PCSK9 TARGETING OLIGONUCLEOTIDES FOR TREATING HYPERCHOLESTEROLEMIA AND RELATED CONDITIONS

Information

  • Patent Application
  • 20210238606
  • Publication Number
    20210238606
  • Date Filed
    April 01, 2019
    5 years ago
  • Date Published
    August 05, 2021
    3 years ago
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
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 D080070015WO00-SEQ-ZJG.txt created on Apr. 1, 2019 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 (Hnf1a), 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 No. 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 (Tm) of an adjacent stem duplex that is higher than the Tm 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, p163-176 (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, p193-198), 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-52, 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 No. 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 Tm 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 Tm than a duplex formed with the nucleic acid comprising the mismatched base.


Non-limiting examples of universal-binding nucleotides include inosine, 1-β-D-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 ID


mRNA
Sequence
NO.





746-783
CGACCTGCTGGAGCTGGCCTTGAA
1233



GTTGCCCCATGTCG






2602-2639
AGCCTCCTTGCCTGGAACTCACTC
1234



ACTCTGGGTGCCTC






2737-2792
CAATGTGCCGATGTCCGTGGGCAG
1235



AATGACTTTTATTGAGCTCTTGTT




CCGTGCCA






2880-2923
CGTTGGGGGGTGAGTGTGAAAGGT
1236



GCTGATGGCCCTCATCTCCA






2956-2996
GATTAATGGAGGCTTAGCTTTCTG
1237



GATGGCATCTAGCCAGA






3015-3075
CCCTGGTGGTCACAGGCTGTGCCT
1238



TGGTTTCCTGAGCCACCTTTACTC




TGCTCTATGCCAG






3099-3178
TGGCCTGCGGGGAGCCATCACCTA
1239



GGACTGACTCGGCAGTGTGCAGTG




GTGCATGCACTGTCTCAGCCAACC




CGCTCCAC






3190-3244
GTACACATTCGCACCCCTACTTCA
1240



CAGAGGAAGAAACCTGGAACCAGA




GGGGGCG






3297-3359
GCTCTGAAGCCAAGCCTCTTCTTA
1241



CTTCACCCGGCTGGGCTCCTCATT




TTTACGGGTAACAGT






3469-3446
AACGATGCCTGCAGGCATGGAACT
1242



TTTTCCGTTATCACCCAGGCCT






3457-3499
TTCACTGGCCTGGCGGAGATGCTT
1243



CTAAGGCATGGTCGGGGGA






3532-3715
GCCCCACCCAAGCAAGCAGACATT
1244



TATCTTTTGGGTCTGTCCTCTCTG




TTGCCTTTTTACAGCCAACTTTTC




TAGACCTGTTTTGCTTTTGTAACT




TGAAGATATTTATTCTGGGTTTTG




TAGCATTTTTATTAATATGGTGAC




TTTTTAAAATAAAAACAAACAAAC




GTTGTCCTAACAAAAA









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:




embedded image


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′→3′ 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







Oligonucleotide Sequences













S SEQ

AS SEQ


App Name
Sense Sequence/mRNA seq
ID NO
Antisense Sequence
ID NO














S1-AS454-M1
AAGCACCCACACCCUAGAAUGUUTC
1
GAAACAUUCUAGGGUGUGGGUGCUUGA
454





S2-AS455-M1
AGCACCCACACCCUAGAAGUUUUCC
2
GGAAAACUUCUAGGGUGUGGGUGCUUG
455





S3-AS456-M1
GCACCCACACCCUAGAAGGUUUCCG
3
CGGAAACCUUCUAGGGUGUGGGUGCUU
456





S4-AS457-M1
ACCCACACCCUAGAAGGUUUCCGCA
4
UGCGGAAACCUUCUAGGGUGUGGGUGC
457





S5-AS458-M1
CCCACACCCUAGAAGGUUUUCGCAG
5
CUGCGAAAACCUUCUAGGGUGUGGGUG
458





S6-AS459-M1
AGUUCAGGGUCUGAGCCUGUAGGAG
6
CUCCUACAGGCUCAGACCCUGAACUGA
459





S7-AS460-M1
GUUCAGGGUCUGAGCCUGGAGGAGT
7
ACUCCUCCAGGCUCAGACCCUGAACUG
460





S8-AS461-M1
UUCAGGGUCUGAGCCUGGAUGAGTG
8
CACUCAUCCAGGCUCAGACCCUGAACU
461





S9-AS462-M1
UCAGGGUCUGAGCCUGGAGUAGUGA
9
UCACUACUCCAGGCUCAGACCCUGAAC
462





S10-AS463-M1
AGGGUCUGAGCCUGGAGGAUUGAGC
10
GCUCAAUCCUCCAGGCUCAGACCCUGA
463





S11-AS464-M1
GGUCUGAGCCUGGAGGAGUUAGCCA
11
UGGCUAACUCCUCCAGGCUCAGACCCU
464





S12-AS465-M1
AGGAUUCCGCGCGCCCCUUUACGCG
12
CGCGUAAAGGGGCGCGCGGAAUCCUGG
465





S13-AS466-M1
GGAUUCCGCGCGCCCCUUCACGCGC
13
GCGCGUGAAGGGGCGCGCGGAAUCCUG
466





S14-AS467-M1
UCACGCGCCCUGCUCCUGAACUUCA
14
UGAAGUUCAGGAGCAGGGCGCGUGAAG
467





S15-AS468-M1
CACGCGCCCUGCUCCUGAAUUUCAG
15
CUGAAAUUCAGGAGCAGGGCGCGUGAA
468





S16-AS469-M1
CCCUGCUCCUGAACUUCAGUUCCTG
16
CAGGAACUGAAGUUCAGGAGCAGGGCG
469





S17-AS470-M1
CUGCUCCUGAACUUCAGCUUCUGCA
17
UGCAGAAGCUGAAGUUCAGGAGCAGGG
470





S18-AS471-M1
UGCUCCUGAACUUCAGCUCUUGCAC
18
GUGCAAGAGCUGAAGUUCAGGAGCAGG
471





S19-AS472-M1
GCUCCUGAACUUCAGCUCCUGCACA
19
UGUGCAGGAGCUGAAGUUCAGGAGCAG
472





S20-AS473-M1
CUCCUGAACUUCAGCUCCUUCACAG
20
CUGUGAAGGAGCUGAAGUUCAGGAGCA
473





S21-AS474-M1
UCCUGAACUUCAGCUCCUGUACAGT
21
ACUGUACAGGAGCUGAAGUUCAGGAGC
474





S22-AS475-M1
CCUGAACUUCAGCUCCUGCACAGTC
22
GACUGUGCAGGAGCUGAAGUUCAGGAG
475





S23-AS476-M1
CUGAACUUCAGCUCCUGCAUAGUCC
23
GGACUAUGCAGGAGCUGAAGUUCAGGA
476





S24-AS477-M1
UGAACUUCAGCUCCUGCACAGUCCT
24
AGGACUGUGCAGGAGCUGAAGUUCAGG
477





S25-AS478-M1
GAACUUCAGCUCCUGCACAUUCCTC
25
GAGGAAUGUGCAGGAGCUGAAGUUCAG
478





S26-AS479-M1
AACUUCAGCUCCUGCACAGUCCUCC
26
GGAGGACUGUGCAGGAGCUGAAGUUCA
479





S27-AS480-M1
ACUUCAGCUCCUGCACAGUUCUCCC
27
GGGAGAACUGUGCAGGAGCUGAAGUUC
480





S28-AS481-M1
CUUCAGCUCCUGCACAGUCUUCCCC
28
GGGGAAGACUGUGCAGGAGCUGAAGUU
481





S29-AS482-M1
ACAGUCCUCCCCACCGCAAUGCUCA
29
UGAGCAUUGCGGUGGGGAGGACUGUGC
482





S30-AS483-M1
CAGUCCUCCCCACCGCAAGUCUCAA
30
UUGAGACUUGCGGUGGGGAGGACUGUG
483





S31-AS484-M1
GCCUCUAGGUCUCCUCGCCAGGACA
31
UGUCCUGGCGAGGAGACCUAGAGGCCG
484





S32-AS485-M1
GCCAGGACAGCAACCUCUCUCCUGG
32
CCAGGAGAGAGGUUGCUGUCCUGGCGA
485





S33-AS486-M1
GGACAGCAACCUCUCCCCUUGCCCT
33
AGGGCAAGGGGAGAGGUUGCUGUCCUG
486





S34-AS487-M1
CCCCUGGCCCUCAUGGGCAUCGUCA
34
UGACGAUGCCCAUGAGGGCCAGGGGAG
487





S35-AS488-M1
UGGCCCUCAUGGGCACCGUUAGCTC
35
GAGCUAACGGUGCCCAUGAGGGCCAGG
488





S36-AS489-M1
GGCCCUCAUGGGCACCGUCAGCUCC
36
GGAGCUGACGGUGCCCAUGAGGGCCAG
489





S37-AS490-M1
GCCCUCAUGGGCACCGUCAUCUCCA
37
UGGAGAUGACGGUGCCCAUGAGGGCCA
490





S38-AS491-M1
GCGGUCCUGGUGGCCGCUGUCACTG
38
CAGUGACAGCGGCCACCAGGACCGCCU
491





S39-AS492-M1
GGCCUGGCCGAAGCACCCGAGCACG
39
CGUGCUCGGGUGCUUCGGCCAGGCCGU
492





S40-AS493-M1
ACCCGAGCACGGAACCACAUCCACC
40
GGUGGAUGUGGUUCCGUGCUCGGGUGC
493





S41-AS494-M1
AGCACGGAACCACAGCCACUUUCCA
41
UGGAAAGUGGCUGUGGUUCCGUGCUCG
494





S42-AS495-M1
CACGGAACCACAGCCACCUUCCACC
42
GGUGGAAGGUGGCUGUGGUUCCGUGCU
495





S43-AS496-M1
ACGGAACCACAGCCACCUUUCACCG
43
CGGUGAAAGGUGGCUGUGGUUCCGUGC
496





S44-AS497-M1
GCCAAGGAUCCGUGGAGGUUGCCTG
44
CAGGCAACCUCCACGGAUCCUUGGCGC
497





S45-AS498-M1
CCAAGGAUCCGUGGAGGUUUCCUGG
45
CCAGGAAACCUCCACGGAUCCUUGGCG
498





S46-AS499-M1
AAGGAUCCGUGGAGGUUGCUUGGCA
46
UGCCAAGCAACCUCCACGGAUCCUUGG
499





S47-AS500-M1
GGAUCCGUGGAGGUUGCCUUGCACC
47
GGUGCAAGGCAACCUCCACGGAUCCUU
500





S48-AS501-M1
UGGAGGUUGCCUGGCACCUACGUGG
48
CCACGUAGGUGCCAGGCAACCUCCACG
501





S49-AS502-M1
UGCCUGGCACCUACGUGGUUGUGCT
49
AGCACAACCACGUAGGUGCCAGGCAAC
502





S50-AS503-M1
GCCUGGCACCUACGUGGUGUUGCTG
50
CAGCAACACCACGUAGGUGCCAGGCAA
503





S51-AS504-M1
AGGAGGAGACCCACCUCUCUCAGTC
51
GACUGAGAGAGGUGGGUCUCCUCCUUC
504





S52-AS505-M1
CCUGCAUGUCUUCCAUGGCUUUCTT
52
AAGAAAGCCAUGGAAGACAUGCAGGAU
505





S53-AS506-M1
UGCAUGUCUUCCAUGGCCUUCUUCC
53
GGAAGAAGGCCAUGGAAGACAUGCAGG
506





S54-AS507-M1
ACCUGCUGGAGCUGGCCUUUAAGTT
54
AACUUAAAGGCCAGCUCCAGCAGGUCG
507





S55-AS508-M1
CUGCUGGAGCUGGCCUUGAAGUUGC
55
GCAACUUCAAGGCCAGCUCCAGCAGGU
508





S56-AS509-M1
UGCUGGAGCUGGCCUUGAAUUUGCC
56
GGCAAAUUCAAGGCCAGCUCCAGCAGG
509





S57-AS510-M1
UGGAGCUGGCCUUGAAGUUUCCCCA
57
UGGGGAAACUUCAAGGCCAGCUCCAGC
510





S58-AS511-M1
GGCCUUGAAGUUGCCCCAUUUCGAC
58
GUCGAAAUGGGGCAACUUCAAGGCCAG
511





S59-AS512-M1
GCCUUGAAGUUGCCCCAUGUCGACT
59
AGUCGACAUGGGGCAACUUCAAGGCCA
512





S60-AS513-M1
CCUUGAAGUUGCCCCAUGUUGACTA
60
UAGUCAACAUGGGGCAACUUCAAGGCC
513





S61-AS514-M1
CUUGAAGUUGCCCCAUGUCUACUAC
61
GUAGUAGACAUGGGGCAACUUCAAGGC
514





S62-AS515-M1
ACUCCUCUGUCUUUGCCCAUAGCAT
62
AUGCUAUGGGCAAAGACAGAGGAGUCC
515





S63-AS516-M1
CUCCUCUGUCUUUGCCCAGAGCATC
63
GAUGCUCUGGGCAAAGACAGAGGAGUC
516





S64-AS517-M1
UCCUCUGUCUUUGCCCAGAUCAUCC
64
GGAUGAUCUGGGCAAAGACAGAGGAGU
517





S65-AS518-M1
CCUCUGUCUUUGCCCAGAGUAUCCC
65
GGGAUACUCUGGGCAAAGACAGAGGAG
518





S66-AS519-M1
UCUGUCUUUGCCCAGAGCAUCCCGT
66
ACGGGAUGCUCUGGGCAAAGACAGAGG
519





S67-AS520-M1
CUGUCUUUGCCCAGAGCAUUCCGTG
67
CACGGAAUGCUCUGGGCAAAGACAGAG
520





S68-AS521-M1
GUCUUUGCCCAGAGCAUCCUGUGGA
68
UCCACAGGAUGCUCUGGGCAAAGACAG
521





S69-AS522-M1
UCUUUGCCCAGAGCAUCCCUUGGAA
69
UUCCAAGGGAUGCUCUGGGCAAAGACA
522





S70-AS523-M1
UUUGCCCAGAGCAUCCCGUUGAACC
70
GGUUCAACGGGAUGCUCUGGGCAAAGA
523





S71-AS524-M1
AGAGCAUCCCGUGGAACCUUGAGCG
71
CGCUCAAGGUUCCACGGGAUGCUCUGG
524





S72-AS525-M1
GAGCAUCCCGUGGAACCUGUAGCGG
72
CCGCUACAGGUUCCACGGGAUGCUCUG
525





S73-AS526-M1
AGCAUCCCGUGGAACCUGGAGCGGA
73
UCCGCUCCAGGUUCCACGGGAUGCUCU
526





S74-AS527-M1
GCAUCCCGUGGAACCUGGAUCGGAT
74
AUCCGAUCCAGGUUCCACGGGAUGCUC
527





S75-AS528-M1
CAUCCCGUGGAACCUGGAGUGGATT
75
AAUCCACUCCAGGUUCCACGGGAUGCU
528





S76-AS529-M1
AUCCCGUGGAACCUGGAGCUGAUTA
76
UAAUCAGCUCCAGGUUCCACGGGAUGC
529





S77-AS530-M1
UCCCGUGGAACCUGGAGCGUAUUAC
77
GUAAUACGCUCCAGGUUCCACGGGAUG
530





S78-AS531-M1
CCCGUGGAACCUGGAGCGGAUUACC
78
GGUAAUCCGCUCCAGGUUCCACGGGAU
531





S79-AS532-M1
CCGUGGAACCUGGAGCGGAUUACCC
79
GGGUAAUCCGCUCCAGGUUCCACGGGA
532





S80-AS533-M1
CUGGAGCGGAUUACCCCUCUACGGT
80
ACCGUAGAGGGGUAAUCCGCUCCAGGU
533





S81-AS534-M1
UGGAGCGGAUUACCCCUCCACGGTA
81
UACCGUGGAGGGGUAAUCCGCUCCAGG
534





S82-AS535-M1
GGAGCGGAUUACCCCUCCAUGGUAC
82
GUACCAUGGAGGGGUAAUCCGCUCCAG
535





S83-AS536-M1
GAGCGGAUUACCCCUCCACUGUACC
83
GGUACAGUGGAGGGGUAAUCCGCUCCA
536





S84-AS537-M1
AGCGGAUUACCCCUCCACGUUACCG
84
CGGUAACGUGGAGGGGUAAUCCGCUCC
537





S85-AS538-M1
CGGAUUACCCCUCCACGGUACCGGG
85
CCCGGUACCGUGGAGGGGUAAUCCGCU
538





S86-AS539-M1
GGAUUACCCCUCCACGGUAUCGGGC
86
GCCCGAUACCGUGGAGGGGUAAUCCGC
539





S87-AS540-M1
UCCACGGUACCGGGCGGAUUAAUAC
87
GUAUUAAUCCGCCCGGUACCGUGGAGG
540





S88-AS541-M1
CGGAGGCAGCCUGGUGGAGUUGUAT
88
AUACAACUCCACCAGGCUGCCUCCGUC
541





S89-AS542-M1
AGACACCAGCAUACAGAGUUACCAC
89
GUGGUAACUCUGUAUGCUGGUGUCUAG
542





S90-AS543-M1
GCAUACAGAGUGACCACCGUGAAAT
90
AUUUCACGGUGGUCACUCUGUAUGCUG
543





S91-AS544-M1
CGAGAAUGUGCCCGAGGAGUACGGG
91
CCCGUACUCCUCGGGCACAUUCUCGAA
544





S92-AS545-M1
GAGAAUGUGCCCGAGGAGGACGGGA
92
UCCCGUCCUCCUCGGGCACAUUCUCGA
545





S93-AS546-M1
AGAAUGUGCCCGAGGAGGAUGGGAC
93
GUCCCAUCCUCCUCGGGCACAUUCUCG
546





S94-AS547-M1
GCAAGUGUGACAGUCAUGGUACCCA
94
UGGGUACCAUGACUGUCACACUUGCUG
547





S95-AS548-M1
CAAGUGUGACAGUCAUGGCACCCAC
95
GUGGGUGCCAUGACUGUCACACUUGCU
548





S96-AS549-M1
AAGUGUGACAGUCAUGGCAUCCACC
96
GGUGGAUGCCAUGACUGUCACACUUGC
549





S97-AS550-M1
CGCAGCCUGCGCGUGCUCAACUGCC
97
GGCAGUUGAGCACGCGCAGGCUGCGCA
550





S98-AS551-M1
GCAGCCUGCGCGUGCUCAAUUGCCA
98
UGGCAAUUGAGCACGCGCAGGCUGCGC
551





S99-AS552-M1
AGCCUGUGGGGCCACUGGUUGUGCT
99
AGCACAACCAGUGGCCCCACAGGCUGG
552





S100-AS553-M1
CCUCUACUCCCCAGCCUCAUCUCCC
100
GGGAGAUGAGGCUGGGGAGUAGAGGCA
553





S101-AS554-M1
CAGCCUCAGCUCCCGAGGUUAUCAC
101
GUGAUAACCUCGGGAGCUGAGGCUGGG
554





S102-AS555-M1
GCCACCAAUGCCCAAGACCAGCCGG
102
CCGGCUGGUCUUGGGCAUUGGUGGCCC
555





S103-AS556-M1
AUGCCCAAGACCAGCCGGUUACCCT
103
AGGGUAACCGGCUGGUCUUGGGCAUUG
556





S104-AS557-M1
UGCCCAAGACCAGCCGGUGACCCTG
104
CAGGGUCACCGGCUGGUCUUGGGCAUU
557





S105-AS558-M1
GUCACAGAGUGGGACAUCAUAGGCT
105
AGCCUAUGAUGUCCCACUCUGUGACAC
558





S106-AS559-M1
GAGUGGGACAUCACAGGCUUCUGCC
106
GGCAGAAGCCUGUGAUGUCCCACUCUG
559





S107-AS560-M1
UGGGACAUCACAGGCUGCUUCCCAC
107
GUGGGAAGCAGCCUGUGAUGUCCCACU
560





S108-AS561-M1
GGGACAUCACAGGCUGCUGUCCACG
108
CGUGGACAGCAGCCUGUGAUGUCCCAC
561





S109-AS562-M1
CUCACCCUGGCCGAGUUGAUGCAGA
109
UCUGCAUCAACUCGGCCAGGGUGAGCU
562





S110-AS563-M1
ACCCUGGCCGAGUUGAGGCAGAGAC
110
GUCUCUGCCUCAACUCGGCCAGGGUGA
563





S111-AS564-M1
ACUUCUCUGCCAAAGAUGUUAUCAA
111
UUGAUAACAUCUUUGGCAGAGAAGUGG
564





S112-AS565-M1
CCCAUGGGGCAGGUUGGCAUCUGTT
112
AACAGAUGCCAACCUGCCCCAUGGGUG
565





S113-AS566-M1
UGGGGCAGGUUGGCAGCUGUUUUGC
113
GCAAAACAGCUGCCAACCUGCCCCAUG
566





S114-AS567-M1
CUGUUUUGCAGGACUGUAUUGUCAG
114
CUGACAAUACAGUCCUGCAAAACAGCU
567





S115-AS568-M1
UUUUGCAGGACUGUAUGGUUAGCAC
115
GUGCUAACCAUACAGUCCUGCAAAACA
568





S116-AS569-M1
CAGGACUGUAUGGUCAGCAUACUCG
116
CGAGUAUGCUGACCAUACAGUCCUGCA
569





S117-AS570-M1
GGACUGUAUGGUCAGCACAUUCGGG
117
CCCGAAUGUGCUGACCAUACAGUCCUG
570





S118-AS571-M1
CGCUGCGCCCCAGAUGAGGAGCUGC
118
GCAGCUCCUCAUCUGGGGCGCAGCGGG
571





S119-AS572-M1
GCGCCCCAGAUGAGGAGCUUCUGAG
119
CUCAGAAGCUCCUCAUCUGGGGCGCAG
572





S120-AS573-M1
CCCCAGAUGAGGAGCUGCUUAGCTG
120
CAGCUAAGCAGCUCCUCAUCUGGGGCG
573





S121-AS574-M1
CCCAGAUGAGGAGCUGCUGAGCUGC
121
GCAGCUCAGCAGCUCCUCAUCUGGGGC
574





S122-AS575-M1
CCAGAUGAGGAGCUGCUGAUCUGCT
122
AGCAGAUCAGCAGCUCCUCAUCUGGGG
575





S123-AS576-M1
CGGCGGGGCGAGCGCAUGGAGGCCC
123
GGGCCUCCAUGCGCUCGCCCCGCCGCU
576





S124-AS577-M1
GGCGGGGCGAGCGCAUGGAUGCCCA
124
UGGGCAUCCAUGCGCUCGCCCCGCCGC
577





S125-AS578-M1
GGCGAGCGCAUGGAGGCCCAAGGGG
125
CCCCUUGGGCCUCCAUGCGCUCGCCCC
578





S126-AS579-M1
CUGGUCUGCCGGGCCCACAACGCTT
126
AAGCGUUGUGGGCCCGGCAGACCAGCU
579





S127-AS580-M1
UGCCUGCUACCCCAGGCCAACUGCA
127
UGCAGUUGGCCUGGGGUAGCAGGCAGC
580





S128-AS581-M1
GCCUGCUACCCCAGGCCAAUUGCAG
128
CUGCAAUUGGCCUGGGGUAGCAGGCAG
581





S129-AS582-M1
CCCAGGCCAACUGCAGCGUUCACAC
129
GUGUGAACGCUGCAGUUGGCCUGGGGU
582





S130-AS583-M1
GGCCCCUCAGGAGCAGGUGACCGTG
130
CACGGUCACCUGCUCCUGAGGGGCCGG
583





S131-AS584-M1
UGACCGUGGCCUGCGAGGAUGGCTG
131
CAGCCAUCCUCGCAGGCCACGGUCACC
584





S132-AS585-M1
GCGAGGAGGGCUGGACCCUUACUGG
132
CCAGUAAGGGUCCAGCCCUCCUCGCAG
585





S133-AS586-M1
CGAGGAGGGCUGGACCCUGACUGGC
133
GCCAGUCAGGGUCCAGCCCUCCUCGCA
586





S134-AS587-M1
GGGCUGGACCCUGACUGGCUGCAGT
134
ACUGCAGCCAGUCAGGGUCCAGCCCUC
587





S135-AS588-M1
GGCUGGACCCUGACUGGCUUCAGTG
135
CACUGAAGCCAGUCAGGGUCCAGCCCU
588





S136-AS589-M1
UGGACCCUGACUGGCUGCAUUGCCC
136
GGGCAAUGCAGCCAGUCAGGGUCCAGC
589





S137-AS590-M1
GGCUGCAGUGCCCUCCCUGUGACCT
137
AGGUCACAGGGAGGGCACUGCAGCCAG
590





S138-AS591-M1
UCCCUGGGACCUCCCACGUUCUGGG
138
CCCAGAACGUGGGAGGUCCCAGGGAGG
591





S139-AS592-M1
CCCUGGGACCUCCCACGUCUUGGGG
139
CCCCAAGACGUGGGAGGUCCCAGGGAG
592





S140-AS593-M1
GGGCCUACGCCGUAGACAAUACGTG
140
CACGUAUUGUCUACGGCGUAGGCCCCC
593





S141-AS594-M1
GACGUCAGCACUACAGGCAUCACCA
141
UGGUGAUGCCUGUAGUGCUGACGUCCC
594





S142-AS595-M1
CAGCACUACAGGCAGCACCAGCGAA
142
UUCGCUGGUGCUGCCUGUAGUGCUGAC
595





S143-AS596-M1
AGCACUACAGGCAGCACCAUCGAAG
143
CUUCGAUGGUGCUGCCUGUAGUGCUGA
596





S144-AS597-M1
GCACUACAGGCAGCACCAGUGAAGG
144
CCUUCACUGGUGCUGCCUGUAGUGCUG
597





S145-AS598-M1
GGGGCCGUGACAGCCGUUGUCAUCT
145
AGAUGACAACGGCUGUCACGGCCCCUU
598





S146-AS599-M1
GGAGCUCCAGUGACAGCCCUAUCCC
146
GGGAUAGGGCUGUCACUGGAGCUCCUG
599





S147-AS600-M1
AGGAUGGGUGUCUGGGGAGUGUCAA
147
UUGACACUCCCCAGACACCCAUCCUGG
600





S148-AS601-M1
UGGGUGUCUGGGGAGGGUCAAGGGC
148
GCCCUUGACCCUCCCCAGACACCCAUC
601





S149-AS602-M1
GGGUGUCUGGGGAGGGUCAAGGGCT
149
AGCCCUUGACCCUCCCCAGACACCCAU
602





S150-AS603-M1
GGUGUCUGGGGAGGGUCAAUGGCTG
150
CAGCCAUUGACCCUCCCCAGACACCCA
603





S151-AS604-M1
AGGGUCAAGGGCUGGGGCUUAGCTT
151
AAGCUAAGCCCCAGCCCUUGACCCUCC
604





S152-AS605-M1
GGGUCAAGGGCUGGGGCUGAGCUTT
152
AAAGCUCAGCCCCAGCCCUUGACCCUC
605





S153-AS606-M1
GACUUGUCCCUCUCUCAGCUCUCCA
153
UGGAGAGCUGAGAGAGGGACAAGUCGG
606





S154-AS607-M1
ACUUGUCCCUCUCUCAGCCUUCCAT
154
AUGGAAGGCUGAGAGAGGGACAAGUCG
607





S155-AS608-M1
CUUGUCCCUCUCUCAGCCCUCCATG
155
CAUGGAGGGCUGAGAGAGGGACAAGUC
608





S156-AS609-M1
UUGUCCCUCUCUCAGCCCUUCAUGG
156
CCAUGAAGGGCUGAGAGAGGGACAAGU
609





S157-AS610-M1
UCCCUCUCUCAGCCCUCCAUGGCCT
157
AGGCCAUGGAGGGCUGAGAGAGGGACA
610





S158-AS611-M1
UGGCCUGGCACGAGGGGAUUGGGAT
158
AUCCCAAUCCCCUCGUGCCAGGCCAUG
611





S159-AS612-M1
UGGCACGAGGGGAUGGGGAUGCUTC
159
GAAGCAUCCCCAUCCCCUCGUGCCAGG
612





S160-AS613-M1
CGAGGGGAUGGGGAUGCUUUCGCCT
160
AGGCGAAAGCAUCCCCAUCCCCUCGUG
613





S161-AS614-M1
GAGGGGAUGGGGAUGCUUCUGCCTT
161
AAGGCAGAAGCAUCCCCAUCCCCUCGU
614





S162-AS615-M1
GGGAUGGGGAUGCUUCCGCUUUUCC
162
GGAAAAGCGGAAGCAUCCCCAUCCCCU
615





S163-AS616-M1
AUGGGGAUGCUUCCGCCUUUCCGGG
163
CCCGGAAAGGCGGAAGCAUCCCCAUCC
616





S164-AS617-M1
UGGGGAUGCUUCCGCCUUUUCGGGG
164
CCCCGAAAAGGCGGAAGCAUCCCCAUC
617





S165-AS618-M1
GGGGAUGCUUCCGCCUUUCUGGGGC
165
GCCCCAGAAAGGCGGAAGCAUCCCCAU
618





S166-AS619-M1
GGGAUGCUUCCGCCUUUCCUGGGCT
166
AGCCCAGGAAAGGCGGAAGCAUCCCCA
619





S167-AS620-M1
CCCUUGAGUGGGGCAGCCUUCUUGC
167
GCAAGAAGGCUGCCCCACUCAAGGGCC
620





S168-AS621-M1
UGAGUGGGGCAGCCUCCUUUCCUGG
168
CCAGGAAAGGAGGCUGCCCCACUCAAG
621





S169-AS622-M1
GGGGCAGCCUCCUUGCCUGUAACTC
169
GAGUUACAGGCAAGGAGGCUGCCCCAC
622





S170-AS623-M1
GGCAGCCUCCUUGCCUGGAACUCAC
170
GUGAGUUCCAGGCAAGGAGGCUGCCCC
623





S171-AS624-M1
GCAGCCUCCUUGCCUGGAAUUCACT
171
AGUGAAUUCCAGGCAAGGAGGCUGCCC
624





S172-AS625-M1
AGCCUCCUUGCCUGGAACUUACUCA
172
UGAGUAAGUUCCAGGCAAGGAGGCUGC
625





S173-AS626-M1
GCCUCCUUGCCUGGAACUCACUCAC
173
GUGAGUGAGUUCCAGGCAAGGAGGCUG
626





S174-AS627-M1
CCUCCUUGCCUGGAACUCAUUCACT
174
AGUGAAUGAGUUCCAGGCAAGGAGGCU
627





S175-AS628-M1
CUCCUUGCCUGGAACUCACUCACTC
175
GAGUGAGUGAGUUCCAGGCAAGGAGGC
628





S176-AS629-M1
UCCUUGCCUGGAACUCACUUACUCT
176
AGAGUAAGUGAGUUCCAGGCAAGGAGG
629





S177-AS630-M1
CCUUGCCUGGAACUCACUCACUCTG
177
CAGAGUGAGUGAGUUCCAGGCAAGGAG
630





S178-AS631-M1
CUUGCCUGGAACUCACUCAUUCUGG
178
CCAGAAUGAGUGAGUUCCAGGCAAGGA
631





S179-AS632-M1
UUGCCUGGAACUCACUCACUCUGGG
179
CCCAGAGUGAGUGAGUUCCAGGCAAGG
632





S180-AS633-M1
UGCCUGGAACUCACUCACUUUGGGT
180
ACCCAAAGUGAGUGAGUUCCAGGCAAG
633





S181-AS634-M1
UCUGGGUGCCUCCUCCCCAUGUGGA
181
UCCACAUGGGGAGGAGGCACCCAGAGU
634





S182-AS635-M1
CCCAGGUGGAGGUGCCAGGAAGCTC
182
GAGCUUCCUGGCACCUCCACCUGGGGA
635





S183-AS636-M1
CCAGGAAGCUCCCUCCCUCACUGTG
183
CACAGUGAGGGAGGGAGCUUCCUGGCA
636





S184-AS637-M1
GGAAGCUCCCUCCCUCACUUUGGGG
184
CCCCAAAGUGAGGGAGGGAGCUUCCUG
637





S185-AS638-M1
AGCUCCCUCCCUCACUGUGUGGCAT
185
AUGCCACACAGUGAGGGAGGGAGCUUC
638





S186-AS639-M1
GCUCCCUCCCUCACUGUGGUGCATT
186
AAUGCACCACAGUGAGGGAGGGAGCUU
639





S187-AS640-M1
GGGGCAUUUCACCAUUCAAACAGGT
187
ACCUGUUUGAAUGGUGAAAUGCCCCAC
640





S188-AS641-M1
GGGCAUUUCACCAUUCAAAUAGGTC
188
GACCUAUUUGAAUGGUGAAAUGCCCCA
641





S189-AS642-M1
CACCAUUCAAACAGGUCGAUCUGTG
189
CACAGAUCGACCUGUUUGAAUGGUGAA
642





S190-AS643-M1
ACCAUUCAAACAGGUCGAGUUGUGC
190
GCACAACUCGACCUGUUUGAAUGGUGA
643





S191-AS644-M1
UGCUCGGGUGCUGCCAGCUUCUCCC
191
GGGAGAAGCUGGCAGCACCCGAGCACA
644





S192-AS645-M1
CGGGUGCUGCCAGCUGCUCUCAATG
192
CAUUGAGAGCAGCUGGCAGCACCCGAG
645





S193-AS646-M1
GGGUGCUGCCAGCUGCUCCUAAUGT
193
ACAUUAGGAGCAGCUGGCAGCACCCGA
646





S194-AS647-M1
GCCAGCUGCUCCCAAUGUGUCGATG
194
CAUCGACACAUUGGGAGCAGCUGGCAG
647





S195-AS648-M1
CCAGCUGCUCCCAAUGUGCUGAUGT
195
ACAUCAGCACAUUGGGAGCAGCUGGCA
648





S196-AS649-M1
UGCCGAUGUCCGUGGGCAGAAUGAC
196
GUCAUUCUGCCCACGGACAUCGGCACA
649





S197-AS650-M1
GCAGAAUGACUUUUAUUGAUCUCTT
197
AAGAGAUCAAUAAAAGUCAUUCUGCCC
650





S198-AS651-M1
CAGAAUGACUUUUAUUGAGUUCUTG
198
CAAGAACUCAAUAAAAGUCAUUCUGCC
651





S199-AS652-M1
AGAAUGACUUUUAUUGAGCUCUUGT
199
ACAAGAGCUCAAUAAAAGUCAUUCUGC
652





S200-AS653-M1
GAAUGACUUUUAUUGAGCUUUUGTT
200
AACAAAAGCUCAAUAAAAGUCAUUCUG
653





S201-AS654-M1
AAUGACUUUUAUUGAGCUCUUGUTC
201
GAACAAGAGCUCAAUAAAAGUCAUUCU
654





S202-AS655-M1
AUGACUUUUAUUGAGCUCUUGUUCC
202
GGAACAAGAGCUCAAUAAAAGUCAUUC
655





S203-AS656-M1
UGACUUUUAUUGAGCUCUUUUUCCG
203
CGGAAAAAGAGCUCAAUAAAAGUCAUU
656





S204-AS657-M1
CUUGUUCCGUGCCAGGCAUUCAATC
204
GAUUGAAUGCCUGGCACGGAACAAGAG
657





S205-AS658-M1
CCAGGCAUUCAAUCCUCAGUUCUCC
205
GGAGAACUGAGGAUUGAAUGCCUGGCA
658





S206-AS659-M1
CAUUCAAUCCUCAGGUCUCUACCAA
206
UUGGUAGAGACCUGAGGAUUGAAUGCC
659





S207-AS660-M1
AUUCAAUCCUCAGGUCUCCACCAAG
207
CUUGGUGGAGACCUGAGGAUUGAAUGC
660





S208-AS661-M1
UUCAAUCCUCAGGUCUCCAUCAAGG
208
CCUUGAUGGAGACCUGAGGAUUGAAUG
661





S209-AS662-M1
CCUCAGGUCUCCACCAAGGAGGCAG
209
CUGCCUCCUUGGUGGAGACCUGAGGAU
662





S210-AS663-M1
CUCAGGUCUCCACCAAGGAUGCAGG
210
CCUGCAUCCUUGGUGGAGACCUGAGGA
663





S211-AS664-M1
GCGGUAGGGGCUGCAGGGAUAAACA
211
UGUUUAUCCCUGCAGCCCCUACCGCCC
664





S212-AS665-M1
CGGUAGGGGCUGCAGGGACAAACAT
212
AUGUUUGUCCCUGCAGCCCCUACCGCC
665





S213-AS666-M1
GGUAGGGGCUGCAGGGACAAACATC
213
GAUGUUUGUCCCUGCAGCCCCUACCGC
666





S214-AS667-M1
UAGGGGCUGCAGGGACAAAUAUCGT
214
ACGAUAUUUGUCCCUGCAGCCCCUACC
667





S215-AS668-M1
AGGGGCUGCAGGGACAAACAUCGTT
215
AACGAUGUUUGUCCCUGCAGCCCCUAC
668





S216-AS669-M1
GGGGCUGCAGGGACAAACAUCGUTG
216
CAACGAUGUUUGUCCCUGCAGCCCCUA
669





S217-AS670-M1
GGGCUGCAGGGACAAACAUUGUUGG
217
CCAACAAUGUUUGUCCCUGCAGCCCCU
670





S218-AS671-M1
GGCUGCAGGGACAAACAUCUUUGGG
218
CCCAAAGAUGUUUGUCCCUGCAGCCCC
671





S219-AS672-M1
GGGGUGAGUGUGAAAGGUGUUGATG
219
CAUCAACACCUUUCACACUCACCCCCC
672





S220-AS673-M1
GGGUGAGUGUGAAAGGUGCUGAUGG
220
CCAUCAGCACCUUUCACACUCACCCCC
673





S221-AS674-M1
GGUGAGUGUGAAAGGUGCUUAUGGC
221
GCCAUAAGCACCUUUCACACUCACCCC
674





S222-AS675-M1
GUGAGUGUGAAAGGUGCUGAUGGCC
222
GGCCAUCAGCACCUUUCACACUCACCC
675





S223-AS676-M1
UGAGUGUGAAAGGUGCUGAUGGCCC
223
GGGCCAUCAGCACCUUUCACACUCACC
676





S224-AS677-M1
GAGUGUGAAAGGUGCUGAUUGCCCT
224
AGGGCAAUCAGCACCUUUCACACUCAC
677





S225-AS678-M1
AGUGUGAAAGGUGCUGAUGUCCCTC
225
GAGGGACAUCAGCACCUUUCACACUCA
678





S226-AS679-M1
GUGUGAAAGGUGCUGAUGGUCCUCA
226
UGAGGACCAUCAGCACCUUUCACACUC
679





S227-AS680-M1
UGUGAAAGGUGCUGAUGGCUCUCAT
227
AUGAGAGCCAUCAGCACCUUUCACACU
680





S228-AS681-M1
GUGAAAGGUGCUGAUGGCCUUCATC
228
GAUGAAGGCCAUCAGCACCUUUCACAC
681





S229-AS682-M1
UGAAAGGUGCUGAUGGCCCUCAUCT
229
AGAUGAGGGCCAUCAGCACCUUUCACA
682





S230-AS683-M1
GAAAGGUGCUGAUGGCCCUUAUCTC
230
GAGAUAAGGGCCAUCAGCACCUUUCAC
683





S231-AS684-M1
CUCAUCUCCAGCUAACUGUUGAGAA
231
UUCUCAACAGUUAGCUGGAGAUGAGGG
684





S232-AS685-M1
CCAGCUAACUGUGGAGAAGUCCCTG
232
CAGGGACUUCUCCACAGUUAGCUGGAG
685





S233-AS686-M1
CAGCUAACUGUGGAGAAGCUCCUGG
233
CCAGGAGCUUCUCCACAGUUAGCUGGA
686





S234-AS687-M1
AGCUAACUGUGGAGAAGCCUCUGGG
234
CCCAGAGGCUUCUCCACAGUUAGCUGG
687





S235-AS688-M1
GCUAACUGUGGAGAAGCCCUUGGGG
235
CCCCAAGGGCUUCUCCACAGUUAGCUG
688





S236-AS689-M1
GGGCUCCCUGAUUAAUGGAUGCUTA
236
UAAGCAUCCAUUAAUCAGGGAGCCCCC
689





S237-AS690-M1
AUGGAGGCUUAGCUUUCUGUAUGGC
237
GCCAUACAGAAAGCUAAGCCUCCAUUA
690





S238-AS691-M1
UGGAGGCUUAGCUUUCUGGAUGGCA
238
UGCCAUCCAGAAAGCUAAGCCUCCAUU
691





S239-AS692-M1
GGAGGCUUAGCUUUCUGGAUGGCAT
239
AUGCCAUCCAGAAAGCUAAGCCUCCAU
692





S240-AS693-M1
GAGGCUUAGCUUUCUGGAUUGCATC
240
GAUGCAAUCCAGAAAGCUAAGCCUCCA
693





S241-AS694-M1
AGGCUUAGCUUUCUGGAUGUCAUCT
241
AGAUGACAUCCAGAAAGCUAAGCCUCC
694





S242-AS695-M1
GGCUUAGCUUUCUGGAUGGUAUCTA
242
UAGAUACCAUCCAGAAAGCUAAGCCUC
695





S243-AS696-M1
GCUUAGCUUUCUGGAUGGCAUCUAG
243
CUAGAUGCCAUCCAGAAAGCUAAGCCU
696





S244-AS697-M1
GACAGGUGCGCCCCUGGUGUUCACA
244
UGUGAACACCAGGGGCGCACCUGUCUC
697





S245-AS698-M1
GCGCCCCUGGUGGUCACAGUCUGTG
245
CACAGACUGUGACCACCAGGGGCGCAC
698





S246-AS699-M1
CCCCUGGUGGUCACAGGCUUUGCCT
246
AGGCAAAGCCUGUGACCACCAGGGGCG
699





S247-AS700-M1
CCCUGGUGGUCACAGGCUGUGCCTT
247
AAGGCACAGCCUGUGACCACCAGGGGC
700





S248-AS701-M1
GUGGUCACAGGCUGUGCCUUGGUTT
248
AAACCAAGGCACAGCCUGUGACCACCA
701





S249-AS702-M1
UGGUCACAGGCUGUGCCUUUGUUTC
249
GAAACAAAGGCACAGCCUGUGACCACC
702





S250-AS703-M1
GGUCACAGGCUGUGCCUUGUUUUCC
250
GGAAAACAAGGCACAGCCUGUGACCAC
703





S251-AS704-M1
GUCACAGGCUGUGCCUUGGUUUCCT
251
AGGAAACCAAGGCACAGCCUGUGACCA
704





S252-AS705-M1
GGCUGUGCCUUGGUUUCCUUAGCCA
252
UGGCUAAGGAAACCAAGGCACAGCCUG
705





S253-AS706-M1
GCUGUGCCUUGGUUUCCUGAGCCAC
253
GUGGCUCAGGAAACCAAGGCACAGCCU
706





S254-AS707-M1
CUGUGCCUUGGUUUCCUGAUCCACC
254
GGUGGAUCAGGAAACCAAGGCACAGCC
707





S255-AS708-M1
UGUGCCUUGGUUUCCUGAGUCACCT
255
AGGUGACUCAGGAAACCAAGGCACAGC
708





S256-AS709-M1
GUGCCUUGGUUUCCUGAGCUACCTT
256
AAGGUAGCUCAGGAAACCAAGGCACAG
709





S257-AS710-M1
UGCCUUGGUUUCCUGAGCCACCUTT
257
AAAGGUGGCUCAGGAAACCAAGGCACA
710





S258-AS711-M1
GCCUUGGUUUCCUGAGCCAUCUUTA
258
UAAAGAUGGCUCAGGAAACCAAGGCAC
711





S259-AS712-M1
CCUUGGUUUCCUGAGCCACUUUUAC
259
GUAAAAGUGGCUCAGGAAACCAAGGCA
712





S260-AS713-M1
CUUGGUUUCCUGAGCCACCUUUACT
260
AGUAAAGGUGGCUCAGGAAACCAAGGC
713





S261-AS714-M1
UUGGUUUCCUGAGCCACCUUUACTC
261
GAGUAAAGGUGGCUCAGGAAACCAAGG
714





S262-AS715-M1
UGGUUUCCUGAGCCACCUUUACUCT
262
AGAGUAAAGGUGGCUCAGGAAACCAAG
715





S263-AS716-M1
GGUUUCCUGAGCCACCUUUACUCTG
263
CAGAGUAAAGGUGGCUCAGGAAACCAA
716





S264-AS717-M1
GUUUCCUGAGCCACCUUUAUUCUGC
264
GCAGAAUAAAGGUGGCUCAGGAAACCA
717





S265-AS718-M1
CUGAGCCACCUUUACUCUGUUCUAT
265
AUAGAACAGAGUAAAGGUGGCUCAGGA
718





S266-AS719-M1
CCAGGCUGUGCUAGCAACAUCCAAA
266
UUUGGAUGUUGCUAGCACAGCCUGGCA
719





S267-AS720-M1
CUGCGGGGAGCCAUCACCUAGGACT
267
AGUCCUAGGUGAUGGCUCCCCGCAGGC
720





S268-AS721-M1
UGCGGGGAGCCAUCACCUAUGACTG
268
CAGUCAUAGGUGAUGGCUCCCCGCAGG
721





S269-AS722-M1
GCGGGGAGCCAUCACCUAGUACUGA
269
UCAGUACUAGGUGAUGGCUCCCCGCAG
722





S270-AS723-M1
CGGGGAGCCAUCACCUAGGACUGAC
270
GUCAGUCCUAGGUGAUGGCUCCCCGCA
723





S271-AS724-M1
GGGGAGCCAUCACCUAGGAUUGACT
271
AGUCAAUCCUAGGUGAUGGCUCCCCGC
724





S272-AS725-M1
GCCAUCACCUAGGACUGACUCGGCA
272
UGCCGAGUCAGUCCUAGGUGAUGGCUC
725





S273-AS726-M1
CCAUCACCUAGGACUGACUUGGCAG
273
CUGCCAAGUCAGUCCUAGGUGAUGGCU
726





S274-AS727-M1
CAUCACCUAGGACUGACUCUGCAGT
274
ACUGCAGAGUCAGUCCUAGGUGAUGGC
727





S275-AS728-M1
CUAGGACUGACUCGGCAGUUUGCAG
275
CUGCAAACUGCCGAGUCAGUCCUAGGU
728





S276-AS729-M1
UGACUCGGCAGUGUGCAGUUGUGCA
276
UGCACAACUGCACACUGCCGAGUCAGU
729





S277-AS730-M1
GACUCGGCAGUGUGCAGUGUUGCAT
277
AUGCAACACUGCACACUGCCGAGUCAG
730





S278-AS731-M1
CUCGGCAGUGUGCAGUGGUUCAUGC
278
GCAUGAACCACUGCACACUGCCGAGUC
731





S279-AS732-M1
UCGGCAGUGUGCAGUGGUGUAUGCA
279
UGCAUACACCACUGCACACUGCCGAGU
732





S280-AS733-M1
CGGCAGUGUGCAGUGGUGCAUGCAC
280
GUGCAUGCACCACUGCACACUGCCGAG
733





S281-AS734-M1
GUGUGCAGUGGUGCAUGCAUUGUCT
281
AGACAAUGCAUGCACCACUGCACACUG
734





S282-AS735-M1
UGUGCAGUGGUGCAUGCACUGUCTC
282
GAGACAGUGCAUGCACCACUGCACACU
735





S283-AS736-M1
GUGCAGUGGUGCAUGCACUUUCUCA
283
UGAGAAAGUGCAUGCACCACUGCACAC
736





S284-AS737-M1
UGCAGUGGUGCAUGCACUGUCUCAG
284
CUGAGACAGUGCAUGCACCACUGCACA
737





S285-AS738-M1
GCAGUGGUGCAUGCACUGUUUCAGC
285
GCUGAAACAGUGCAUGCACCACUGCAC
738





S286-AS739-M1
CAGUGGUGCAUGCACUGUCUCAGCC
286
GGCUGAGACAGUGCAUGCACCACUGCA
739





S287-AS740-M1
AGUGGUGCAUGCACUGUCUUAGCCA
287
UGGCUAAGACAGUGCAUGCACCACUGC
740





S288-AS741-M1
UGCAUGCACUGUCUCAGCCAACCCG
288
CGGGUUGGCUGAGACAGUGCAUGCACC
741





S289-AS742-M1
GCAUGCACUGUCUCAGCCAACCCGC
289
GCGGGUUGGCUGAGACAGUGCAUGCAC
742





S290-AS743-M1
CAUUCGCACCCCUACUUCAUAGAGG
290
CCUCUAUGAAGUAGGGGUGCGAAUGUG
743





S291-AS744-M1
AUUCGCACCCCUACUUCACAGAGGA
291
UCCUCUGUGAAGUAGGGGUGCGAAUGU
744





S292-AS745-M1
UUCGCACCCCUACUUCACAUAGGAA
292
UUCCUAUGUGAAGUAGGGGUGCGAAUG
745





S293-AS746-M1
UCGCACCCCUACUUCACAGAGGAAG
293
CUUCCUCUGUGAAGUAGGGGUGCGAAU
746





S294-AS747-M1
CGCACCCCUACUUCACAGAUGAAGA
294
UCUUCAUCUGUGAAGUAGGGGUGCGAA
747





S295-AS748-M1
GCACCCCUACUUCACAGAGUAAGAA
295
UUCUUACUCUGUGAAGUAGGGGUGCGA
748





S296-AS749-M1
CACCCCUACUUCACAGAGGAAGAAA
296
UUUCUUCCUCUGUGAAGUAGGGGUGCG
749





S297-AS750-M1
ACCCCUACUUCACAGAGGAAGAAAC
297
GUUUCUUCCUCUGUGAAGUAGGGGUGC
750





S298-AS751-M1
CCCCUACUUCACAGAGGAAUAAACC
298
GGUUUAUUCCUCUGUGAAGUAGGGGUG
751





S299-AS752-M1
CCCUACUUCACAGAGGAAGAAACCT
299
AGGUUUCUUCCUCUGUGAAGUAGGGGU
752





S300-AS753-M1
CUUCACAGAGGAAGAAACCUGGAAC
300
GUUCCAGGUUUCUUCCUCUGUGAAGUA
753





S301-AS754-M1
UUCACAGAGGAAGAAACCUUGAACC
301
GGUUCAAGGUUUCUUCCUCUGUGAAGU
754





S302-AS755-M1
UCACAGAGGAAGAAACCUGUAACCA
302
UGGUUACAGGUUUCUUCCUCUGUGAAG
755





S303-AS756-M1
CACAGAGGAAGAAACCUGGAACCAG
303
CUGGUUCCAGGUUUCUUCCUCUGUGAA
756





S304-AS757-M1
ACAGAGGAAGAAACCUGGAACCAGA
304
UCUGGUUCCAGGUUUCUUCCUCUGUGA
757





S305-AS758-M1
CAGAGGAAGAAACCUGGAAUCAGAG
305
CUCUGAUUCCAGGUUUCUUCCUCUGUG
758





S306-AS759-M1
AGAGGAAGAAACCUGGAACUAGAGG
306
CCUCUAGUUCCAGGUUUCUUCCUCUGU
759





S307-AS760-M1
GAGGAAGAAACCUGGAACCAGAGGG
307
CCCUCUGGUUCCAGGUUUCUUCCUCUG
760





S308-AS761-M1
AGGAAGAAACCUGGAACCAUAGGGG
308
CCCCUAUGGUUCCAGGUUUCUUCCUCU
761





S309-AS762-M1
GCAGAUUGGGCUGGCUCUGAAGCCA
309
UGGCUUCAGAGCCAGCCCAAUCUGCGU
762





S310-AS763-M1
CAGAUUGGGCUGGCUCUGAAGCCAA
310
UUGGCUUCAGAGCCAGCCCAAUCUGCG
763





S311-AS764-M1
AGAUUGGGCUGGCUCUGAAUCCAAG
311
CUUGGAUUCAGAGCCAGCCCAAUCUGC
764





S312-AS765-M1
UGGGCUGGCUCUGAAGCCAAGCCTC
312
GAGGCUUGGCUUCAGAGCCAGCCCAAU
765





S313-AS766-M1
GGGCUGGCUCUGAAGCCAAUCCUCT
313
AGAGGAUUGGCUUCAGAGCCAGCCCAA
766





S314-AS767-M1
GAAGCCAAGCCUCUUCUUAUUUCAC
314
GUGAAAUAAGAAGAGGCUUGGCUUCAG
767





S315-AS768-M1
AAGCCUCUUCUUACUUCACUCGGCT
315
AGCCGAGUGAAGUAAGAAGAGGCUUGG
768





S316-AS769-M1
AGCCUCUUCUUACUUCACCUGGCTG
316
CAGCCAGGUGAAGUAAGAAGAGGCUUG
769





S317-AS770-M1
GCCUCUUCUUACUUCACCCUGCUGG
317
CCAGCAGGGUGAAGUAAGAAGAGGCUU
770





S318-AS771-M1
CCCGGCUGGGCUCCUCAUUUUUACG
318
CGUAAAAAUGAGGAGCCCAGCCGGGUG
771





S319-AS772-M1
CCGGCUGGGCUCCUCAUUUUUACGG
319
CCGUAAAAAUGAGGAGCCCAGCCGGGU
772





S320-AS773-M1
CGGCUGGGCUCCUCAUUUUUACGGG
320
CCCGUAAAAAUGAGGAGCCCAGCCGGG
773





S321-AS774-M1
GGCUGGGCUCCUCAUUUUUACGGGT
321
ACCCGUAAAAAUGAGGAGCCCAGCCGG
774





S322-AS775-M1
GCUGGGCUCCUCAUUUUUAUGGGTA
322
UACCCAUAAAAAUGAGGAGCCCAGCCG
775





S323-AS776-M1
ACGGGUAACAGUGAGGCUGUGAAGG
323
CCUUCACAGCCUCACUGUUACCCGUAA
776





S324-AS777-M1
AGCUCGGUGAGUGAUGGCAUAACGA
324
UCGUUAUGCCAUCACUCACCGAGCUUC
777





S325-AS778-M1
GCUCGGUGAGUGAUGGCAGAACGAT
325
AUCGUUCUGCCAUCACUCACCGAGCUU
778





S326-AS779-M1
CUCGGUGAGUGAUGGCAGAACGATG
326
CAUCGUUCUGCCAUCACUCACCGAGCU
779





S327-AS780-M1
UCGGUGAGUGAUGGCAGAAUGAUGC
327
GCAUCAUUCUGCCAUCACUCACCGAGC
780





S328-AS781-M1
CGGUGAGUGAUGGCAGAACUAUGCC
328
GGCAUAGUUCUGCCAUCACUCACCGAG
781





S329-AS782-M1
AUGCCUGCAGGCAUGGAACUUUUTC
329
GAAAAAGUUCCAUGCCUGCAGGCAUCG
782





S330-AS783-M1
UGCCUGCAGGCAUGGAACUUUUUCC
330
GGAAAAAGUUCCAUGCCUGCAGGCAUC
783





S331-AS784-M1
GCCUGCAGGCAUGGAACUUUUUCCG
331
CGGAAAAAGUUCCAUGCCUGCAGGCAU
784





S332-AS785-M1
CCUGCAGGCAUGGAACUUUUUCCGT
332
ACGGAAAAAGUUCCAUGCCUGCAGGCA
785





S333-AS786-M1
CUGCAGGCAUGGAACUUUUUCCGTT
333
AACGGAAAAAGUUCCAUGCCUGCAGGC
786





S334-AS787-M1
AUGGAACUUUUUCCGUUAUUACCCA
334
UGGGUAAUAACGGAAAAAGUUCCAUGC
787





S335-AS788-M1
UUUUUCCGUUAUCACCCAGUCCUGA
335
UCAGGACUGGGUGAUAACGGAAAAAGU
788





S336-AS789-M1
UUUUCCGUUAUCACCCAGGUCUGAT
336
AUCAGACCUGGGUGAUAACGGAAAAAG
789





S337-AS790-M1
UUUCCGUUAUCACCCAGGCUUGATT
337
AAUCAAGCCUGGGUGAUAACGGAAAAA
790





S338-AS791-M1
UUCCGUUAUCACCCAGGCCUGAUTC
338
GAAUCAGGCCUGGGUGAUAACGGAAAA
791





S339-AS792-M1
UCCGUUAUCACCCAGGCCUUAUUCA
339
UGAAUAAGGCCUGGGUGAUAACGGAAA
792





S340-AS793-M1
CCGUUAUCACCCAGGCCUGAUUCAC
340
GUGAAUCAGGCCUGGGUGAUAACGGAA
793





S341-AS794-M1
CGUUAUCACCCAGGCCUGAUUCACT
341
AGUGAAUCAGGCCUGGGUGAUAACGGA
794





S342-AS795-M1
CACCCAGGCCUGAUUCACUUGCCTG
342
CAGGCAAGUGAAUCAGGCCUGGGUGAU
795





S343-AS796-M1
ACCCAGGCCUGAUUCACUGUCCUGG
343
CCAGGACAGUGAAUCAGGCCUGGGUGA
796





S344-AS797-M1
UGGCCUGGCGGAGAUGCUUUUAAGG
344
CCUUAAAAGCAUCUCCGCCAGGCCAGU
797





S345-AS798-M1
GGCCUGGCGGAGAUGCUUCUAAGGC
345
GCCUUAGAAGCAUCUCCGCCAGGCCAG
798





S346-AS799-M1
GCCUGGCGGAGAUGCUUCUAAGGCA
346
UGCCUUAGAAGCAUCUCCGCCAGGCCA
799





S347-AS800-M1
CCUGGCGGAGAUGCUUCUAAGGCAT
347
AUGCCUUAGAAGCAUCUCCGCCAGGCC
800





S348-AS801-M1
CUGGCGGAGAUGCUUCUAAUGCATG
348
CAUGCAUUAGAAGCAUCUCCGCCAGGC
801





S349-AS802-M1
UGGCGGAGAUGCUUCUAAGUCAUGG
349
CCAUGACUUAGAAGCAUCUCCGCCAGG
802





S350-AS803-M1
GGCGGAGAUGCUUCUAAGGUAUGGT
350
ACCAUACCUUAGAAGCAUCUCCGCCAG
803





S351-AS804-M1
GCGGAGAUGCUUCUAAGGCAUGGTC
351
GACCAUGCCUUAGAAGCAUCUCCGCCA
804





S352-AS805-M1
CGGAGAUGCUUCUAAGGCAUGGUCG
352
CGACCAUGCCUUAGAAGCAUCUCCGCC
805





S353-AS806-M1
GGAGAUGCUUCUAAGGCAUUGUCGG
353
CCGACAAUGCCUUAGAAGCAUCUCCGC
806





S354-AS807-M1
GAGAUGCUUCUAAGGCAUGUUCGGG
354
CCCGAACAUGCCUUAGAAGCAUCUCCG
807





S355-AS808-M1
GGAGAGGGCCAACAACUGUUCCUCC
355
GGAGGAACAGUUGUUGGCCCUCUCCCC
808





S356-AS809-M1
GCCAACAACUGUCCCUCCUUGAGCA
356
UGCUCAAGGAGGGACAGUUGUUGGCCC
809





S357-AS810-M1
CCAACAACUGUCCCUCCUUUAGCAC
357
GUGCUAAAGGAGGGACAGUUGUUGGCC
810





S358-AS811-M1
UUGAGCACCAGCCCCACCCAAGCAA
358
UUGCUUGGGUGGGGCUGGUGCUCAAGG
811





S359-AS812-M1
UGAGCACCAGCCCCACCCAAGCAAG
359
CUUGCUUGGGUGGGGCUGGUGCUCAAG
812





S360-AS813-M1
GAGCACCAGCCCCACCCAAUCAAGC
360
GCUUGAUUGGGUGGGGCUGGUGCUCAA
813





S361-AS814-M1
AGCACCAGCCCCACCCAAGUAAGCA
361
UGCUUACUUGGGUGGGGCUGGUGCUCA
814





S362-AS815-M1
ACCCAAGCAAGCAGACAUUUAUCTT
362
AAGAUAAAUGUCUGCUUGCUUGGGUGG
815





S363-AS816-M1
CCCAAGCAAGCAGACAUUUAUCUTT
363
AAAGAUAAAUGUCUGCUUGCUUGGGUG
816





S364-AS817-M1
CCAAGCAAGCAGACAUUUAUCUUTT
364
AAAAGAUAAAUGUCUGCUUGCUUGGGU
817





S365-AS818-M1
CAAGCAAGCAGACAUUUAUUUUUTG
365
CAAAAAAUAAAUGUCUGCUUGCUUGGG
818





S366-AS819-M1
AAGCAAGCAGACAUUUAUCUUUUGG
366
CCAAAAGAUAAAUGUCUGCUUGCUUGG
819





S367-AS820-M1
AGCAAGCAGACAUUUAUCUUUUGGG
367
CCCAAAAGAUAAAUGUCUGCUUGCUUG
820





S368-AS821-M1
GCAAGCAGACAUUUAUCUUUUGGGT
368
ACCCAAAAGAUAAAUGUCUGCUUGCUU
821





S369-AS822-M1
AAGCAGACAUUUAUCUUUUUGGUCT
369
AGACCAAAAAGAUAAAUGUCUGCUUGC
822





S370-AS823-M1
AGCAGACAUUUAUCUUUUGUGUCTG
370
CAGACACAAAAGAUAAAUGUCUGCUUG
823





S371-AS824-M1
GCAGACAUUUAUCUUUUGGUUCUGT
371
ACAGAACCAAAAGAUAAAUGUCUGCUU
824





S372-AS825-M1
UGUUGCCUUUUUACAGCCAACUUTT
372
AAAAGUUGGCUGUAAAAAGGCAACAGA
825





S373-AS826-M1
GUUGCCUUUUUACAGCCAAUUUUTC
373
GAAAAAUUGGCUGUAAAAAGGCAACAG
826





S374-AS827-M1
UUUACAGCCAACUUUUCUAUACCTG
374
CAGGUAUAGAAAAGUUGGCUGUAAAAA
827





S375-AS828-M1
UUACAGCCAACUUUUCUAGACCUGT
375
ACAGGUCUAGAAAAGUUGGCUGUAAAA
828





S376-AS829-M1
UUUUCUAGACCUGUUUUGCUUUUGT
376
ACAAAAGCAAAACAGGUCUAGAAAAGU
829





S377-AS830-M1
UUUCUAGACCUGUUUUGCUUUUGTA
377
UACAAAAGCAAAACAGGUCUAGAAAAG
830





S378-AS831-M1
UUCUAGACCUGUUUUGCUUUUGUAA
378
UUACAAAAGCAAAACAGGUCUAGAAAA
831





S379-AS832-M1
UCUAGACCUGUUUUGCUUUUGUAAC
379
GUUACAAAAGCAAAACAGGUCUAGAAA
832





S380-AS833-M1
CUAGACCUGUUUUGCUUUUUUAACT
380
AGUUAAAAAAGCAAAACAGGUCUAGAA
833





S381-AS834-M1
UAGACCUGUUUUGCUUUUGUAACTT
381
AAGUUACAAAAGCAAAACAGGUCUAGA
834





S382-AS835-M1
AGACCUGUUUUGCUUUUGUAACUTG
382
CAAGUUACAAAAGCAAAACAGGUCUAG
835





S383-AS836-M1
GACCUGUUUUGCUUUUGUAACUUGA
383
UCAAGUUACAAAAGCAAAACAGGUCUA
836





S384-AS837-M1
ACCUGUUUUGCUUUUGUAAUUUGAA
384
UUCAAAUUACAAAAGCAAAACAGGUCU
837





S385-AS838-M1
CCUGUUUUGCUUUUGUAACUUGAAG
385
CUUCAAGUUACAAAAGCAAAACAGGUC
838





S386-AS839-M1
CUGUUUUGCUUUUGUAACUUGAAGA
386
UCUUCAAGUUACAAAAGCAAAACAGGU
839





S387-AS840-M1
UGUUUUGCUUUUGUAACUUUAAGAT
387
AUCUUAAAGUUACAAAAGCAAAACAGG
840





S388-AS841-M1
GUUUUGCUUUUGUAACUUGAAGATA
388
UAUCUUCAAGUUACAAAAGCAAAACAG
841





S389-AS842-M1
UUUUGCUUUUGUAACUUGAAGAUAT
389
AUAUCUUCAAGUUACAAAAGCAAAACA
842





S390-AS843-M1
UUUGCUUUUGUAACUUGAAUAUATT
390
AAUAUAUUCAAGUUACAAAAGCAAAAC
843





S391-AS844-M1
UUGCUUUUGUAACUUGAAGAUAUTT
391
AAAUAUCUUCAAGUUACAAAAGCAAAA
844





S392-AS845-M1
UGCUUUUGUAACUUGAAGAUAUUTA
392
UAAAUAUCUUCAAGUUACAAAAGCAAA
845





S393-AS846-M1
GCUUUUGUAACUUGAAGAUAUUUAT
393
AUAAAUAUCUUCAAGUUACAAAAGCAA
846





S394-AS847-M1
CUUUUGUAACUUGAAGAUAUUUATT
394
AAUAAAUAUCUUCAAGUUACAAAAGCA
847





S395-AS848-M1
UUUUGUAACUUGAAGAUAUUUAUTC
395
GAAUAAAUAUCUUCAAGUUACAAAAGC
848





S396-AS849-M1
UUUGUAACUUGAAGAUAUUUAUUCT
396
AGAAUAAAUAUCUUCAAGUUACAAAAG
849





S397-AS850-M1
UUGUAACUUGAAGAUAUUUAUUCTG
397
CAGAAUAAAUAUCUUCAAGUUACAAAA
850





S398-AS851-M1
UGUAACUUGAAGAUAUUUAUUCUGG
398
CCAGAAUAAAUAUCUUCAAGUUACAAA
851





S399-AS852-M1
GUAACUUGAAGAUAUUUAUUCUGGG
399
CCCAGAAUAAAUAUCUUCAAGUUACAA
852





S400-AS853-M1
UAACUUGAAGAUAUUUAUUUUGGGT
400
ACCCAAAAUAAAUAUCUUCAAGUUACA
853





S401-AS854-M1
ACUUGAAGAUAUUUAUUCUUGGUTT
401
AAACCAAGAAUAAAUAUCUUCAAGUUA
854





S402-AS855-M1
CUUGAAGAUAUUUAUUCUGUGUUTT
402
AAAACACAGAAUAAAUAUCUUCAAGUU
855





S403-AS856-M1
UUGAAGAUAUUUAUUCUGGUUUUTG
403
CAAAAACCAGAAUAAAUAUCUUCAAGU
856





S404-AS857-M1
GAAGAUAUUUAUUCUGGGUUUUGTA
404
UACAAAACCCAGAAUAAAUAUCUUCAA
857





S405-AS858-M1
AAGAUAUUUAUUCUGGGUUUUGUAG
405
CUACAAAACCCAGAAUAAAUAUCUUCA
858





S406-AS859-M1
AUAUUUAUUCUGGGUUUUGUAGCAT
406
AUGCUACAAAACCCAGAAUAAAUAUCU
859





S407-AS860-M1
UAUUUAUUCUGGGUUUUGUAGCATT
407
AAUGCUACAAAACCCAGAAUAAAUAUC
860





S408-AS861-M1
AUUUAUUCUGGGUUUUGUAUCAUTT
408
AAAUGAUACAAAACCCAGAAUAAAUAU
861





S409-AS862-M1
UUUAUUCUGGGUUUUGUAGUAUUTT
409
AAAAUACUACAAAACCCAGAAUAAAUA
862





S410-AS863-M1
AUUCUGGGUUUUGUAGCAUUUUUAT
410
AUAAAAAUGCUACAAAACCCAGAAUAA
863





S411-AS864-M1
UUCUGGGUUUUGUAGCAUUUUUATT
411
AAUAAAAAUGCUACAAAACCCAGAAUA
864





S412-AS865-M1
UCUGGGUUUUGUAGCAUUUUUAUTA
412
UAAUAAAAAUGCUACAAAACCCAGAAU
865





S413-AS866-M1
CUGGGUUUUGUAGCAUUUUUAUUAA
413
UUAAUAAAAAUGCUACAAAACCCAGAA
866





S414-AS867-M1
UGGGUUUUGUAGCAUUUUUAUUAAT
414
AUUAAUAAAAAUGCUACAAAACCCAGA
867





S415-AS868-M1
GGGUUUUGUAGCAUUUUUAUUAATA
415
UAUUAAUAAAAAUGCUACAAAACCCAG
868





S416-AS869-M1
GGUUUUGUAGCAUUUUUAUUAAUAT
416
AUAUUAAUAAAAAUGCUACAAAACCCA
869





S417-AS870-M1
GUUUUGUAGCAUUUUUAUUAAUATG
417
CAUAUUAAUAAAAAUGCUACAAAACCC
870





S418-AS871-M1
UUUUGUAGCAUUUUUAUUAAUAUGG
418
CCAUAUUAAUAAAAAUGCUACAAAACC
871





S419-AS872-M1
UUUGUAGCAUUUUUAUUAAUAUGGT
419
ACCAUAUUAAUAAAAAUGCUACAAAAC
872





S420-AS873-M1
UUGUAGCAUUUUUAUUAAUAUGGTG
420
CACCAUAUUAAUAAAAAUGCUACAAAA
873





S421-AS874-M1
UGUAGCAUUUUUAUUAAUAUGGUGA
421
UCACCAUAUUAAUAAAAAUGCUACAAA
874





S422-AS875-M1
GUAGCAUUUUUAUUAAUAUUGUGAC
422
GUCACAAUAUUAAUAAAAAUGCUACAA
875





S423-AS876-M1
UAGCAUUUUUAUUAAUAUGUUGACT
423
AGUCAACAUAUUAAUAAAAAUGCUACA
876





S424-AS877-M1
AGCAUUUUUAUUAAUAUGGUGACTT
424
AAGUCACCAUAUUAAUAAAAAUGCUAC
877





S425-AS878-M1
GCAUUUUUAUUAAUAUGGUUACUTT
425
AAAGUAACCAUAUUAAUAAAAAUGCUA
878





S426-AS879-M1
CAUUUUUAUUAAUAUGGUGACUUTT
426
AAAAGUCACCAUAUUAAUAAAAAUGCU
879





S427-AS880-M1
AUUUUUAUUAAUAUGGUGAUUUUTT
427
AAAAAAUCACCAUAUUAAUAAAAAUGC
880





S428-AS881-M1
UUUUUAUUAAUAUGGUGACUUUUTA
428
UAAAAAGUCACCAUAUUAAUAAAAAUG
881





S429-AS882-M1
UUUUAUUAAUAUGGUGACUUUUUAA
429
UUAAAAAGUCACCAUAUUAAUAAAAAU
882





S430-AS883-M1
UUUAUUAAUAUGGUGACUUUUUAAA
430
UUUAAAAAGUCACCAUAUUAAUAAAAA
883





S431-AS884-M1
UUAUUAAUAUGGUGACUUUUUAAAA
431
UUUUAAAAAGUCACCAUAUUAAUAAAA
884





S432-AS885-M1
UAUUAAUAUGGUGACUUUUUAAAAT
432
AUUUUAAAAAGUCACCAUAUUAAUAAA
885





S433-AS886-M1
AUUAAUAUGGUGACUUUUUAAAATA
433
UAUUUUAAAAAGUCACCAUAUUAAUAA
886





S434-AS887-M1
UUAAUAUGGUGACUUUUUAAAAUAA
434
UUAUUUUAAAAAGUCACCAUAUUAAUA
887





S435-AS888-M1
UAAUAUGGUGACUUUUUAAAAUAAA
435
UUUAUUUUAAAAAGUCACCAUAUUAAU
888





S436-AS889-M1
AAUAUGGUGACUUUUUAAAAUAAAA
436
UUUUAUUUUAAAAAGUCACCAUAUUAA
889





S437-AS890-M1
AUAUGGUGACUUUUUAAAAUAAAAA
437
UUUUUAUUUUAAAAAGUCACCAUAUUA
890





S438-AS891-M1
UAUGGUGACUUUUUAAAAUAAAAAC
438
GUUUUUAUUUUAAAAAGUCACCAUAUU
891





S439-AS892-M1
AUGGUGACUUUUUAAAAUAAAAACA
439
UGUUUUUAUUUUAAAAAGUCACCAUAU
892





S440-AS893-M1
UGGUGACUUUUUAAAAUAAAAACAA
440
UUGUUUUUAUUUUAAAAAGUCACCAUA
893





S441-AS894-M1
GGUGACUUUUUAAAAUAAAAACAAA
441
UUUGUUUUUAUUUUAAAAAGUCACCAU
894





S442-AS895-M1
GUGACUUUUUAAAAUAAAAACAAAC
442
GUUUGUUUUUAUUUUAAAAAGUCACCA
895





S443-AS896-M1
UGACUUUUUAAAAUAAAAAUAAACA
443
UGUUUAUUUUUAUUUUAAAAAGUCACC
896





S444-AS897-M1
GACUUUUUAAAAUAAAAACAAACAA
444
UUGUUUGUUUUUAUUUUAAAAAGUCAC
897





S445-AS898-M1
ACUUUUUAAAAUAAAAACAAACAAA
445
UUUGUUUGUUUUUAUUUUAAAAAGUCA
898





S446-AS899-M1
UUUUAAAAUAAAAACAAACAAACGT
446
ACGUUUGUUUGUUUUUAUUUUAAAAAG
899





S447-AS900-M1
UUUAAAAUAAAAACAAACAAACGTT
447
AACGUUUGUUUGUUUUUAUUUUAAAAA
900





S448-AS901-M1
UUAAAAUAAAAACAAACAAACGUTG
448
CAACGUUUGUUUGUUUUUAUUUUAAAA
901





S449-AS902-M1
UAAAAUAAAAACAAACAAAUGUUGT
449
ACAACAUUUGUUUGUUUUUAUUUUAAA
902





S450-AS903-M1
AAAAACAAACAAACGUUGUUCUAAC
450
GUUAGAACAACGUUUGUUUGUUUUUAU
903





S451-AS904-M1
CAAACAAACGUUGUCCUAAUAAAAA
451
UUUUUAUUAGGACAACGUUUGUUUGUU
904





S452-AS905-M1
AAACAAACGUUGUCCUAACAAAAAA
452
UUUUUUGUUAGGACAACGUUUGUUUGU
905





S453-AS906-M1
AACAAACGUUGUCCUAACAAAAAAA
453
UUUUUUUGUUAGGACAACGUUUGUUUG
906





S907-AS1030-M1
CUCCAGGCGGUCCUGGUGGUCGCTG
907
CAGCGACCACCAGGACCGCCUGGAGCU
1030





S908-AS1031-M1
UCCAGGCGGUCCUGGUGGCUGCUGC
908
GCAGCAGCCACCAGGACCGCCUGGAGC
1031





S909-AS1032-M1
GCCGCUGCCACUGCUGCUGUUGCTG
909
CAGCAACAGCAGCAGUGGCAGCGGCCA
1032





S910-AS1033-M1
CCGCUGCCACUGCUGCUGCUGCUGC
910
GCAGCAGCAGCAGCAGUGGCAGCGGCC
1033





S911-AS1034-M1
GCCCGUGCGCAGGAGGACGAGGACG
911
CGUCCUCGUCCUCCUGCGCACGGGCGC
1034





S912-AS1035-M1
CCCGUGCGCAGGAGGACGAUGACGG
912
CCGUCAUCGUCCUCCUGCGCACGGGCG
1035





S913-AS1036-M1
CCGUGCGCAGGAGGACGAGUACGGC
913
GCCGUACUCGUCCUCCUGCGCACGGGC
1036





S914-AS1037-M1
CGUGCGCAGGAGGACGAGGACGGCG
914
CGCCGUCCUCGUCCUCCUGCGCACGGG
1037





S915-AS1038-M1
GUGCGCAGGAGGACGAGGAUGGCGA
915
UCGCCAUCCUCGUCCUCCUGCGCACGG
1038





S916-AS1039-M1
UGCGCAGGAGGACGAGGACUGCGAC
916
GUCGCAGUCCUCGUCCUCCUGCGCACG
1039





S917-AS1040-M1
GCGCAGGAGGACGAGGACGUCGACT
917
AGUCGACGUCCUCGUCCUCCUGCGCAC
1040





S918-AS1041-M1
GGAGGACGAGGACGGCGACUACGAG
918
CUCGUAGUCGCCGUCCUCGUCCUCCUG
1041





S919-AS1042-M1
GCGUUCCGAGGAGGACGGCUUGGCC
919
GGCCAAGCCGUCCUCCUCGGAACGCAA
1042





S920-AS1043-M1
CGUUCCGAGGAGGACGGCCUGGCCG
920
CGGCCAGGCCGUCCUCCUCGGAACGCA
1043





S921-AS1044-M1
GUUCCGAGGAGGACGGCCUUGCCGA
921
UCGGCAAGGCCGUCCUCCUCGGAACGC
1044





S922-AS1045-M1
UUCCGAGGAGGACGGCCUGUCCGAA
922
UUCGGACAGGCCGUCCUCCUCGGAACG
1045





S923-AS1046-M1
UCCGAGGAGGACGGCCUGGUCGAAG
923
CUUCGACCAGGCCGUCCUCCUCGGAAC
1046





S924-AS1047-M1
CCGAGGAGGACGGCCUGGCUGAAGC
924
GCUUCAGCCAGGCCGUCCUCCUCGGAA
1047





S925-AS1048-M1
CGAGGAGGACGGCCUGGCCUAAGCA
925
UGCUUAGGCCAGGCCGUCCUCCUCGGA
1048





S926-AS1049-M1
GAGGAGGACGGCCUGGCCGAAGCAC
926
GUGCUUCGGCCAGGCCGUCCUCCUCGG
1049





S927-AS1050-M1
GCCACCUUCCACCGCUGCGUCAAGG
927
CCUUGACGCAGCGGUGGAAGGUGGCUG
1050





S928-AS1051-M1
CCACCUUCCACCGCUGCGCUAAGGA
928
UCCUUAGCGCAGCGGUGGAAGGUGGCU
1051





S929-AS1052-M1
CACCUUCCACCGCUGCGCCAAGGAT
929
AUCCUUGGCGCAGCGGUGGAAGGUGGC
1052





S930-AS1053-M1
ACCUUCCACCGCUGCGCCAAGGATC
930
GAUCCUUGGCGCAGCGGUGGAAGGUGG
1053





S931-AS1054-M1
AGCGCACUGCCCGCCGCCUUCAGGC
931
GCCUGAAGGCGGCGGGCAGUGCGCUCU
1054





S932-AS1055-M1
GCGCACUGCCCGCCGCCUGUAGGCC
932
GGCCUACAGGCGGCGGGCAGUGCGCUC
1055





S933-AS1056-M1
CGCACUGCCCGCCGCCUGCAGGCCC
933
GGGCCUGCAGGCGGCGGGCAGUGCGCU
1056





S934-AS1057-M1
GCACUGCCCGCCGCCUGCAUGCCCA
934
UGGGCAUGCAGGCGGCGGGCAGUGCGC
1057





S935-AS1058-M1
CACUGCCCGCCGCCUGCAGUCCCAG
935
CUGGGACUGCAGGCGGCGGGCAGUGCG
1058





S936-AS1059-M1
ACUGCCCGCCGCCUGCAGGUCCAGG
936
CCUGGACCUGCAGGCGGCGGGCAGUGC
1059





S937-AS1060-M1
CUGCCCGCCGCCUGCAGGCUCAGGC
937
GCCUGAGCCUGCAGGCGGCGGGCAGUG
1060





S938-AS1061-M1
UGCCCGCCGCCUGCAGGCCUAGGCT
938
AGCCUAGGCCUGCAGGCGGCGGGCAGU
1061





S939-AS1062-M1
GCCCGCCGCCUGCAGGCCCAGGCTG
939
CAGCCUGGGCCUGCAGGCGGCGGGCAG
1062





S940-AS1063-M1
CCCGCCGCCUGCAGGCCCAUGCUGC
940
GCAGCAUGGGCCUGCAGGCGGCGGGCA
1063





S941-AS1064-M1
UGGCGACCUGCUGGAGCUGUCCUTG
941
CAAGGACAGCUCCAGCAGGUCGCCACU
1064





S942-AS1065-M1
GGCGACCUGCUGGAGCUGGUCUUGA
942
UCAAGACCAGCUCCAGCAGGUCGCCAC
1065





S943-AS1066-M1
GCGACCUGCUGGAGCUGGCUUUGAA
943
UUCAAAGCCAGCUCCAGCAGGUCGCCA
1066





S944-AS1067-M1
CGACCUGCUGGAGCUGGCCUUGAAG
944
CUUCAAGGCCAGCUCCAGCAGGUCGCC
1067





S945-AS1068-M1
GAGGCAGCCUGGUGGAGGUUUAUCT
945
AGAUAAACCUCCACCAGGCUGCCUCCG
1068





S946-AS1069-M1
AGGCAGCCUGGUGGAGGUGUAUCTC
946
GAGAUACACCUCCACCAGGCUGCCUCC
1069





S947-AS1070-M1
UGUGCCCGAGGAGGACGGGACCCGC
947
GCGGGUCCCGUCCUCCUCGGGCACAUU
1070





S948-AS1071-M1
GUGCCCGAGGAGGACGGGAUCCGCT
948
AGCGGAUCCCGUCCUCCUCGGGCACAU
1071





S949-AS1072-M1
UGCCCGAGGAGGACGGGACUCGCTT
949
AAGCGAGUCCCGUCCUCCUCGGGCACA
1072





S950-AS1073-M1
GCCCGAGGAGGACGGGACCUGCUTC
950
GAAGCAGGUCCCGUCCUCCUCGGGCAC
1073





S951-AS1074-M1
CCCGAGGAGGACGGGACCCUCUUCC
951
GGAAGAGGGUCCCGUCCUCCUCGGGCA
1074





S952-AS1075-M1
CCGAGGAGGACGGGACCCGUUUCCA
952
UGGAAACGGGUCCCGUCCUCCUCGGGC
1075





S953-AS1076-M1
CGAGGAGGACGGGACCCGCUUCCAC
953
GUGGAAGCGGGUCCCGUCCUCCUCGGG
1076





S954-AS1077-M1
GGCAGGGGUGGUCAGCGGCUGGGAT
954
AUCCCAGCCGCUGACCACCCCUGCCAG
1077





S955-AS1078-M1
GCAGGGGUGGUCAGCGGCCUGGATG
955
CAUCCAGGCCGCUGACCACCCCUGCCA
1078





S956-AS1079-M1
CAGGGGUGGUCAGCGGCCGUGAUGC
956
GCAUCACGGCCGCUGACCACCCCUGCC
1079





S957-AS1080-M1
GUGCUGCUGCCCCUGGCGGUUGGGT
957
ACCCAACCGCCAGGGGCAGCAGCACCA
1080





S958-AS1081-M1
UGCUGCUGCCCCUGGCGGGUGGGTA
958
UACCCACCCGCCAGGGGCAGCAGCACC
1081





S959-AS1082-M1
GCUGCUGCCCCUGGCGGGUUGGUAC
959
GUACCAACCCGCCAGGGGCAGCAGCAC
1082





S960-AS1083-M1
CUGCUGCCCCUGGCGGGUGUGUACA
960
UGUACACACCCGCCAGGGGCAGCAGCA
1083





S961-AS1084-M1
UGCUGCCCCUGGCGGGUGGUUACAG
961
CUGUAACCACCCGCCAGGGGCAGCAGC
1084





S962-AS1085-M1
GCUGCCCCUGGCGGGUGGGUACAGC
962
GCUGUACCCACCCGCCAGGGGCAGCAG
1085





S963-AS1086-M1
CUGCCCCUGGCGGGUGGGUACAGCC
963
GGCUGUACCCACCCGCCAGGGGCAGCA
1086





S964-AS1087-M1
UGCCCCUGGCGGGUGGGUAUAGCCG
964
CGGCUAUACCCACCCGCCAGGGGCAGC
1087





S965-AS1088-M1
GCCCCUGGCGGGUGGGUACAGCCGC
965
GCGGCUGUACCCACCCGCCAGGGGCAG
1088





S966-AS1089-M1
UCAACGCCGCCUGCCAGCGUCUGGC
966
GCCAGACGCUGGCAGGCGGCGUUGAGG
1089





S967-AS1090-M1
CAACGCCGCCUGCCAGCGCUUGGCG
967
CGCCAAGCGCUGGCAGGCGGCGUUGAG
1090





S968-AS1091-M1
AACGCCGCCUGCCAGCGCCUGGCGA
968
UCGCCAGGCGCUGGCAGGCGGCGUUGA
1091





S969-AS1092-M1
ACGCCGCCUGCCAGCGCCUUGCGAG
969
CUCGCAAGGCGCUGGCAGGCGGCGUUG
1092





S970-AS1093-M1
CGCCGCCUGCCAGCGCCUGUCGAGG
970
CCUCGACAGGCGCUGGCAGGCGGCGUU
1093





S971-AS1094-M1
GCCGCCUGCCAGCGCCUGGUGAGGG
971
CCCUCACCAGGCGCUGGCAGGCGGCGU
1094





S972-AS1095-M1
CCGCCUGCCAGCGCCUGGCUAGGGC
972
GCCCUAGCCAGGCGCUGGCAGGCGGCG
1095





S973-AS1096-M1
CGCCUGCCAGCGCCUGGCGAGGGCT
973
AGCCCUCGCCAGGCGCUGGCAGGCGGC
1096





S974-AS1097-M1
GCCUGCCAGCGCCUGGCGAUGGCTG
974
CAGCCAUCGCCAGGCGCUGGCAGGCGG
1097





S975-AS1098-M1
CCAGCGCCUGGCGAGGGCUUGGGTC
975
GACCCAAGCCCUCGCCAGGCGCUGGCA
1098





S976-AS1099-M1
CAGCGCCUGGCGAGGGCUGUGGUCG
976
CGACCACAGCCCUCGCCAGGCGCUGGC
1099





S977-AS1100-M1
AGCGCCUGGCGAGGGCUGGUGUCGT
977
ACGACACCAGCCCUCGCCAGGCGCUGG
1100





S978-AS1101-M1
GCGCCUGGCGAGGGCUGGGUUCGTG
978
CACGAACCCAGCCCUCGCCAGGCGCUG
1101





S979-AS1102-M1
CGCCUGGCGAGGGCUGGGGUCGUGC
979
GCACGACCCCAGCCCUCGCCAGGCGCU
1102





S980-AS1103-M1
GCGAGGGCUGGGGUCGUGCUGGUCA
980
UGACCAGCACGACCCCAGCCCUCGCCA
1103





S981-AS1104-M1
AUGCCUGCCUCUACUCCCCAGCCTC
981
GAGGCUGGGGAGUAGAGGCAGGCAUCG
1104





S982-AS1105-M1
GCCUCUACUCCCCAGCCUCAGCUCC
982
GGAGCUGAGGCUGGGGAGUAGAGGCAG
1105





S983-AS1106-M1
GACCUCUUUGCCCCAGGGGAGGACA
983
UGUCCUCCCCUGGGGCAAAGAGGUCCA
1106





S984-AS1107-M1
CUUUGCCCCAGGGGAGGACAUCATT
984
AAUGAUGUCCUCCCCUGGGGCAAAGAG
1107





S985-AS1108-M1
UUUGCCCCAGGGGAGGACAUCAUTG
985
CAAUGAUGUCCUCCCCUGGGGCAAAGA
1108





S986-AS1109-M1
UUGCCCCAGGGGAGGACAUUAUUGG
986
CCAAUAAUGUCCUCCCCUGGGGCAAAG
1109





S987-AS1110-M1
UGCCCCAGGGGAGGACAUCAUUGGT
987
ACCAAUGAUGUCCUCCCCUGGGGCAAA
1110





S988-AS1111-M1
GCCCCAGGGGAGGACAUCAUUGGTG
988
CACCAAUGAUGUCCUCCCCUGGGGCAA
1111





S989-AS1112-M1
ACACGGAUGGCCACAGCCGUCGCCC
989
GGGCGACGGCUGUGGCCAUCCGUGUAG
1112





S990-AS1113-M1
CUCCAGGAGUGGGAAGCGGUGGGGC
990
GCCCCACCGCUUCCCACUCCUGGAGAA
1113





S991-AS1114-M1
UCCAGGAGUGGGAAGCGGCUGGGCG
991
CGCCCAGCCGCUUCCCACUCCUGGAGA
1114





S992-AS1115-M1
CCAGGAGUGGGAAGCGGCGUGGCGA
992
UCGCCACGCCGCUUCCCACUCCUGGAG
1115





S993-AS1116-M1
CAGGAGUGGGAAGCGGCGGUGCGAG
993
CUCGCACCGCCGCUUCCCACUCCUGGA
1116





S994-AS1117-M1
AGGAGUGGGAAGCGGCGGGUCGAGC
994
GCUCGACCCGCCGCUUCCCACUCCUGG
1117





S995-AS1118-M1
GGAGUGGGAAGCGGCGGGGUGAGCG
995
CGCUCACCCCGCCGCUUCCCACUCCUG
1118





S996-AS1119-M1
GAGUGGGAAGCGGCGGGGCUAGCGC
996
GCGCUAGCCCCGCCGCUUCCCACUCCU
1119





S997-AS1120-M1
AGUGGGAAGCGGCGGGGCGAGCGCA
997
UGCGCUCGCCCCGCCGCUUCCCACUCC
1120





S998-AS1121-M1
GAAGCGGCGGGGCGAGCGCAUGGAG
998
CUCCAUGCGCUCGCCCCGCCGCUUCCC
1121





S999-AS1122-M1
AAGCGGCGGGGCGAGCGCAUGGAGG
999
CCUCCAUGCGCUCGCCCCGCCGCUUCC
1122





S1000-AS1123-M1
AGCGGCGGGGCGAGCGCAUUGAGGC
1000
GCCUCAAUGCGCUCGCCCCGCCGCUUC
1123





S1001-AS1124-M1
GGUGCUGCCUGCUACCCCAUGCCAA
1001
UUGGCAUGGGGUAGCAGGCAGCACCUG
1124





S1002-AS1125-M1
GUGCUGCCUGCUACCCCAGUCCAAC
1002
GUUGGACUGGGGUAGCAGGCAGCACCU
1125





S1003-AS1126-M1
UGCUGCCUGCUACCCCAGGUCAACT
1003
AGUUGACCUGGGGUAGCAGGCAGCACC
1126





S1004-AS1127-M1
GGGCCACGUCCUCACAGGCUGCAGC
1004
GCUGCAGCCUGUGAGGACGUGGCCCUG
1127





S1005-AS1128-M1
GGCCACGUCCUCACAGGCUUCAGCT
1005
AGCUGAAGCCUGUGAGGACGUGGCCCU
1128





S1006-AS1129-M1
GCCACGUCCUCACAGGCUGUAGCTC
1006
GAGCUACAGCCUGUGAGGACGUGGCCC
1129





S1007-AS1130-M1
GGCUGCAGCUCCCACUGGGAGGUGG
1007
CCACCUCCCAGUGGGAGCUGCAGCCUG
1130





S1008-AS1131-M1
GCUGCAGCUCCCACUGGGAUGUGGA
1008
UCCACAUCCCAGUGGGAGCUGCAGCCU
1131





S1009-AS1132-M1
CUGCAGCUCCCACUGGGAGUUGGAG
1009
CUCCAACUCCCAGUGGGAGCUGCAGCC
1132





S1010-AS1133-M1
UGCAGCUCCCACUGGGAGGUGGAGG
1010
CCUCCACCUCCCAGUGGGAGCUGCAGC
1133





S1011-AS1134-M1
GCAGCUCCCACUGGGAGGUUGAGGA
1011
UCCUCAACCUCCCAGUGGGAGCUGCAG
1134





S1012-AS1135-M1
CAGCUCCCACUGGGAGGUGUAGGAC
1012
GUCCUACACCUCCCAGUGGGAGCUGCA
1135





S1013-AS1136-M1
AGCUCCCACUGGGAGGUGGAGGACC
1013
GGUCCUCCACCUCCCAGUGGGAGCUGC
1136





S1014-AS1137-M1
GCUCCCACUGGGAGGUGGAUGACCT
1014
AGGUCAUCCACCUCCCAGUGGGAGCUG
1137





S1015-AS1138-M1
CUCCCACUGGGAGGUGGAGUACCTT
1015
AAGGUACUCCACCUCCCAGUGGGAGCU
1138





S1016-AS1139-M1
UCCCACUGGGAGGUGGAGGACCUTG
1016
CAAGGUCCUCCACCUCCCAGUGGGAGC
1139





S1017-AS1140-M1
UGGCACCCACAAGCCGCCUUUGCTG
1017
CAGCAAAGGCGGCUUGUGGGUGCCAAG
1140





S1018-AS1141-M1
GGCACCCACAAGCCGCCUGUGCUGA
1018
UCAGCACAGGCGGCUUGUGGGUGCCAA
1141





S1019-AS1142-M1
AGCCGCCUGUGCUGAGGCCACGAGG
1019
CCUCGUGGCCUCAGCACAGGCGGCUUG
1142





S1020-AS1143-M1
GCCGCCUGUGCUGAGGCCAUGAGGT
1020
ACCUCAUGGCCUCAGCACAGGCGGCUU
1143





S1021-AS1144-M1
CCGCCUGUGCUGAGGCCACUAGGTC
1021
GACCUAGUGGCCUCAGCACAGGCGGCU
1144





S1022-AS1145-M1
GGGCCACAGGGAGGCCAGCAUCCAC
1022
GUGGAUGCUGGCCUCCCUGUGGCCCAC
1145





S1023-AS1146-M1
GGCCACAGGGAGGCCAGCAUCCACG
1023
CGUGGAUGCUGGCCUCCCUGUGGCCCA
1146





S1024-AS1147-M1
GCCACAGGGAGGCCAGCAUUCACGC
1024
GCGUGAAUGCUGGCCUCCCUGUGGCCC
1147





S1025-AS1148-M1
CGGCCCCUCAGGAGCAGGUUACCGT
1025
ACGGUAACCUGCUCCUGAGGGGCCGGG
1148





S1026-AS1149-M1
UGCUGCCGGAGCCGGCACCUGGCGC
1026
GCGCCAGGUGCCGGCUCCGGCAGCAGA
1149





S1027-AS1150-M1
UCACAGGCUGCUGCCCACGUGGCTG
1027
CAGCCACGUGGGCAGCAGCCUGUGAUG
1150





S1028-AS1151-M1
CACAGGCUGCUGCCCACGUUGCUGG
1028
CCAGCAACGUGGGCAGCAGCCUGUGAU
1151





S1029-AS1152-M1
GCUUCCUGCUGCCAUGCCCUAGGTC
1029
GACCUAGGGCAUGGCAGCAGGAAGCGU
1152





S1153-AS1193-M2
AACUUCAGCUCCUGCACAGUGCAGC
1153
ACUGUGCAGGAGCUGAAGUUCA
1193



CGAAAGGCUGC








S1154-AS1194-M2
UGGCCCUCAUGGGCACCGUUGCAGC
1154
AACGGUGCCCAUGAGGGCCAGG
1194



CGAAAGGCUGC








S1155-AS1195-M2
AGGAGGAGACCCACCUCUCUGCAGC
1155
AGAGAGGUGGGUCUCCUCCUUC
1195



CGAAAGGCUGC








S1156-AS1196-M2
UGCUGGAGCUGGCCUUGAAUGCAGC
1156
AUUCAAGGCCAGCUCCAGCAGG
1196



CGAAAGGCUGC








S1157-AS1197-M2
UCUGUCUUUGCCCAGAGCAUGCAGC
1157
AUGCUCUGGGCAAAGACAGAGG
1197



CGAAAGGCUGC








S1158-AS1198-M2
CUGUCUUUGCCCAGAGCAUUGCAGC
1158
AAUGCUCUGGGCAAAGACAGAG
1198



CGAAAGGCUGC








S1159-AS1199-M2
CUUGCCUGGAACUCACUCAUGCAGC
1159
AUGAGUGAGUUCCAGGCAAGGA
1199



CGAAAGGCUGC








S1160-AS1200-M2
UUGCCUGGAACUCACUCACUGCAGC
1160
AGUGAGUGAGUUCCAGGCAAGG
1200



CGAAAGGCUGC








S1161-AS1201-M2
AGAAUGACUUUUAUUGAGCUGCAGC
1161
AGCUCAAUAAAAGUCAUUCUGC
1201



CGAAAGGCUGC








S1162-AS1202-M2
GAAUGACUUUUAUUGAGCUUGCAGC
1162
AAGCUCAAUAAAAGUCAUUCUG
1202



CGAAAGGCUGC








S1163-AS1203-M2
AUGACUUUUAUUGAGCUCUUGCAGC
1163
AAGAGCUCAAUAAAAGUCAUUC
1203



CGAAAGGCUGC








S1164-AS1204-M2
UGACUUUUAUUGAGCUCUUUGCAGC
1164
AAAGAGCUCAAUAAAAGUCAUU
1204



CGAAAGGCUGC








S1165-AS1205-M2
CUUGUUCCGUGCCAGGCAUUGCAGC
1165
AAUGCCUGGCACGGAACAAGAG
1205



CGAAAGGCUGC








S1166-AS1206-M2
UGUGAAAGGUGCUGAUGGCUGCAGC
1166
AGCCAUCAGCACCUUUCACACU
1206



CGAAAGGCUGC








S1167-AS1207-M2
AUGGAGGCUUAGCUUUCUGUGCAGC
1167
ACAGAAAGCUAAGCCUCCAUUA
1207



CGAAAGGCUGC








S1168-AS1208-M2
GAGGCUUAGCUUUCUGGAUUGCAGC
1168
AAUCCAGAAAGCUAAGCCUCCA
1208



CGAAAGGCUGC








S1169-AS1209-M2
AGGCUUAGCUUUCUGGAUGUGCAGC
1169
ACAUCCAGAAAGCUAAGCCUCC
1209



CGAAAGGCUGC








S1170-AS1210-M2
GCUUAGCUUUCUGGAUGGCAGCAGC
1170
UGCCAUCCAGAAAGCUAAGCCU
1210



CGAAAGGCUGC








S1171-AS1211-M2
CCAGGCUGUGCUAGCAACAUGCAGC
1171
AUGUUGCUAGCACAGCCUGGCA
1211



CGAAAGGCUGC








S1172-AS1212-M2
UGCGGGGAGCCAUCACCUAUGCAGC
1172
AUAGGUGAUGGCUCCCCGCAGG
1212



CGAAAGGCUGC








S1173-AS1213-M2
CGGCAGUGUGCAGUGGUGCAGCAGC
1173
UGCACCACUGCACACUGCCGAG
1213



CGAAAGGCUGC








S1174-AS1214-M2
ACAGAGGAAGAAACCUGGAAGCAGC
1174
UUCCAGGUUUCUUCCUCUGUGA
1214



CGAAAGGCUGC








S1175-AS1215-M2
CAGAGGAAGAAACCUGGAAUGCAGC
1175
AUUCCAGGUUUCUUCCUCUGUG
1215



CGAAAGGCUGC








S1176-AS1216-M2
AGAGGAAGAAACCUGGAACUGCAGC
1176
AGUUCCAGGUUUCUUCCUCUGU
1216



CGAAAGGCUGC








S1177-AS1217-M2
UGGCGGAGAUGCUUCUAAGUGCAGC
1177
ACUUAGAAGCAUCUCCGCCAGG
1217



CGAAAGGCUGC








S1178-AS1218-M2
UUACAGCCAACUUUUCUAGAGCAGC
1178
UCUAGAAAAGUUGGCUGUAAAA
1218



CGAAAGGCUGC








S1179-AS1219-M2
CUGUUUUGCUUUUGUAACUUGCAGC
1179
AAGUUACAAAAGCAAAACAGGU
1219



CGAAAGGCUGC








S1180-AS1220-M2
UGUUUUGCUUUUGUAACUUUGCAGC
1180
AAAGUUACAAAAGCAAAACAGG
1220



CGAAAGGCUGC








S1181-AS1221-M2
UUUGCUUUUGUAACUUGAAUGCAGC
1181
AUUCAAGUUACAAAAGCAAAAC
1221



CGAAAGGCUGC








S1182-AS1222-M2
UUUGUAGCAUUUUUAUUAAUGCAGC
1182
AUUAAUAAAAAUGCUACAAAAC
1222



CGAAAGGCUGC








S1183-AS1223-M2
UGUAGCAUUUUUAUUAAUAUGCAGC
1183
AUAUUAAUAAAAAUGCUACAAA
1223



CGAAAGGCUGC








S1184-AS1224-M2
GUAGCAUUUUUAUUAAUAUUGCAGC
1184
AAUAUUAAUAAAAAUGCUACAA
1224



CGAAAGGCUGC








S1185-AS1225-M2
AUUAAUAUGGUGACUUUUUAGCAGC
1185
UAAAAAGUCACCAUAUUAAUAA
1225



CGAAAGGCUGC








S1186-AS1226-M2
UUAAUAUGGUGACUUUUUAAGCAGC
1186
UUAAAAAGUCACCAUAUUAAUA
1226



CGAAAGGCUGC








S1187-AS1227-M2
AAUAUGGUGACUUUUUAAAAGCAGC
1187
UUUUAAAAAGUCACCAUAUUAA
1227



CGAAAGGCUGC








S1188-AS1228-M2
AUAUGGUGACUUUUUAAAAUGCAGC
1188
AUUUUAAAAAGUCACCAUAUUA
1228



CGAAAGGCUGC








S1189-AS1229-M2
UAUGGUGACUUUUUAAAAUAGCAGC
1189
UAUUUUAAAAAGUCACCAUAUU
1229



CGAAAGGCUGC








S1190-AS1230-M2
AUGGUGACUUUUUAAAAUAAGCAGC
1190
UUAUUUUAAAAAGUCACCAUAU
1230



CGAAAGGCUGC








S1191-AS1231-M2
UGGUGACUUUUUAAAAUAAAGCAGC
1191
UUUAUUUUAAAAAGUCACCAUA
1231



CGAAAGGCUGC








S1192-AS1232-M2
GUGACUUUUUAAAAUAAAAAGCAGC
1192
UUUUUAUUUUAAAAAGUCACCA
1232



CGAAAGGCUGC








S1153-AS1193-M3
AACUUCAGCUCCUGCACAGUGCAGC
1153
ACUGUGCAGGAGCUGAAGUUCA
1193



CGAAAGGCUGC








S1154-AS1194-M3
UGGCCCUCAUGGGCACCGUUGCAGC
1154
AACGGUGCCCAUGAGGGCCAGG
1194



CGAAAGGCUGC








S1155-AS1195-M3
AGGAGGAGACCCACCUCUCUGCAGC
1155
AGAGAGGUGGGUCUCCUCCUUC
1195



CGAAAGGCUGC








S1156-AS1196-M3
UGCUGGAGCUGGCCUUGAAUGCAGC
1156
AUUCAAGGCCAGCUCCAGCAGG
1196



CGAAAGGCUGC








S1157-AS1197-M3
UCUGUCUUUGCCCAGAGCAUGCAGC
1157
AUGCUCUGGGCAAAGACAGAGG
1197



CGAAAGGCUGC








S1158-AS1198-M3
CUGUCUUUGCCCAGAGCAUUGCAGC
1158
AAUGCUCUGGGCAAAGACAGAG
1198



CGAAAGGCUGC








S1159-AS1199-M3
CUUGCCUGGAACUCACUCAUGCAGC
1159
AUGAGUGAGUUCCAGGCAAGGA
1199



CGAAAGGCUGC








S1160-AS1200-M3
UUGCCUGGAACUCACUCACUGCAGC
1160
AGUGAGUGAGUUCCAGGCAAGG
1200



CGAAAGGCUGC








S1161-AS1201-M3
AGAAUGACUUUUAUUGAGCUGCAGC
1161
AGCUCAAUAAAAGUCAUUCUGC
1201



CGAAAGGCUGC








S1162-AS1202-M3
GAAUGACUUUUAUUGAGCUUGCAGC
1162
AAGCUCAAUAAAAGUCAUUCUG
1202



CGAAAGGCUGC








S1163-AS1203-M3
AUGACUUUUAUUGAGCUCUUGCAGC
1163
AAGAGCUCAAUAAAAGUCAUUC
1203



CGAAAGGCUGC








S1164-AS1204-M3
UGACUUUUAUUGAGCUCUUUGCAGC
1164
AAAGAGCUCAAUAAAAGUCAUU
1204



CGAAAGGCUGC








S1165-AS1205-M3
CUUGUUCCGUGCCAGGCAUUGCAGC
1165
AAUGCCUGGCACGGAACAAGAG
1205



CGAAAGGCUGC








S1166-AS1206-M3
UGUGAAAGGUGCUGAUGGCUGCAGC
1166
AGCCAUCAGCACCUUUCACACU
1206



CGAAAGGCUGC








S1167-AS1207-M3
AUGGAGGCUUAGCUUUCUGUGCAGC
1167
ACAGAAAGCUAAGCCUCCAUUA
1207



CGAAAGGCUGC








S1168-AS1208-M3
GAGGCUUAGCUUUCUGGAUUGCAGC
1168
AAUCCAGAAAGCUAAGCCUCCA
1208



CGAAAGGCUGC








S1169-AS1209-M3
AGGCUUAGCUUUCUGGAUGUGCAGC
1169
ACAUCCAGAAAGCUAAGCCUCC
1209



CGAAAGGCUGC








S1170-AS1210-M3
GCUUAGCUUUCUGGAUGGCAGCAGC
1170
UGCCAUCCAGAAAGCUAAGCCU
1210



CGAAAGGCUGC








S1171-AS1211-M3
CCAGGCUGUGCUAGCAACAUGCAGC
1171
AUGUUGCUAGCACAGCCUGGCA
1211



CGAAAGGCUGC








S1172-AS1212-M3
UGCGGGGAGCCAUCACCUAUGCAGC
1172
AUAGGUGAUGGCUCCCCGCAGG
1212



CGAAAGGCUGC








S1173-AS1213-M3
CGGCAGUGUGCAGUGGUGCAGCAGC
1173
UGCACCACUGCACACUGCCGAG
1213



CGAAAGGCUGC








S1174-AS1214-M3
ACAGAGGAAGAAACCUGGAAGCAGC
1174
UUCCAGGUUUCUUCCUCUGUGA
1214



CGAAAGGCUGC








S1175-AS1215-M3
CAGAGGAAGAAACCUGGAAUGCAGC
1175
AUUCCAGGUUUCUUCCUCUGUG
1215



CGAAAGGCUGC








S1176-AS1216-M3
AGAGGAAGAAACCUGGAACUGCAGC
1176
AGUUCCAGGUUUCUUCCUCUGU
1216



CGAAAGGCUGC








S1177-AS1217-M3
UGGCGGAGAUGCUUCUAAGUGCAGC
1177
ACUUAGAAGCAUCUCCGCCAGG
1217



CGAAAGGCUGC








S1178-AS1218-M3
UUACAGCCAACUUUUCUAGAGCAGC
1178
UCUAGAAAAGUUGGCUGUAAAA
1218



CGAAAGGCUGC








S1179-AS1219-M3
CUGUUUUGCUUUUGUAACUUGCAGC
1179
AAGUUACAAAAGCAAAACAGGU
1219



CGAAAGGCUGC








S1180-AS1220-M3
UGUUUUGCUUUUGUAACUUUGCAGC
1180
AAAGUUACAAAAGCAAAACAGG
1220



CGAAAGGCUGC








S1181-AS1221-M3
UUUGCUUUUGUAACUUGAAUGCAGC
1181
AUUCAAGUUACAAAAGCAAAAC
1221



CGAAAGGCUGC








S1182-AS1222-M3
UUUGUAGCAUUUUUAUUAAUGCAGC
1182
AUUAAUAAAAAUGCUACAAAAC
1222



CGAAAGGCUGC








S1183-AS1223-M3
UGUAGCAUUUUUAUUAAUAUGCAGC
1183
AUAUUAAUAAAAAUGCUACAAA
1223



CGAAAGGCUGC








S1184-AS1224-M3
GUAGCAUUUUUAUUAAUAUUGCAGC
1184
AAUAUUAAUAAAAAUGCUACAA
1224



CGAAAGGCUGC








S1185-AS1225-M3
AUUAAUAUGGUGACUUUUUAGCAGC
1185
UAAAAAGUCACCAUAUUAAUAA
1225



CGAAAGGCUGC








S1186-AS1226-M3
UUAAUAUGGUGACUUUUUAAGCAGC
1186
UUAAAAAGUCACCAUAUUAAUA
1226



CGAAAGGCUGC








S1187-AS1227-M3
AAUAUGGUGACUUUUUAAAAGCAGC
1187
UUUUAAAAAGUCACCAUAUUAA
1227



CGAAAGGCUGC








S1188-AS1228-M3
AUAUGGUGACUUUUUAAAAUGCAGC
1188
AUUUUAAAAAGUCACCAUAUUA
1228



CGAAAGGCUGC








S1189-AS1229-M3
UAUGGUGACUUUUUAAAAUAGCAGC
1189
UAUUUUAAAAAGUCACCAUAUU
1229



CGAAAGGCUGC








S1190-AS1230-M3
AUGGUGACUUUUUAAAAUAAGCAGC
1190
UUAUUUUAAAAAGUCACCAUAU
1230



CGAAAGGCUGC








S1191-AS1231-M3
UGGUGACUUUUUAAAAUAAAGCAGC
1191
UUUAUUUUAAAAAGUCACCAUA
1231



CGAAAGGCUGC








S1192-AS1232-M3
GUGACUUUUUAAAAUAAAAAGCAGC
1192
UUUUUAUUUUAAAAAGUCACCA
1232



CGAAAGGCUGC








S1180-AS1220-M4
UGUUUUGCUUUUGUAACUUUGCAGC
1180
AAAGUUACAAAAGCAAAACAGG
1220



CGAAAGGCUGC








S1163-AS1203-M4
AUGACUUUUAUUGAGCUCUUGCAGC
1163
AAGAGCUCAAUAAAAGUCAUUC
1203



CGAAAGGCUGC








S1181-AS1221-M4
UUUGCUUUUGUAACUUGAAUGCAGC
1181
AUUCAAGUUACAAAAGCAAAAC
1221



CGAAAGGCUGC








S1248-AS1257-M4
GCUGGGCUCCUCAUUUUUAUGCAGC
1248
AUAAAAAUGAGGAGCCCAGCGG
1257



CGAAAGGCUGC








S1249-AS1258-M4
GCUGGCGGAGAUGCUUCUAAGCAGC
1249
UUAGAAGCAUCUCCGCCAGCGG
1258



CGAAAGGCUGC








S1250-AS1259-M4
UUUACAGCCAACUUUUCUAUGCAGC
1250
AUAGAAAAGUUGGCUGUAAAGG
1259



CGAAAGGCUGC








S1251-AS1260-M4
GGCUGGGCUCCUCAUUUUUAGCAGC
1251
UAAAAAUGAGGAGCCCAGCCGG
1260



CGAAAGGCUGC








S1252-AS1261-M4
AGCACGGAACCACAGCCACUGCAGC
1252
AGUGGCUGUGGUUCCGUGCUGG
1261



CGAAAGGCUGC








S1253-AS1262-M4
AAUGACUUUUAUUGAGCUCUGCAGC
1253
AGAGCUCAAUAAAAGUCAUUGG
1262



CGAAAGGCUGC








S1254-AS1263-M4
UUUUGUAGCAUUUUUAUUAAGCAGC
1254
UUAAUAAAAAUGCUACAAAAGG
1263



CGAAAGGCUGC








S1255-AS1264-M4
GCUUGCCUGGAACUCACUCAGCAGC
1255
UGAGUGAGUUCCAGGCAAGCGG
1264



CGAAAGGCUGC








S1256-AS1265-M4
UGGAGGCUUAGCUUUCUGGAGCAGC
1256
UCCAGAAAGCUAAGCCUCCAGG
1265



CGAAAGGCUGC








S1180-AS1220-M4
UGUUUUGCUUUUGUAACUUUGCAGC
1180
AAAGUUACAAAAGCAAAACAGG
1220



CGAAAGGCUGC








S1180-AS1220-M5
UGUUUUGCUUUUGUAACUUUGCAGC
1180
AAAGUUACAAAAGCAAAACAGG
1220



CGAAAGGCUGC








S1164-AS1204-M5
UGACUUUUAUUGAGCUCUUUGCAGC
1164
AAAGAGCUCAAUAAAAGUCAUU
1204



CGAAAGGCUGC








S1178-AS1218-M6
UUACAGCCAACUUUUCUAGAGCAGC
1178
UCUAGAAAAGUUGGCUGUAAAA
1218



CGAAAGGCUGC








S1178-AS1218-M5
UUACAGCCAACUUUUCUAGAGCAGC
1178
UCUAGAAAAGUUGGCUGUAAAA
1218



CGAAAGGCUGC








S1179-AS1219-M6
CUGUUUUGCUUUUGUAACUUGCAGC
1179
AAGUUACAAAAGCAAAACAGGU
1219



CGAAAGGCUGC








S1179-AS1219-M5
CUGUUUUGCUUUUGUAACUUGCAGC
1179
AAGUUACAAAAGCAAAACAGGU
1219



CGAAAGGCUGC








S1181-AS1221-M5
UUUGCUUUUGUAACUUGAAUGCAGC
1181
AUUCAAGUUACAAAAGCAAAAC
1221



CGAAAGGCUGC








S1182-AS1222-M5
UUUGUAGCAUUUUUAUUAAUGCAGC
1182
AUUAAUAAAAAUGCUACAAAAC
1222



CGAAAGGCUGC








S1183-AS1223-M5
UGUAGCAUUUUUAUUAAUAUGCAGC
1183
AUAUUAAUAAAAAUGCUACAAA
1223



CGAAAGGCUGC








S1187-AS1227-M5
AAUAUGGUGACUUUUUAAAAGCAGC
1187
UUUUAAAAAGUCACCAUAUUAA
1227



CGAAAGGCUGC








S1188-AS1228-M5
AUAUGGUGACUUUUUAAAAUGCAGC
1188
AUUUUAAAAAGUCACCAUAUUA
1228



CGAAAGGCUGC








S1189-AS1229-M5
UAUGGUGACUUUUUAAAAUAGCAGC
1189
UAUUUUAAAAAGUCACCAUAUU
1229



CGAAAGGCUGC








S1158-AS1198-M5
CUGUCUUUGCCCAGAGCAUUGCAGC
1158
AAUGCUCUGGGCAAAGACAGAG
1198



CGAAAGGCUGC








S1159-AS1199-M5
CUUGCCUGGAACUCACUCAUGCAGC
1159
AUGAGUGAGUUCCAGGCAAGGA
1199



CGAAAGGCUGC








S1160-AS1200-M5
UUGCCUGGAACUCACUCACUGCAGC
1160
AGUGAGUGAGUUCCAGGCAAGG
1200



CGAAAGGCUGC








S1161-AS1201-M5
AGAAUGACUUUUAUUGAGCUGCAGC
1161
AGCUCAAUAAAAGUCAUUCUGC
1201



CGAAAGGCUGC








S1163-AS1203-M5
AUGACUUUUAUUGAGCUCUUGCAGC
1163
AAGAGCUCAAUAAAAGUCAUUC
1203



CGAAAGGCUGC








S1184-AS1224-M5
GUAGCAUUUUUAUUAAUAUUGCAGC
1184
AAUAUUAAUAAAAAUGCUACAA
1224



CGAAAGGCUGC








S1185-AS1225-M5
AUUAAUAUGGUGACUUUUUAGCAGC
1185
UAAAAAGUCACCAUAUUAAUAA
1225



CGAAAGGCUGC








S1186-AS1226-M6
UUAAUAUGGUGACUUUUUAAGCAGC
1186
UUAAAAAGUCACCAUAUUAAUA
1226



CGAAAGGCUGC








S1186-AS1226-M5
UUAAUAUGGUGACUUUUUAAGCAGC
1186
UUAAAAAGUCACCAUAUUAAUA
1226



CGAAAGGCUGC








S1190-AS1230-M5
AUGGUGACUUUUUAAAAUAAGCAGC
1190
UUAUUUUAAAAAGUCACCAUAU
1230



CGAAAGGCUGC








S1191-AS1231-M5
UGGUGACUUUUUAAAAUAAAGCAGC
1191
UUUAUUUUAAAAAGUCACCAUA
1231



CGAAAGGCUGC








S1192-AS1232-M5
GUGACUUUUUAAAAUAAAAAGCAGC
1192
UUUUUAUUUUAAAAAGUCACCA
1232



CGAAAGGCUGC








S1266-AS1269-M7
UGUUUUGCUUUUGUAACUU[U/A]G
1266
[U/A]AAGUUACAAAAGCAAAA
1269



CAGCCGAAAGGCUGC

CAGG






S1266-AS1269-M8
UGUUUUGCUUUUGUAACUU[U/A]G
1266
[U/A]AAGUUACAAAAGCAAAA
1269



CAGCCGAAAGGCUGC

CAGG






S1266-AS1269-M9
UGUUUUGCUUUUGUAACUU[U/A]G
1266
[U/A]AAGUUACAAAAGCAAAA
1269



CAGCCGAAAGGCUGC

CAGG






S1267-AS1270-M10
UUUUGUAACUUGAAGAUAUAGCAGC
1267
UAUAUCUUCAAGUUACAAAAGG
1270



CGAAAGGCUGC








S1268-AS1271-M11
CUGGGUUUUGUAGCAUUUUAGCAGC
1268
UAAAAUGCUACAAAACCCAGGG
1271



CGAAAGGCUGC








S1268-AS1271-M9
CUGGGUUUUGUAGCAUUUUAGCAGC
1268
UAAAAUGCUACAAAACCCAGGG
1271



CGAAAGGCUGC








S1266-AS1269-M12
UGUUUUGCUUUUGUAACUU[U/A]G
1266
[U/A]AAGUUACAAAAGCAAAA
1269



CAGCCGAAAGGCUGC

CAGG






S1266-AS1269-M13
UGUUUUGCUUUUGUAACUU[U/A]G
1266
[U/A]AAGUUACAAAAGCAAAA
1269



CAGCCGAAAGGCUGC

CAGG









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. 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: 1193-1232, 1257-1265 and 1269-1271.
  • 2. The oligonucleotide of claim 1, further comprising a sense strand that comprises a sequence as set forth in any one of SEQ ID NOs: 1153-1192, 1248-1256, and 1266-1268.
  • 3. The oligonucleotide of claim 1 or 2, wherein the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1193-1232, 1257-1265 and 1269-1271.
  • 4. The oligonucleotide of claim 2 or 3, wherein the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1153-1192, 1248-1256, and 1266-1268.
  • 5. An oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, wherein 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, wherein the region of complementarity is at least 15 contiguous nucleotides in length.
  • 6. The oligonucleotide of claim 5, wherein the region of complementarity is fully complementary to the target sequence of PCSK9.
  • 7. The oligonucleotide of any one of claims 1 to 6, wherein the antisense strand is 19 to 27 nucleotides in length.
  • 8. The oligonucleotide of any one of claims 1 to 7, wherein the antisense strand is 21 to 27 nucleotides in length.
  • 9. The oligonucleotide of any one of claims 1 to 8, further comprising a sense strand of 15 to 40 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand.
  • 10. The oligonucleotide of claim 9, wherein the sense strand is 19 to 40 nucleotides in length.
  • 11. The oligonucleotide of claim 9 or 10, wherein the duplex region is at least 19 nucleotides in length.
  • 12. The oligonucleotide of any one of claims 9 to 11, wherein the duplex region is at least 21 nucleotides in length.
  • 13. The oligonucleotide of any one of claims 5 to 12, wherein the region of complementarity to PCSK9 is at least 19 contiguous nucleotides in length.
  • 14. The oligonucleotide of any one of claims 5 to 13, wherein the region of complementarity to PCSK9 is at least 21 contiguous nucleotides in length.
  • 15. The oligonucleotide of any one of claims 9 to 14, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1153-1192, 1248-1256, and 1266-1268.
  • 16. The oligonucleotide of any one of claims 5 to 15, wherein the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1193-1232, 1257-1265 and 1269-1271.
  • 17. The oligonucleotide of any one of claims 9 to 16, wherein the sense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1153-1192, 1248-1256, and 1266-1268.
  • 18. The oligonucleotide of any one of claims 5 to 17, wherein the antisense strand consists of a sequence as set forth in any one of SEQ ID NOs: 1193-1232, 1257-1265 and 1269-1271.
  • 19. The oligonucleotide of any one of claims 9 to 18, 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.
  • 20. An oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides in length 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.
  • 21. The oligonucleotide of claim 20, wherein the region of complementarity is fully complementary to at least 19 contiguous nucleotides of PCSK9 mRNA.
  • 22. The oligonucleotide of any one of claims 19 to 21, wherein L is a tetraloop.
  • 23. The oligonucleotide of any one of claims 19 to 22, wherein L is 4 nucleotides in length.
  • 24. The oligonucleotide of any one of claims 19 to 23, wherein L comprises a sequence set forth as GAAA.
  • 25. The oligonucleotide of any one of claims 9 to 18, wherein the antisense strand is 27 nucleotides in length and the sense strand is 25 nucleotides in length.
  • 26. The oligonucleotide of claim 25, wherein the antisense strand and sense strand form a duplex region of 25 nucleotides in length.
  • 27. The oligonucleotide of any one of claims 20 to 24, further comprising a 3′-overhang sequence on the antisense strand of two nucleotides in length.
  • 28. The oligonucleotide of any one of claims 9 to 18, wherein the oligonucleotide comprises an antisense strand and a sense strand that are each in a range of 21 to 23 nucleotides in length.
  • 29. The oligonucleotide of claim 28, wherein the oligonucleotide comprises a duplex structure in a range of 19 to 21 nucleotides in length.
  • 30. The oligonucleotide of claim 28 or 29, wherein the oligonucleotide comprises a 3′-overhang sequence of one or more nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand, the sense strand, or the antisense strand and sense strand.
  • 31. The oligonucleotide of claim 28 or 29, wherein the oligonucleotide comprises a 3′-overhang sequence of two nucleotides in length, wherein the 3′-overhang sequence is present on the antisense strand, and wherein 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.
  • 32. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified nucleotide.
  • 33. The oligonucleotide of claim 32, wherein the modified nucleotide comprises a 2′-modification.
  • 34. The oligonucleotide of claim 33, wherein 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.
  • 35. The oligonucleotide of any one of claims 32 to 34, wherein all of the nucleotides of the oligonucleotide are modified.
  • 36. The oligonucleotide of any one of the preceding claims, wherein the oligonucleotide comprises at least one modified internucleotide linkage.
  • 37. The oligonucleotide of claim 36, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • 38. The oligonucleotide of any one of the preceding claims, wherein the 4′-carbon of the sugar of the 5′-nucleotide of the antisense strand comprises a phosphate analog.
  • 39. The oligonucleotide of claim 38, wherein the phosphate analog is oxymethylphosphonate, vinylphosphonate, or malonylphosphonate.
  • 40. The oligonucleotide of any one of the preceding claims, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.
  • 41. The oligonucleotide of claim 40, wherein each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide or lipid.
  • 42. The oligonucleotide of claim 41, wherein each targeting ligand comprises a N-acetylgalactosamine (GalNAc) moiety.
  • 43. The oligonucleotide of claim 42, wherein the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.
  • 44. The oligonucleotide of any one of claims 19 to 24, wherein up to 4 nucleotides of L of the stem-loop are each conjugated to a monovalent GalNAc moiety.
  • 45. The oligonucleotide of claim 40, wherein the targeting ligand comprises an aptamer.
  • 46. A composition comprising an oligonucleotide of any one of the preceding claims and an excipient.
  • 47. A method of delivering an oligonucleotide to a subject, the method comprising administering the composition of claim 46 to the subject.
  • 48. A method of decreasing one or more symptoms of hypercholesterolemia, atherosclerosis, and/or one or more symptoms or complications thereof in a subject, the method comprising administering the composition of claim 46 to the subject.
  • 49. An oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising a sense strand of 15 to 50 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 1 to 453, 907-1029, 1153-1192, 1248-1256, and 1266-1268 and wherein the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 454-906, 1030-1152, 1193-1232, 1257-1265 and 1269-1271.
  • 50. An oligonucleotide for reducing expression of PCSK9, the oligonucleotide comprising a pair of sense and antisense strands selected from a row of the table set forth in Table 4.
RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/659,693, filed Apr. 18, 2018, and entitled “PCSK9 TARGETING OLIGONUCLEOTIDES FOR TREATING HYPERCHOLESTEROLEMIA AND RELATED CONDITIONS,” and U.S. Provisional Application No. 62/820,558, filed Mar. 19, 2019, and entitled “PCSK9 TARGETING OLIGONUCLEOTIDES FOR TREATING HYPERCHOLESTEROLEMIA AND RELATED CONDITIONS,” the entire contents of which are incorporated herein by reference.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2019/025253 4/1/2019 WO 00
Provisional Applications (2)
Number Date Country
62659693 Apr 2018 US
62820558 Mar 2019 US