
AS1113-M1			CUGGAGAA
S991-	UCCAGGAGUGGGAAGCGGCUGGGCG	991	CGCCCAGCCGCUUCCCACU	1114
AS1114-M1			CCUGGAGA
S992-	CCAGGAGUGGGAAGCGGCGUGGCGA	992	UCGCCACGCCGCUUCCCAC	1115
AS1115-M1			UCCUGGAG
S993-	CAGGAGUGGGAAGCGGCGGUGCGAG	993	CUCGCACCGCCGCUUCCCA	1116
AS1116-M1			CUCCUGGA
S994-	AGGAGUGGGAAGCGGCGGGUCGAGC	994	GCUCGACCCGCCGCUUCCC	1117
AS1117-M1			ACUCCUGG
S995-	GGAGUGGGAAGCGGCGGGGUGAGCG	995	CGCUCACCCCGCCGCUUCC	1118
AS1118-M1			CACUCCUG
S996-	GAGUGGGAAGCGGCGGGGCUAGCGC	996	GCGCUAGCCCCGCCGCUUC	1119
AS1119-M1			CCACUCCU
S997-	AGUGGGAAGCGGCGGGGCGAGCGCA	997	UGCGCUCGCCCCGCCGCUU	1120
AS1120-M1			CCCACUCC
S998-	GAAGCGGCGGGGCGAGCGCAUGGAG	998	CUCCAUGCGCUCGCCCCGC	1121
AS1121-M1			CGCUUCCC
S999-	AAGCGGCGGGGCGAGCGCAUGGAGG	999	CCUCCAUGCGCUCGCCCCG	1122
AS1122-M1			CCGCUUCC
S1000-	AGCGGCGGGGCGAGCGCAUUGAGGC	1000	GCCUCAAUGCGCUCGCCCC	1123
AS1123-M1			GCCGCUUC
S1001-	GGUGCUGCCUGCUACCCCAUGCCAA	1001	UUGGCAUGGGGUAGCAGGC	1124
AS1124-M1			AGCACCUG
S1002-	GUGCUGCCUGCUACCCCAGUCCAAC	1002	GUUGGACUGGGGUAGCAGG	1125
AS1125-M1			CAGCACCU
S1003-	UGCUGCCUGCUACCCCAGGUCAACT	1003	AGUUGACCUGGGGUAGCAG	1126
AS1126-M1			GCAGCACC
S1004-	GGGCCACGUCCUCACAGGCUGCAGC	1004	GCUGCAGCCUGUGAGGACG	1127
AS1127-M1			UGGCCCUG
S1005-	GGCCACGUCCUCACAGGCUUCAGCT	1005	AGCUGAAGCCUGUGAGGAC	1128
AS1128-M1			GUGGCCCU
S1006-	GCCACGUCCUCACAGGCUGUAGCTC	1006	GAGCUACAGCCUGUGAGGA	1129
AS1129-M1			CGUGGCCC
S1007-	GGCUGCAGCUCCCACUGGGAGGUGG	1007	CCACCUCCCAGUGGGAGCU	1130
AS1130-M1			GCAGCCUG
S1008-	GCUGCAGCUCCCACUGGGAUGUGGA	1008	UCCACAUCCCAGUGGGAGC	1131
AS1131-M1			UGCAGCCU
S1009-	CUGCAGCUCCCACUGGGAGUUGGAG	1009	CUCCAACUCCCAGUGGGAG	1132
AS1132-M1			CUGCAGCC
S1010-	UGCAGCUCCCACUGGGAGGUGGAGG	1010	CCUCCACCUCCCAGUGGGA	1133
AS1133-M1			GCUGCAGC
S1011-	GCAGCUCCCACUGGGAGGUUGAGGA	1011	UCCUCAACCUCCCAGUGGG	1134
AS1134-M1			AGCUGCAG
S1012-	CAGCUCCCACUGGGAGGUGUAGGAC	1012	GUCCUACACCUCCCAGUGG	1135
AS1135-M1			GAGCUGCA
S1013-	AGCUCCCACUGGGAGGUGGAGGACC	1013	GGUCCUCCACCUCCCAGUG	1136
AS1136-M1			GGAGCUGC
S1014-	GCUCCCACUGGGAGGUGGAUGACCT	1014	AGGUCAUCCACCUCCCAGU	1137
AS1137-M1			GGGAGCUG
S1015-	CUCCCACUGGGAGGUGGAGUACCTT	1015	AAGGUACUCCACCUCCCAG	1138
AS1138-M1			UGGGAGCU
S1016-	UCCCACUGGGAGGUGGAGGACCUTG	1016	CAAGGUCCUCCACCUCCCA	1139
AS1139-M1			GUGGGAGC
S1017-	UGGCACCCACAAGCCGCCUUUGCTG	1017	CAGCAAAGGCGGCUUGUGG	1140
AS1140-M1			GUGCCAAG
S1018-	GGCACCCACAAGCCGCCUGUGCUGA	1018	UCAGCACAGGCGGCUUGUG	1141
AS1141-M1			GGUGCCAA
S1019-	AGCCGCCUGUGCUGAGGCCACGAGG	1019	CCUCGUGGCCUCAGCACAG	1142
AS1142-M1			GCGGCUUG
S1020-	GCCGCCUGUGCUGAGGCCAUGAGGT	1020	ACCUCAUGGCCUCAGCACA	1143
AS1143-M1			GGCGGCUU
S1021-	CCGCCUGUGCUGAGGCCACUAGGTC	1021	GACCUAGUGGCCUCAGCAC	1144
AS1144-M1			AGGCGGCU
S1022-	GGGCCACAGGGAGGCCAGCAUCCAC	1022	GUGGAUGCUGGCCUCCCUG	1145
AS1145-M1			UGGCCCAC
S1023-	GGCCACAGGGAGGCCAGCAUCCACG	1023	CGUGGAUGCUGGCCUCCCU	1146
AS1146-M1			GUGGCCCA
S1024-	GCCACAGGGAGGCCAGCAUUCACGC	1024	GCGUGAAUGCUGGCCUCCC	1147
AS1147-M1			UGUGGCCC
S1025-	CGGCCCCUCAGGAGCAGGUUACCGT	1025	ACGGUAACCUGCUCCUGAG	1148
AS1148-M1			GGGCCGGG
S1026-	UGCUGCCGGAGCCGGCACCUGGCGC	1026	GCGCCAGGUGCCGGCUCCG	1149
AS1149-M1			GCAGCAGA
S1027-	UCACAGGCUGCUGCCCACGUGGCTG	1027	CAGCCACGUGGGCAGCAGC	1150
AS1150-M1			CUGUGAUG
S1028-	CACAGGCUGCUGCCCACGUUGCUGG	1028	CCAGCAACGUGGGCAGCAG	1151
AS1151-M1			CCUGUGAU
S1029-	GCUUCCUGCUGCCAUGCCCUAGGTC	1029	GACCUAGGGCAUGGCAGCA	1152
AS1152-M1			GGAAGCGU
S1153-	AACUUCAGCUCCUGCACAGUGCAGC	1153	ACUGUGCAGGAGCUGAAGU	1193
AS1193-M2	CGAAAGGCUGC		UCA
S1154-	UGGCCCUCAUGGGCACCGUUGCAGC	1154	AACGGUGCCCAUGAGGGCC	1194
AS1194-M2	CGAAAGGCUGC		AGG
S1155-	AGGAGGAGACCCACCUCUCUGCAGC	1155	AGAGAGGUGGGUCUCCUCC	1195
AS1195-M2	CGAAAGGCUGC		UUC
S1156-	UGCUGGAGCUGGCCUUGAAUGCAGC	1156	AUUCAAGGCCAGCUCCAGC	1196
AS1196-M2	CGAAAGGCUGC		AGG
S1157-	UCUGUCUUUGCCCAGAGCAUGCAGC	1157	AUGCUCUGGGCAAAGACAG	1197
AS1197-M2	CGAAAGGCUGC		AGG
S1158-	CUGUCUUUGCCCAGAGCAUUGCAGC	1158	AAUGCUCUGGGCAAAGACA	1198
AS1198-M2	CGAAAGGCUGC		GAG
S1159-	CUUGCCUGGAACUCACUCAUGCAGC	1159	AUGAGUGAGUUCCAGGCAA	1199
AS1199-M2	CGAAAGGCUGC		GGA
S1160-	UUGCCUGGAACUCACUCACUGCAGC	1160	AGUGAGUGAGUUCCAGGCA	1200
AS1200-M2	CGAAAGGCUGC		AGG
S1161-	AGAAUGACUUUUAUUGAGCUGCAGC	1161	AGCUCAAUAAAAGUCAUUC	1201
AS1201-M2	CGAAAGGCUGC		UGC
S1162-	GAAUGACUUUUAUUGAGCUUGCAGC	1162	AAGCUCAAUAAAAGUCAUU	1202
AS1202-M2	CGAAAGGCUGC		CUG
S1163-	AUGACUUUUAUUGAGCUCUUGCAGC	1163	AAGAGCUCAAUAAAAGUCA	1203
AS1203-M2	CGAAAGGCUGC		UUC
S1164-	UGACUUUUAUUGAGCUCUUUGCAGC	1164	AAAGAGCUCAAUAAAAGUC	1204
AS1204-M2	CGAAAGGCUGC		AUU
S1165-	CUUGUUCCGUGCCAGGCAUUGCAGC	1165	AAUGCCUGGCACGGAACAA	1205
AS1205-M2	CGAAAGGCUGC		GAG
S1166-	UGUGAAAGGUGCUGAUGGCUGCAGC	1166	AGCCAUCAGCACCUUUCAC	1206
AS1206-M2	CGAAAGGCUGC		ACU
S1167-	AUGGAGGCUUAGCUUUCUGUGCAGC	1167	ACAGAAAGCUAAGCCUCCA	1207
AS1207-M2	CGAAAGGCUGC		UUA
S1168-	GAGGCUUAGCUUUCUGGAUUGCAGC	1168	AAUCCAGAAAGCUAAGCCU	1208
AS1208-M2	CGAAAGGCUGC		CCA
S1169-	AGGCUUAGCUUUCUGGAUGUGCAGC	1169	ACAUCCAGAAAGCUAAGCC	1209
AS1209-M2	CGAAAGGCUGC		UCC
S1170-	GCUUAGCUUUCUGGAUGGCAGCAGC	1170	UGCCAUCCAGAAAGCUAAG	1210
AS1210-M2	CGAAAGGCUGC		CCU
S1171-	CCAGGCUGUGCUAGCAACAUGCAGC	1171	AUGUUGCUAGCACAGCCUG	1211
AS1211-M2	CGAAAGGCUGC		GCA
S1172-	UGCGGGGAGCCAUCACCUAUGCAGC	1172	AUAGGUGAUGGCUCCCCGC	1212
AS1212-M2	CGAAAGGCUGC		AGG
S1173-	CGGCAGUGUGCAGUGGUGCAGCAGC	1173	UGCACCACUGCACACUGCC	1213
AS1213-M2	CGAAAGGCUGC		GAG
S1174-	ACAGAGGAAGAAACCUGGAAGCAGC	1174	UUCCAGGUUUCUUCCUCUG	1214
AS1214-M2	CGAAAGGCUGC		UGA
S1175-	CAGAGGAAGAAACCUGGAAUGCAGC	1175	AUUCCAGGUUUCUUCCUCU	1215
AS1215-M2	CGAAAGGCUGC		GUG
S1176-	AGAGGAAGAAACCUGGAACUGCAGC	1176	AGUUCCAGGUUUCUUCCUC	1216
AS1216-M2	CGAAAGGCUGC		UGU
S1177-	UGGCGGAGAUGCUUCUAAGUGCAGC	1177	ACUUAGAAGCAUCUCCGCC	1217
AS1217-M2	CGAAAGGCUGC		AGG
S1178-	UUACAGCCAACUUUUCUAGAGCAGC	1178	UCUAGAAAAGUUGGCUGUA	1218
AS1218-M2	CGAAAGGCUGC		AAA
S1179-	CUGUUUUGCUUUUGUAACUUGCAGC	1179	AAGUUACAAAAGCAAAACA	1219
AS1219-M2	CGAAAGGCUGC		GGU
S1180-	UGUUUUGCUUUUGUAACUUUGCAGC	1180	AAAGUUACAAAAGCAAAAC	1220
AS1220-M2	CGAAAGGCUGC		AGG
S1181-	UUUGCUUUUGUAACUUGAAUGCAGC	1181	AUUCAAGUUACAAAAGCAA	1221
AS1221-M2	CGAAAGGCUGC		AAC
S1182-	UUUGUAGCAUUUUUAUUAAUGCAGC	1182	AUUAAUAAAAAUGCUACAA	1222
AS1222-M2	CGAAAGGCUGC		AAC
S1183-	UGUAGCAUUUUUAUUAAUAUGCAGC	1183	AUAUUAAUAAAAAUGCUAC	1223
AS1223-M2	CGAAAGGCUGC		AAA
S1184-	GUAGCAUUUUUAUUAAUAUUGCAGC	1184	AAUAUUAAUAAAAAUGCUA	1224
AS1224-M2	CGAAAGGCUGC		CAA
S1185-	AUUAAUAUGGUGACUUUUUAGCAGC	1185	UAAAAAGUCACCAUAUUAA	1225
AS1225-M2	CGAAAGGCUGC		UAA
S1186-	UUAAUAUGGUGACUUUUUAAGCAGC	1186	UUAAAAAGUCACCAUAUUA	1226
AS1226-M2	CGAAAGGCUGC		AUA
S1187-	AAUAUGGUGACUUUUUAAAAGCAGC	1187	UUUUAAAAAGUCACCAUAU	1227
AS1227-M2	CGAAAGGCUGC		UAA
S1188-	AUAUGGUGACUUUUUAAAAUGCAGC	1188	AUUUUAAAAAGUCACCAUA	1228
AS1228-M2	CGAAAGGCUGC		UUA
S1189-	UAUGGUGACUUUUUAAAAUAGCAGC	1189	UAUUUUAAAAAGUCACCAU	1229
AS1229-M2	CGAAAGGCUGC		AUU
S1190-	AUGGUGACUUUUUAAAAUAAGCAGC	1190	UUAUUUUAAAAAGUCACCA	1230
AS1230-M2	CGAAAGGCUGC		UAU
S1191-	UGGUGACUUUUUAAAAUAAAGCAGC	1191	UUUAUUUUAAAAAGUCACC	1231
AS1231-M2	CGAAAGGCUGC		AUA
S1192-	GUGACUUUUUAAAAUAAAAAGCAGC	1192	UUUUUAUUUUAAAAAGUCA	1232
AS1232-M2	CGAAAGGCUGC		CCA
S1153-	AACUUCAGCUCCUGCACAGUGCAGC	1153	ACUGUGCAGGAGCUGAAGU	1193
AS1193-M3	CGAAAGGCUGC		UCA
S1154-	UGGCCCUCAUGGGCACCGUUGCAGC	1154	AACGGUGCCCAUGAGGGCC	1194
AS1194-M3	CGAAAGGCUGC		AGG
S1155-	AGGAGGAGACCCACCUCUCUGCAGC	1155	AGAGAGGUGGGUCUCCUCC	1195
AS1195-M3	CGAAAGGCUGC		UUC
S1156-	UGCUGGAGCUGGCCUUGAAUGCAGC	1156	AUUCAAGGCCAGCUCCAGC	1196
AS1196-M3	CGAAAGGCUGC		AGG
S1157-	UCUGUCUUUGCCCAGAGCAUGCAGC	1157	AUGCUCUGGGCAAAGACAG	1197
AS1197-M3	CGAAAGGCUGC		AGG
S1158-	CUGUCUUUGCCCAGAGCAUUGCAGC	1158	AAUGCUCUGGGCAAAGACA	1198
AS1198-M3	CGAAAGGCUGC		GAG
S1159-	CUUGCCUGGAACUCACUCAUGCAGC	1159	AUGAGUGAGUUCCAGGCAA	1199
AS1199-M3	CGAAAGGCUGC		GGA
S1160-	UUGCCUGGAACUCACUCACUGCAGC	1160	AGUGAGUGAGUUCCAGGCA	1200
AS1200-M3	CGAAAGGCUGC		AGG
S1161-	AGAAUGACUUUUAUUGAGCUGCAGC	1161	AGCUCAAUAAAAGUCAUUC	1201
AS1201-M3	CGAAAGGCUGC		UGC
S1162-	GAAUGACUUUUAUUGAGCUUGCAGC	1162	AAGCUCAAUAAAAGUCAUU	1202
AS1202-M3	CGAAAGGCUGC		CUG
S1163-	AUGACUUUUAUUGAGCUCUUGCAGC	1163	AAGAGCUCAAUAAAAGUCA	1203
AS1203-M3	CGAAAGGCUGC		UUC
S1164-	UGACUUUUAUUGAGCUCUUUGCAGC	1164	AAAGAGCUCAAUAAAAGUC	1204
AS1204-M3	CGAAAGGCUGC		AUU
S1165-	CUUGUUCCGUGCCAGGCAUUGCAGC	1165	AAUGCCUGGCACGGAACAA	1205
AS1205-M3	CGAAAGGCUGC		GAG
S1166-	UGUGAAAGGUGCUGAUGGCUGCAGC	1166	AGCCAUCAGCACCUUUCAC	1206
AS1206-M3	CGAAAGGCUGC		CAU
S1167-	AUGGAGGCUUAGCUUUCUGUGCAGC	1167	ACAGAAAGCUAAGCCUCCA	1207
AS1207-M3	CGAAAGGCUGC		UUA
S1168-	GAGGCUUAGCUUUCUGGAUUGCAGC	1168	AAUCCAGAAAGCUAAGCCU	1208
AS1208-M3	CGAAAGGCUGC		CCA
S1169-	AGGCUUAGCUUUCUGGAUGUGCAGC	1169	ACAUCCAGAAAGCUAAGCC	1209
AS1209-M3	CGAAAGGCUGC		UCC
S1170-	GCUUAGCUUUCUGGAUGGCAGCAGC	1170	UGCCAUCCAGAAAGCUAAG	1210
AS1210-M3	CGAAAGGCUGC		CCU
S1171-	CCAGGCUGUGCUAGCAACAUGCAGC	1171	AUGUUGCUAGCACAGCCUG	1211
AS1211-M3	CGAAAGGCUGC		GCA
S1172-	UGCGGGGAGCCAUCACCUAUGCAGC	1172	AUAGGUGAUGGCUCCCCGC	1212
AS1212-M3	CGAAAGGCUGC		AGG
S1173-	CGGCAGUGUGCAGUGGUGCAGCAGC	1173	UGCACCACUGCACACUGCC	1213
AS1213-M3	CGAAAGGCUGC		GAG
S1174-	ACAGAGGAAGAAACCUGGAAGCAGC	1174	UUCCAGGUUUCUUCCUCUG	1214
AS1214-M3	CGAAAGGCUGC		UGA
S1175-	CAGAGGAAGAAACCUGGAAUGCAGC	1175	AUUCCAGGUUUCUUCCUCU	1215
AS1215-M3	CGAAAGGCUGC		GUG
S1176-	AGAGGAAGAAACCUGGAACUGCAGC	1176	AGUUCCAGGUUUCUUCCUC	1216
AS1216-M3	CGAAAGGCUGC		UGU
S1177-	UGGCGGAGAUGCUUCUAAGUGCAGC	1177	ACUUAGAAGCAUCUCCGCC	1217
AS1217-M3	CGAAAGGCUGC		AGG
S1178-	UUACAGCCAACUUUUCUAGAGCAGC	1178	UCUAGAAAAGUUGGCUGUA	1218
AS1218-M3	CGAAAGGCUGC		AAA
S1179-	CUGUUUUGCUUUUGUAACUUGCAGC	1179	AAGUUACAAAAGCAAAACA	1219
AS1219-M3	CGAAAGGCUGC		GGU
S1180-	UGUUUUGCUUUUGUAACUUUGCAGC	1180	AAAGUUACAAAAGCAAAAC	1220
AS1220-M3	CGAAAGGCUGC		AGG
S1181-	UUUGCUUUUGUAACUUGAAUGCAGC	1181	AUUCAAGUUACAAAAGCAA	1221
AS1221-M3	CGAAAGGCUGC		AAC
S1182-	UUUGUAGCAUUUUUAUUAAUGCAGC	1182	AUUAAUAAAAAUGCUACAA	1222
AS1222-M3	CGAAAGGCUGC		AAC
S1183-	UGUAGCAUUUUUAUUAAUAUGCAGC	1183	AUAUUAAUAAAAAUGCUAC	1223
AS1223-M3	CGAAAGGCUGC		AAA
S1184-	GUAGCAUUUUUAUUAAUAUUGCAGC	1184	AAUAUUAAUAAAAAUGCUA	1224
AS1224-M3	CGAAAGGCUGC		CAA
S1185-	AUUAAUAUGGUGACUUUUUAGCAGC	1185	UAAAAAGUCACCAUAUUAA	1225
AS1225-M3	CGAAAGGCUGC		UAA
S1186-	UUAAUAUGGUGACUUUUUAAGCAGC	1186	UUAAAAAGUCACCAUAUUA	1226
AS1226-M3	CGAAAGGCUGC		AUA
S1187-	AAUAUGGUGACUUUUUAAAAGCAGC	1187	UUUUAAAAAGUCACCAUAU	1227
AS1227-M3	CGAAAGGCUGC		UAA
S1188-	AUAUGGUGACUUUUUAAAAUGCAGC	1188	AUUUUAAAAAGUCACCAUA	1228
AS1228-M3	CGAAAGGCUGC		UUA
S1189-	UAUGGUGACUUUUUAAAAUAGCAGC	1189	UAUUUUAAAAAGUCACCAU	1229
AS1229-M3	CGAAAGGCUGC		AUU
S1190-	AUGGUGACUUUUUAAAAUAAGCAGC	1190	UUAUUUUAAAAAGUCACCA	1230
AS1230-M3	CGAAAGGCUGC		UAU
S1191-	UGGUGACUUUUUAAAAUAAAGCAGC	1191	UUUAUUUUAAAAAGUCACC	1231
AS1231-M3	CGAAAGGCUGC		AUA
S1192-	GUGACUUUUUAAAAUAAAAAGCAGC	1192	UUUUUAUUUUAAAAAGUCA	1232
AS1232-M3	CGAAAGGCUGC		CCA
S1180-	UGUUUUGCUUUUGUAACUUUGCAGC	1180	AAAGUUACAAAAGCAAAAC	1220
AS1220-M4	CGAAAGGCUGC		AGG
S1163-	AUGACUUUUAUUGAGCUCUUGCAGC	1163	AAGAGCUCAAUAAAAGUCA	1203
AS1203-M4	CGAAAGGCUGC		UUC
S1181-	UUUGCUUUUGUAACUUGAAUGCAGC	1181	AUUCAAGUUACAAAAGCAA	1221
AS1221-M4	CGAAAGGCUGC		AAC
S1248-	GCUGGGCUCCUCAUUUUUAUGCAGC	1248	AUAAAAAUGAGGAGCCCAG	1257
AS1257-M4	CGAAAGGCUGC		CGG
S1249-	GCUGGCGGAGAUGCUUCUAAGCAGC	1249	UUAGAAGCAUCUCCGCCAG	1258
AS1258-M4	CGAAAGGCUGC		CGG
S1250-	UUUACAGCCAACUUUUCUAUGCAGC	1250	AUAGAAAAGUUGGCUGUAA	1259
AS1259-M4	CGAAAGGCUGC		AGG
S1251-	GGCUGGGCUCCUCAUUUUUAGCAGC	1251	UAAAAAUGAGGAGCCCAGC	1260
AS1260-M4	CGAAAGGCUGC		CGG
S1252-	AGCACGGAACCACAGCCACUGCAGC	1252	AGUGGCUGUGGUUCCGUGC	1261
AS1261-M4	CGAAAGGCUGC		UGG
S1253-	AAUGACUUUUAUUGAGCUCUGCAGC	1253	AGAGCUCAAUAAAAGUCAU	1262
AS1262-M4	CGAAAGGCUGC		UGG
S1254-	UUUUGUAGCAUUUUUAUUAAGCAGC	1254	UUAAUAAAAAUGCUACAAA	1263
AS1263-M4	CGAAAGGCUGC		AGG
S1255-	GCUUGCCUGGAACUCACUCAGCAGC	1255	UGAGUGAGUUCCAGGCAAG	1264
AS1264-M4	CGAAAGGCUGC		CGG
S1256-	UGGAGGCUUAGCUUUCUGGAGCAGC	1256	UCCAGAAAGCUAAGCCUCC	1265
AS1265-M4	CGAAAGGCUGC		AGG
S1180-	UGUUUUGCUUUUGUAACUUUGCAGC	1180	AAAGUUACAAAAGCAAAAC	1220
AS1220-M4	CGAAAGGCUGC		AGG
S1180-	UGUUUUGCUUUUGUAACUUUGCAGC	1180	AAAGUUACAAAAGCAAAAC	1220
AS1220-M5	CGAAAGGCUGC		AGG
S1164-	UGACUUUUAUUGAGCUCUUUGCAGC	1164	AAAGAGCUCAAUAAAAGUC	1204
AS1204-M5	CGAAAGGCUGC		AUU
S1178-	UUACAGCCAACUUUUCUAGAGCAGC	1178	UCUAGAAAAGUUGGCUGUA	1218
AS1218-M6	CGAAAGGCUGC		AAA
S1178-	UUACAGCCAACUUUUCUAGAGCAGC	1178	UCUAGAAAAGUUGGCUGUA	1218
AS1218-M5	CGAAAGGCUGC		AAA
S1179-	CUGUUUUGCUUUUGUAACUUGCAGC	1179	AAGUUACAAAAGCAAAACA	1219
AS1219-M6	CGAAAGGCUGC		GGU
S1179-	CUGUUUUGCUUUUGUAACUUGCAGC	1179	AAGUUACAAAAGCAAAACA	1219
AS1219-M5	CGAAAGGCUGC		GGU
S1181-	UUUGCUUUUGUAACUUGAAUGCAGC	1181	AUUCAAGUUACAAAAGCAA	1221
AS1221-M5	CGAAAGGCUGC		AAC
S1182-	UUUGUAGCAUUUUUAUUAAUGCAGC	1182	AUUAAUAAAAAUGCUACAA	1222
AS1222-M5	CGAAAGGCUGC		AAC
S1183-	UGUAGCAUUUUUAUUAAUAUGCAGC	1183	AUAUUAAUAAAAAUGCUAC	1223
AS1223-M5	CGAAAGGCUGC		AAA
S1187-	AAUAUGGUGACUUUUUAAAAGCAGC	1187	UUUUAAAAAGUCACCAUAU	1227
AS1227-M5	CGAAAGGCUGC		UAA
S1188-	AUAUGGUGACUUUUUAAAAUGCAGC	1188	AUUUUAAAAAGUCACCAUA	1228
AS1228-M5	CGAAAGGCUGC		UUA
S1189-	UAUGGUGACUUUUUAAAAUAGCAGC	1189	UAUUUUAAAAAGUCACCAU	1229
AS1229-M5	CGAAAGGCUGC		AUU
S1158-	CUGUCUUUGCCCAGAGCAUUGCAGC	1158	AAUGCUCUGGGCAAAGACA	1198
AS1198-M5	CGAAAGGCUGC		GAG
S1159-	CUUGCCUGGAACUCACUCAUGCAGC	1159	AUGAGUGAGUUCCAGGCAA	1199
AS1199-M5	CGAAAGGCUGC		GGA
S1160-	UUGCCUGGAACUCACUCACUGCAGC	1160	AGUGAGUGAGUUCCAGGCA	1200
AS1200-M5	CGAAAGGCUGC		AGG
S1161-	AGAAUGACUUUUAUUGAGCUGCAGC	1161	AGCUCAAUAAAAGUCAUUC	1201
AS1201-M5	CGAAAGGCUGC		UGC
S1163-	AUGACUUUUAUUGAGCUCUUGCAGC	1163	AAGAGCUCAAUAAAAGUCA	1203
AS1203-M5	CGAAAGGCUGC		UUC
S1184-	GUAGCAUUUUUAUUAAUAUUGCAGC	1184	AAUAUUAAUAAAAAUGCUA	1224
AS1224-M5	CGAAAGGCUGC		CAA
S1185-	AUUAAUAUGGUGACUUUUUAGCAGC	1185	UAAAAAGUCACCAUAUUAA	1225
AS1225-M5	CGAAAGGCUGC		UAA
S1186-	UUAAUAUGGUGACUUUUUAAGCAGC	1186	UUAAAAAGUCACCAUAUUA	1226
AS1226-M6	CGAAAGGCUGC		AUA
S1186-	UUAAUAUGGUGACUUUUUAAGCAGC	1186	UUAAAAAGUCACCAUAUUA	1226
AS1226-M5	CGAAAGGCUGC		AUA
S1190-	AUGGUGACUUUUUAAAAUAAGCAGC	1190	UUAUUUUAAAAAGUCACCA	1230
AS1230-M5	CGAAAGGCUGC		UAU
S1191-	UGGUGACUUUUUAAAAUAAAGCAGC	1191	UUUAUUUUAAAAAGUCACC	1231
AS1231-M5	CGAAAGGCUGC		AUA
S1192-	GUGACUUUUUAAAAUAAAAAGCAGC	1192	UUUUUAUUUUAAAAAGUCA	1232
AS1232-M5	CGAAAGGCUGC		CCA
S1266-	UGUUUUGCUUUUGUAACUU[U/A]G	1266	[U/A]AAGUUACAAAAGCA	1269
AS1269-M7	CAGCCGAAAGGCUGC		AAACAGG
S1266-	UGUUUUGCUUUUGUAACUU[U/A]G	1266	[U/A]AAGUUACAAAAGCA	1269
AS1269-M8	CAGCCGAAAGGCUGC		AAACAGG
S1266-	UGUUUUGCUUUUGUAACUU[U/A]G	1266	[U/A]AAGUUACAAAAGCA	1269
AS1269-M9	CAGCCGAAAGGCUGC		AAACAGG
S1267-	UUUUGUAACUUGAAGAUAUAGCAGC	1267	UAUAUCUUCAAGUUACAAA	1270
AS1270-M10	CGAAAGGCUGC		AGG
S1268-	CUGGGUUUUGUAGCAUUUUAGCAGC	1268	UAAAAUGCUACAAAACCCA	1271
AS1271-M11	CGAAAGGCUGC		GGG
S1268-	CUGGGUUUUGUAGCAUUUUAGCAGC	1268	UAAAAUGCUACAAAACCCA	1271
AS1271-M9	CGAAAGGCUGC		GGG
S1266-	UGUUUUGCUUUUGUAACUU[U/A]G	1266	[U/A]AAGUUACAAAAGCA	1269
AS1269-M12	CAGCCGAAAGGCUGC		AAACAGG
S1266-	UGUUUUGCUUUUGUAACUU[U/A]G	1266	[U/A]AAGUUACAAAAGCA	1269
AS1269-M13	CAGCCGAAAGGCUGC		AAACAGG

The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

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: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop between S1 and S2 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.