RNAi agent for suppressing PNPLA3 expression, pharmaceutical composition thereof, and method of use

Abstract

The present disclosure relates to RNAi agents capable of inhibiting the expression of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene, such as double-stranded RNAi agents. Also disclosed are pharmaceutical compositions comprising the PNPLA3 RNAi agents and methods of using the same. The PNPLA3 RNAi agents disclosed herein can be conjugated to a targeting ligand to facilitate delivery to cells, including delivery to hepatocytes. In vivo delivery of the PNPLA3 RNAi agents provides inhibition of PNPLA3 gene expression. The RNAi agents can be used in methods for treating PNPLA3-related diseases and disorders, which diseases and disorders include non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, and alcoholic or non-alcoholic liver diseases, including cirrhosis.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 000,137, filed Mar. 26, 2020, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on Mar. 25, 2021, named 103693_002475_PCT_SL.txt, and is 248,025 bytes in size. Technical Field

[0005] The present disclosure relates to RNA interference (RNAi) agents for inhibiting patatin-like phospholipase domain-containing protein 3 (PNPLA3), such as double-stranded RNAi agents, pharmaceutical compositions comprising a PNPLA3 RNAi agent, and methods of using the same. Background Art

[0006] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world and is estimated to affect approximately 20% of the world's population. In some individuals, the accumulation of ectopic fat in the liver (termed steatosis) triggers inflammation and hepatocyte injury, leading to more advanced stages of the disease, termed non-alcoholic steatohepatitis (NASH). Treatment of NAFLD generally involves weight loss and treatment of any secondary conditions, such as insulin resistance or dyslipidemia.

[0007] Patatin-like phospholipase domain-containing 3 (PNPLA3), a type II transmembrane protein, is expressed in a variety of cells including those in the liver. In hepatocytes, PNPLA3 is expressed on the endoplasmic reticulum and lipid membranes and predominantly exhibits triacylglycerol hydrolase activity.

[0008] The present invention represents a novel method for reducing PNPLA3 levels and treating liver diseases such as NAFLD. Summary of the Invention

[0009] Disclosed herein are RNAi agents for inhibiting the expression of the PNPLA3 gene, the RNAi agent comprising an antisense strand comprising at least 17 contiguous nucleotides that differ from any of the sequences of SEQ ID NOs: 46-60, 176, 181, and 188 by 0 or 1 nucleotide; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

[0010] In some embodiments, the antisense strand comprises nucleotides 2-18 of any one of the sequences of SEQ ID NO: 46-60, 176, 181, and 188.

[0011] In some embodiments, the sense strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differs from the sense strand sequence of any one of SEQ ID NO: 2, 3, 4, 9-20, 214, 219, and 220 by 0 or 1 nucleotide, and wherein the sense strand has a region that is at least 85% complementary to the antisense strand over 17 consecutive nucleotides.

[0012] In some embodiments, at least one nucleotide of the RNAi agent is a modified nucleotide or comprises a modified internucleoside bond.

[0013] According to some embodiments, all or substantially all nucleotides of the sense and / or antisense strands of the RNAi agent are modified nucleotides.

[0014] In some embodiments, the modified nucleotides are selected from the group consisting of: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-acyclic nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxy nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, cyclopropyl phosphonate-containing nucleotides, and 3'-O-methyl nucleotides.

[0015] In other embodiments, all or substantially all of the modified nucleotides are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

[0016] In some embodiments, the antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of SEQ ID NO: 90, 95, and 102.

[0017] In some embodiments, the sense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified sense strand sequences of SEQ ID NO: 131, 136, and 137.

[0018] In some embodiments, the antisense strand comprises the nucleotide sequence of any one of the modified sequences of SEQ ID NO: 90, 95, and 102, and the sense strand comprises the nucleotide sequence of any one of the modified sequences of SEQ ID NO: 131, 136, and 137.

[0019] In other embodiments, the RNAi agent is linked to a targeting ligand. In some embodiments, the targeting ligand comprises N-acetyl-galactosamine. In still other embodiments, the targeting ligand comprises the structure of (NAG37) or (NAG37)s. In additional embodiments, the targeting ligand is linked to the sense strand. In some embodiments, the targeting ligand is linked to the 5' end of the sense strand.

[0020] In some embodiments, the length of the targeting sense strand is between 18 and 30 nucleotides, and the length of the antisense strand is between 18 and 30 nucleotides. In other embodiments, the lengths of the targeting sense strand and the antisense strand are each between 18 and 27 nucleotides. In other embodiments, the lengths of the targeting sense strand and the antisense strand are each between 18 and 24 nucleotides. In still other embodiments, the lengths of the sense strand and the antisense strand are each 21 nucleotides.

[0021] In some embodiments, the RNAi agent has two blunt ends.

[0022] In some embodiments, the sense strand comprises one or two terminal caps. In other embodiments, the sense strand comprises one or two inverted abasic residues.

[0023] In some embodiments, the RNAi agent is composed of a sense strand and an antisense strand that form a duplex sequence having SEQ ID NO: (176 and 214); (90 and 131); (181 and 219); (95 and 136); (188 and 220); and / or (102 and 137).

[0024] In some embodiments, the sense strand further comprises an inverted abasic residue at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or at both.

[0025] In some aspects, the RNAi agents provided herein comprise an antisense strand that comprises, consists of, or consists essentially of a modified nucleotide sequence that differs from one of the nucleotide sequences of SEQ ID NOs: 90, 95, and 102 by 0 or 1 nucleotide; wherein a, c, g, and u represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate bond; and wherein all or substantially all of the nucleotides on the sense strand are modified nucleotides.

[0026] In some embodiments, the disclosed sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs from one of the nucleotide sequences of SEQ ID NO: 214, 219, and 256 by 0 or 1 nucleotide; wherein a, c, g, i, and u represent 2'-O-methyladenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate bond; and wherein all or substantially all of the nucleotides on the antisense strand are modified nucleotides. In other embodiments, the sense strand further comprises a reverse abasic residue at the 3'-end, at the 5'-end, or at both ends of the nucleotide sequence. In other embodiments, the sense strand of the RNAi agent is linked to a targeting ligand.

[0027] In some embodiments, the targeting ligand has an affinity for the asialoglycoprotein receptor. In some embodiments, the targeting ligand comprises N-acetyl-galactosamine.

[0028] In additional embodiments, the targeting ligand comprises:

[0029]

[0030] In additional embodiments, the antisense strand consists of the modified nucleotide sequences of SEQ ID NO: 90, 95, and 102, and the sense strand consists of the modified nucleotide sequences of SEQ ID NO: 131, 136, and 137; wherein a, c, g, and u are 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s is a phosphorothioate bond; (invAb) is a reverse abasic deoxyribose residue; and (NAG37)s has the following chemical structure:

[0031]

[0032] Also disclosed herein is a composition comprising the disclosed RNAi agent, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0033] Also provided herein is a method for inhibiting the expression of the PNPLA3 gene in a cell, the method comprising introducing an effective amount of the disclosed RNAi agent or the disclosed composition into the cell.

[0034] In some embodiments, the cell is in a subject. In some embodiments, the subject is a human subject.

[0035] In other embodiments, PNPLA3 gene expression is inhibited by at least about 30%. In some embodiments, PNPLA3 gene expression is inhibited by at least about 50% in the cytoplasm of hepatocytes.

[0036] Also provided herein are methods of treating PNPLA3-related diseases or disorders, the methods comprising administering to a human subject in need thereof a therapeutically effective amount of the disclosed compositions.

[0037] In some embodiments, the disease is NAFLD, NASH, liver fibrosis, alcoholic fatty liver disease, or cirrhosis.

[0038] In some embodiments, the RNAi agent is administered at a dose of from about 0.05 mg / kg to about 5.0 mg / kg of the human subject's body weight.

[0039] In other embodiments, the RNAi agent is administered in two or more doses.

[0040] Also provided herein is the use of the disclosed RNAi agent or the disclosed composition for treating a disease, disorder, or symptom that is at least partially mediated by PNPLA3 gene expression.

[0041] In some embodiments, the symptom is liver cirrhosis.

[0042] Further provided herein is the use of the disclosed RNAi agent or the disclosed composition for preparing a pharmaceutical composition for treating a disease, disorder, or symptom that is at least partially mediated by PNPLA3 gene expression.

[0043] In some embodiments, the disease is NAFLD, NASH, liver fibrosis, or an alcoholic or non-alcoholic liver disease such as cirrhosis. In some embodiments, the RNAi agent is administered at a dose of from about 0.05 mg / kg to about 5.0 mg / kg of the human subject's body weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a graph depicting the baseline relative whole liver expression of PNPLA3 obtained by PCR from non-human primates (NHPs) treated with the RNAi agent. Study #1: Data are geometric mean ± SD (n = 4).

[0045] Figure 2 is a graph depicting the average PNPLA3 mRNA knockdown in the cytoplasm of hepatocytes by quantitative ISH among all test animals (Study #1 and Study #2). Study #2: Data are mean ± SD (n = 14). DETAILED DESCRIPTION

[0046] The method disclosed by the present invention can be more easily understood by combining the accompanying drawings that form a part of the present disclosure and referring to the following detailed description. It should be understood that the method disclosed by the present invention is not limited to the specific methods described and / or illustrated herein, and the terms used herein are only used to describe specific embodiments by way of example and are not intended to limit the method protected by the claims.

[0047] It should be understood that certain features of the method disclosed by the present invention are described in the context of each separate embodiment herein for clarity, but can also be provided in combination in a single embodiment. Conversely, the various features of the method disclosed herein are described in the context of a single embodiment for brevity and can also be provided separately or in any sub-combination.

[0048] Definitions

[0049] As used herein, an "RNAi agent" (also referred to as an "RNAi trigger") refers to a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrading or inhibiting under appropriate conditions) the translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. As used herein, an RNAi agent can act through the RNA interference mechanism (i.e., by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells to induce RNA interference) or through any alternative one or more mechanisms or one or more pathways. It is believed that although the term RNAi agent as used herein acts primarily through the RNA interference mechanism, the RNAi agents disclosed by the present invention are not restricted or limited by any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand and include, but are not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA (i.e., PNPLA3 mRNA). The RNAi agent can comprise one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0050] As used herein, when referring to the expression of a given gene, the terms "silence", "reduce", "inhibit", "downregulate" or "knock down" mean that when a cell, cell population, tissue, organ or subject is treated with an RNAi agent as described herein, the expression of the gene is reduced compared to a second cell, cell population, tissue, organ or subject that has not or has not yet been so treated, as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein or protein subunit translated from the mRNA in the cell, cell population, tissue, organ or subject in which the gene is transcribed.

[0051] As used herein, the terms "sequence" and "nucleotide sequence" mean a succession or order of nucleobases or nucleotides described by a series of letters using standard nomenclature. Nucleic acid molecules can include unmodified and / or modified nucleotides. Nucleotide sequences can include unmodified and / or modified nucleotides.

[0052] As used herein, "base", "nucleobase", "nucleotide" or "nucleobase" is a heterocyclic pyrimidine or purine compound that is a component of a polynucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine and uracil. Nucleotides can be unmodified. Nucleotides can be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed., Wiley-VCH, 2008). The synthesis of such modified nucleotides (including phosphoramidite compounds containing modified nucleotides) is known in the art.

[0053] As used herein, and unless otherwise specified, when used to describe the correlation between a first nucleobase or nucleotide sequence (e.g., the sense strand of an RNAi agent or a target mRNA) and a second nucleobase or nucleotide sequence (e.g., the antisense strand of an RNAi agent or a single-stranded antisense oligonucleotide), the term "complementary" means the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or other suitable in vivo or in vitro conditions)) with an oligonucleotide comprising the second nucleotide sequence and to form a duplex or double helix structure under certain standard conditions. One of ordinary skill in the art will be able to select a set of conditions most suitable for the hybridization test. Complementary sequences include Watson Crick base pairs or non-Watson Crick base pairs and include natural or modified nucleotides or nucleotide mimics to at least the extent that the above hybridization requirements are met. Sequence identity or complementarity is independent of modifications. For example, for purposes of determining identity or complementarity, as defined herein, a and Af are complementary to U (or T) and identical to A.

[0054] As used herein, "fully complementary" or "entirely complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in the continuous sequence of the first oligonucleotide will hybridize with the same number of bases in the continuous sequence of the second oligonucleotide. The continuous sequence may comprise all or part of the first or second nucleotide sequence.

[0055] As used herein, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the continuous sequence of the first oligonucleotide will hybridize with the same number of bases in the continuous sequence of the second oligonucleotide. The continuous sequence may comprise all or part of the first or second nucleotide sequence.

[0056] As used herein, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the continuous sequence of the first oligonucleotide will hybridize with the same number of bases in the continuous sequence of the second oligonucleotide. The continuous sequence may comprise all or part of the first or second nucleotide sequence.

[0057] As used herein, the terms "complementary", "fully complementary", "partially complementary" and "substantially complementary" are used with respect to nucleobase or nucleotide matches between the sense and antisense strands of an RNAi agent or between the antisense strand of an RNAi agent and the sequence of PNPLA3 mRNA.

[0058] As used herein, when applied to a nucleic acid sequence, the terms "substantially identical" or "substantial identity" mean that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% or higher sequence identity compared to a reference sequence, such as at least 90%, at least 95%, or at least 99% identity. The percent sequence identity is determined by comparing two optimally aligned sequences in a comparison window. The percent is calculated by determining the number of positions at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and then multiplying the result by 100 to yield the percent sequence identity. The inventions disclosed herein encompass nucleotide sequences that are substantially identical to those disclosed herein.

[0059] As used herein, the terms "individual," "patient," and "subject" are used interchangeably to refer to a member of any animal species, including but not limited to birds, humans, and other primates, as well as other mammals, including commercially relevant mammals or animal models, such as mice, rats, monkeys, cows, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.

[0060] As used herein, the terms "treat," "treatment," etc. refer to a method or step taken to alleviate or reduce the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" can include prevention, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0061] As used herein, when referring to an RNAi agent, the phrase "introduced into a cell" means functionally delivering the RNAi agent to the cell. The phrase "functional delivery" means delivering the RNAi agent to the cell in a manner such that the RNAi agent has the desired biological activity (e.g., sequence-specific inhibition of gene expression).

[0062] Unless otherwise indicated, the symbols used herein are used to mean that any one or more groups can be attached thereto, which is in accordance with the scope of the inventions described herein.

[0063] As used herein, the term "isomer" refers to a compound having the same molecular formula but different in the nature or order of bonding of its atoms or in the arrangement of its atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereoisomers," and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes "optical isomers." A carbon atom bonded to four different substituents is called a "chiral center."

[0064] As used herein, unless specifically indicated that a structure has a particular conformation, for each structure in which there are asymmetric centers and thus enantiomers, diastereomers or other stereoisomeric configurations are generated, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure forms and racemic forms. For example, the structures disclosed herein are intended to cover mixtures of diastereomers as well as individual stereoisomers.

[0065] As used herein, the terms "comprising", "having", "including", and "characterized by" are interchangeable, inclusive, open-ended, and do not exclude additional, unrecited elements or method steps. Any recitation of the term "comprising" herein, particularly in the description of the components of a composition or the elements of a device, should be understood to cover those compositions and methods consisting essentially of and consisting of the recited components or elements.

[0066] As used in the claims herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. As used in the claims herein, the phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those materials or steps that do not materially affect one or more of the basic and novel features of the claimed invention.

[0067] One of ordinary skill in the art will readily understand and appreciate that, depending on the environment in which a compound or composition is placed, certain atoms (e.g., N, O, or S atoms) of the compounds and compositions disclosed herein may be in a protonated or deprotonated state. Thus, as used herein, the structures disclosed herein contemplate that certain functional groups (e.g., OH, SH, or NH) may be protonated or deprotonated. The present disclosure is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the environment (such as pH), as will be readily understood by one of ordinary skill in the art. Accordingly, compounds described herein having labile protons or basic atoms should also be understood to represent the salt forms of the corresponding compounds. The compounds described herein may be in free acid, free base, or salt form. Pharmaceutically acceptable salts of the compounds described herein are understood to be within the scope of the present invention.

[0068] As used herein, when referring to the connection between two compounds or molecules, the terms "connected" or "conjugated" mean that the two compounds or molecules are connected by a covalent bond. Unless stated otherwise, the terms "connected" and "conjugated" as used herein may refer to the connection between a first compound and a second compound, with or without any intermediate atoms or groups of atoms.

[0069] As used herein, the term "including" is used herein to mean the phrase "including but not limited to" and may be used interchangeably with that phrase. Unless the context clearly indicates otherwise, the term "or" is used herein to mean the term "and / or" and may be used interchangeably with that term.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar to or equivalent to those described herein may be used in the operation or testing of the present invention, the following describes suitable methods and materials. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification and its definitions shall control. In addition, the materials, methods and examples are illustrative only and not intended to be limiting.

[0071] Other objects, features, aspects and advantages of the present invention will be apparent from the following detailed description, the accompanying drawings and the claims.

[0072] Detailed Description

[0073] RNAi Agent

[0074] This text describes RNAi agents (referred to herein as PNPLA3 RNAi agents or PNPLA3 RNAi triggers) for inhibiting the expression of the PNPLA3 gene. Each PNPLA3 RNAi agent comprises a sense strand and an antisense strand. The length of each of the sense strand and the antisense strand can be from 16 to 49 nucleotides. The sense strand and the antisense strand can have the same length or they can be of different lengths. In some embodiments, the length of each of the sense strand and the antisense strand is independently from 18 to 27 nucleotides. In some embodiments, the length of each of the sense strand and the antisense strand is from 21 to 26 nucleotides. In some embodiments, the length of each of the sense strand and the antisense strand is from 21 to 24 nucleotides. In some embodiments, the length of each of the sense strand and the antisense strand is independently from 19 to 21 nucleotides. In some embodiments, the length of the sense strand is about 19 nucleotides and the length of the antisense strand is about 21 nucleotides. In some embodiments, the length of the sense strand is about 21 nucleotides and the length of the antisense strand is about 23 nucleotides. In some embodiments, the length of the sense strand is 23 nucleotides and the length of the antisense strand is 21 nucleotides. In some embodiments, the length of each of the sense strand and the antisense strand is 21 nucleotides. In some embodiments, the length of each of the sense strand and the antisense strand of the RNAi agent is independently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 nucleotides. In some embodiments, the double-stranded RNAi agent has a duplex length of about 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

[0075] Examples of nucleotide sequences for forming PNPLA3 RNAi agents are provided in Tables 2, 3 and 4. Examples of RNAi agent duplexes comprising the sense strand and antisense strand sequences in Tables 2, 3 and 4 are shown in Tables 5A and 5B.

[0076] In some embodiments, the length of the region of complete, substantial or partial complementarity between the sense strand and the antisense strand is from 16 to 26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26) nucleotides and occurs at or near the 5' end of the antisense strand (e.g., the region of incomplete, substantially or partially complementarity can be separated from the 5' end of the antisense strand by 0, 1, 2, 3 or 4 nucleotides).

[0077] The sense strand of the PNPLA3 RNAi agent described herein comprises at least 16 contiguous nucleotides that have at least 85% identity with a core stretch of the same number of nucleotides in PNPLA3 mRNA (also referred to herein as the "core stretch" or "core sequence"). In some embodiments, the sense strand core stretch is 100% (fully) complementary or at least about 85% (substantially) complementary to the core stretch in the antisense strand, such that the sense strand core stretch is generally identical or at least about 85% identical to the nucleotide sequence of the same length present in the PNPLA3 mRNA target (sometimes referred to as, e.g., the target sequence). In some embodiments, the length of the sense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the length of the sense strand core stretch is 17 nucleotides. In some embodiments, the length of the sense strand core stretch is 19 nucleotides.

[0078] The antisense strand of the PNPLA3 RNAi agent described herein comprises at least 16 contiguous nucleotides that have at least 85% complementarity with a core stretch of the same number of nucleotides in PNPLA3 mRNA and with a core stretch of the same number of nucleotides in the corresponding sense strand. In some embodiments, the antisense strand core stretch is 100% (fully) complementary or at least about 85% (substantially) complementary to the nucleotide sequence of the same length present in the PNPLA3 mRNA target (e.g., the target sequence). In some embodiments, the length of the antisense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the length of the antisense strand core stretch is 19 nucleotides. In some embodiments, the length of the antisense strand core stretch is 17 nucleotides. The sense strand core stretch sequence may be the same length as the corresponding antisense core sequence or it may be a different length.

[0079] The sense and antisense strands of the PNPLA3 RNAi agent anneal to form a duplex. The sense and antisense strands of the PNPLA3 RNAi agent may be partially, substantially, or completely complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least 85% complementary or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence comprises a sequence of 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 nucleotides that is at least 85% or 100% complementary to the corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of the PNPLA3 RNAi agent have a region of 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 nucleotides with at least 85% base pairing or 100% base pairing).

[0080] In some embodiments, the antisense strand of the PNPLA3 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 or Table 3 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the PNPLA3 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2 or Table 4 by 0, 1, 2, or 3 nucleotides.

[0081] In some embodiments, the sense and / or antisense strand may optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extensions) at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the core stretch sequence. The additional nucleotides of the antisense strand (if present) may or may not be complementary to the corresponding sequence in the PNPLA3 mRNA. The additional nucleotides of the sense strand (if present) may be the same as or different from the corresponding sequence in the PNPLA3 mRNA. The additional nucleotides of the antisense strand (if present) may or may not be complementary to the additional nucleotides of the corresponding sense strand (if present).

[0082] As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' end of a sense strand core stretch sequence and / or an antisense strand core stretch sequence. Extension nucleotides on the sense strand may or may not be complementary to nucleotides (core stretch sequence nucleotides or extension nucleotides) in the corresponding antisense strand. Conversely, extension nucleotides on the antisense strand may or may not be complementary to nucleotides (core stretch nucleotides or extension nucleotides) in the corresponding sense strand. In some embodiments, both the sense and antisense strands of the RNAi agent comprise 3' and 5' extensions. In some embodiments, one or more of the 3' extension nucleotides of one strand base pair with one or more of the 5' extension nucleotides of the other strand. In other embodiments, one or more of the 3' extension nucleotides of one strand do not base pair with one or more of the 5' extension nucleotides of the other strand. In some embodiments, a PNPLA3 RNAi agent has an antisense strand with a 3' extension and a sense strand with a 5' extension. In some embodiments, the extension nucleotides are unpaired and form overhangs. As used herein, an "overhang" refers to a stretch of one or more unpaired nucleotides located at the end of a sense or antisense strand that is not part of the hybridized or duplexed portion of the RNAi agent disclosed herein.

[0083] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand with a 3' extension that is 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, the PNPLA3 RNAi agent comprises an antisense strand with a 3' extension that is 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding PNPLA3 mRNA sequence. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding PNPLA3 mRNA sequence.

[0084] In some embodiments, the PNPLA3 RNAi agent comprises a sense strand with a 3' extension that is 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise adenosine, uracil, or thymine nucleotides, an AT dinucleotide, or nucleotides corresponding or identical to nucleotides in the PNPLA3 mRNA sequence. In some embodiments, the 3' sense strand extension comprises, but is not limited to, one of the following sequences: T, UT, TT, UU, UUT, TTT, or TTTT (listed 5' to 3'), or consists of them.

[0085] The sense strand may have a 3' extension and / or a 5' extension. In some embodiments, the PNPLA3 RNAi agent comprises a sense strand with a 5' extension that is 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the nucleotides of the sense strand extension comprise nucleotides corresponding to or identical to nucleotides in the PNPLA3 mRNA sequence.

[0086] Examples of sequences for forming the PNPLA3 RNAi agent are provided in Tables 2, 3, and 4. In some embodiments, the antisense strand of the PNPLA3 RNAi agent comprises the sequence of any one of the sequences in Table 2 or Table 3. In certain embodiments, the antisense strand of the PNPLA3 RNAi agent comprises or consists of any one of the modified sequences in Table 3. In some embodiments, the antisense strand of the PNPLA3 RNAi agent includes the nucleotide sequence (from the 5' end → 3' end) 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 of any one of the sequences in Table 2 or Table 3. In some embodiments, the sense strand of the PNPLA3 RNAi agent comprises the sequence of any one of the sequences in Table 2 or Table 4. In some embodiments, the sense strand of the PNPLA3 RNAi agent includes the nucleotide sequence (from the 5' end → 3' end) 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any one of the sequences in Table 2 or Table 4. In certain embodiments, the sense strand of the PNPLA3 RNAi agent comprises or consists of the modified sequence of any one of the modified sequences in Table 4.

[0087] In some embodiments, the sense and antisense strands of the RNAi agents described herein comprise the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein comprise different numbers of nucleotides. In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form blunt ends. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form blunt ends. In some embodiments, both ends of the RNAi agent form blunt ends. In some embodiments, neither end of the RNAi agent is a blunt end. As used herein, "blunt end" refers to the end of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (forming complementary base pairs).

[0088] In some embodiments, the 5'-end of the sense strand and the 3'-end of the antisense strand of the RNAi agent form frayed ends. In some embodiments, the 3'-end of the sense strand and the 5'-end of the antisense strand of the RNAi agent form frayed ends. In some embodiments, both ends of the RNAi agent form frayed ends. In some embodiments, neither end of the RNAi agent is a frayed end. As used herein, a frayed end refers to an end of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed strands form a pair (i.e., do not form an overhang) but are not complementary (i.e., form a non-complementary pair). In some embodiments, one or more unpaired nucleotides at the end of one strand of the double-stranded RNAi agent form an overhang. The unpaired nucleotides can be located on the sense strand or the antisense strand, thereby creating a 3' or 5' overhang. In some embodiments, the RNAi agent comprises: blunt ends and frayed ends, blunt ends and 5' overhang ends, blunt ends and 3' overhang ends, frayed ends and 5' overhang ends, frayed ends and 3' overhang ends, two 5' overhang ends, two 3' overhang ends, 5' overhang ends and 3' overhang ends, two frayed ends or two blunt ends. Generally, when present, the overhangs are located at the 3'-ends of the sense strand, the antisense strand, or both the sense strand and the antisense strand.

[0089] The PNPLA3 RNAi agents disclosed herein can also be composed of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand of the PNPLA3 RNAi agent are modified nucleotides. The PNPLA3 RNAi agents disclosed herein can also be composed of one or more modified internucleoside linkages, such as one or more phosphorothioate linkages. In some embodiments, the PNPLA3 RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, 2'-modified nucleotides are mixed with modified internucleoside linkages.

[0090] In some embodiments, the PNPLA3 RNAi agent is prepared or provided as a salt, mixed salt, or free acid. In some embodiments, the PNPLA3 RNAi agent is prepared as a sodium salt. Such forms, well known in the art, are within the scope of the invention disclosed herein.

[0091] Modified Nucleotide

[0092] When used in various oligonucleotide constructs, modified nucleotides can retain the activity of the compounds in cells while increasing the serum stability of these compounds and also minimizing the likelihood of activating interferon activity in the human body when the oligonucleotide constructs are administered.

[0093] In some embodiments, the PNPLA3 RNAi agent comprises one or more modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxynucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, inverted nucleotides, nucleotides containing modified nucleobases, bridged nucleotides, peptide nucleic acids (PNAs), 2',3'-seco-nucleotide mimics (unlocked nucleobase analogs), locked nucleotides, 3'-O-methoxy (2'-internucleoside linked) nucleotides, 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, morpholino nucleotides, deoxyribonucleotides containing vinyl phosphonates, nucleotides containing vinyl phosphonates, and nucleotides containing cyclopropyl phosphonates. 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2'-position of the pentose sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-fluoronucleotides (also referred to herein as 2'-deoxy-2'-fluoronucleotides), 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (also referred to as 2'-MOE), 2'-aminonucleotides, and 2'-alkyl nucleotides. All sites in a given compound need not be uniformly modified. Instead, more than one modification can be incorporated in a single PNPLA3 RNAi agent or even in a single nucleotide thereof. The sense and antisense strands of the PNPLA3 RNAi agent can be synthesized and / or modified by methods known in the art. Modifications on one nucleotide are independent of modifications on another nucleotide.

[0094] Modified nucleobases include synthetic and natural nucleobases such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azauracil, 6-azacytosine, 6-azathymine, 5-uridine (pseudouridine), 4-thiouracil, 8-halogen, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0095] In some embodiments, the 5' end and / or 3' end of the antisense strand can include abasic residues (Ab), which can also be referred to as "abasic sites" or "abasic nucleotides". An abasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the sugar moiety. (See, e.g., U.S. Patent No. 5,998,203). In some embodiments, abasic residues can be placed internally within a nucleotide sequence. In some embodiments, Ab or AbAb can be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab is added to the 3' end of the sense strand. In some embodiments, the abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.

[0096] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are ribonucleotides (i.e., unmodified) in both the sense and antisense strands. As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the sense strand. As used herein, an antisense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides that are unmodified ribonucleotides in the antisense strand. In some embodiments, one or more of the nucleotides of the RNAi agent are unmodified ribonucleotides.

[0097] Modified Internucleoside Bond

[0098] In some embodiments, one or more of the nucleotides of the PNPLA3 RNAi agent are linked by non-standard bonds or backbones (i.e., modified internucleoside bonds or modified backbones). Modified internucleoside bonds or backbones include, but are not limited to: phosphorothioate groups (represented herein as lowercase "s"), chiral phosphorothioates, phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thiophosphoramidate), thioalkyl-phosphonates, thioalkylphosphates, morpholino bonds, boranophosphates with normal 3'-5' bonds, boranophosphate analogs with 2'-5' bonds, or boranophosphates with reverse polarity in which adjacent nucleotide units are 3'-5' to 5'-3' or 2'-5' to 5'-2' bonds. In some embodiments, the modified internucleoside bond or backbone does not contain a phosphorus atom. Modified internucleoside bonds that do not contain a phosphorus atom include, but are not limited to: short-chain alkyl or cycloalkyl sugar-sugar bonds, mixed heteroatom and alkyl or cycloalkyl sugar-sugar bonds, or one or more short-chain heteroatom or heterocyclic sugar-sugar bonds. In some embodiments, the modified internucleoside backbone includes, but is not limited to: siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, olefin-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazine backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having a mixture of N, O, S, and CH2 components.

[0099] In some embodiments, the sense strand of the PNPLA3 RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, the antisense strand of the PNPLA3 RNAi agent may contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds, and both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate bonds. In some embodiments, the sense strand of the PNPLA3 RNAi agent may contain 1, 2, 3, or 4 phosphorothioate bonds, the antisense strand of the PNPLA3 RNAi agent may contain 1, 2, 3, or 4 phosphorothioate bonds, and both the sense strand and the antisense strand may independently contain 1, 2, 3, or 4 phosphorothioate bonds.

[0100] In some embodiments, the sense strand of the PNPLA3 RNAi agent contains at least two phosphorothioate internucleoside bonds. In some embodiments, the phosphorothioate internucleoside bonds are located between nucleotides at positions 1 - 3 starting from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside bond is at the 5' end of the nucleotide sequence of the sense strand, and another phosphorothioate bond is at the 3' end of the nucleotide sequence of the sense strand. In some embodiments, two phosphorothioate internucleoside bonds are located at the 5' end of the nucleotide sequence of the sense strand, and another phosphorothioate bond is at the 3' end of the sense strand. In some embodiments, the sense strand does not include any phosphorothioate internucleoside bonds between nucleotides, but contains one, two, or three phosphorothioate bonds between the terminal nucleotides at the 5' and 3' ends and an optionally present reverse abasic residue terminal cap. In some embodiments, the targeting ligand is linked to the sense strand via a phosphorothioate bond.

[0101] In some embodiments, the antisense strand of the PNPLA3 RNAi agent contains four phosphorothioate internucleoside bonds. In some embodiments, the four phosphorothioate internucleoside bonds are located between nucleotides at positions 1 - 3 starting from the 5' end of the antisense strand and between nucleotides at positions 19 - 21, 20 - 22, 21 - 23, 22 - 24, 23 - 25, or 24 - 26 starting from the 5' end. In some embodiments, three phosphorothioate internucleoside bonds are located between positions 1 - 4 starting from the 5' end of the antisense strand, and the fourth phosphorothioate internucleoside bond is located between positions 20 - 21 starting from the 5' end of the antisense strand. In some embodiments, the PNPLA3 RNAi agent contains at least three or four phosphorothioate internucleoside bonds in the antisense strand.

[0102] Blocking Residue or Moiety

[0103] In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as "caps", "terminal caps" or "capping residues". As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of the nucleotide sequence of an RNAi agent disclosed herein. In some cases, the capping residue can provide certain beneficial properties to the RNAi agent, such as protection against exonuclease degradation. In some embodiments, an inverted abasic residue (invAb) (also referred to in the art as an "inverted abasic site") is added as a capping residue. (See, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art and include, for example, inverted abasic residues as well as carbon chains such as terminal C3H7 (propyl), C6H 13 (hexyl) or C 12 H 25 (dodecyl) groups. In some embodiments, the capping residue is present at the 5'-terminus, 3'-terminus or both the 5'- and 3'-termini of the sense strand. In some embodiments, the 5'-end and / or 3'-end of the sense strand may contain more than one inverted abasic deoxyribose moiety as a capping residue.

[0104] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3'-end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5'-end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites can be inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, one or more inverted abasic residues or inverted abasic sites are included at or near one or more termini of the sense strand of the RNAi agent, allowing for enhanced activity or other desired properties of the RNAi agent.

[0105] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues may be inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown as (invAb)s herein), or other internucleoside linkages. In some embodiments, inclusion of one or more inverted abasic residues at or near one or more termini of the sense strand of the RNAi agent may permit enhanced activity or other desired properties of the RNAi agent. In some embodiments, the inverted abasic (deoxyribose) residues may be replaced with inverted ribitol (abasic ribose) residues. In some embodiments, the 3' end of the antisense strand core stretch sequence or the 3' end of the antisense strand sequence may include inverted abasic residues. The chemical structure of the inverted abasic deoxyribose residues is shown in Table 6 below.

[0106] PNPLA3 RNAi Agent

[0107] The PNPLA3 RNAi agents disclosed herein are designed to target specific positions on the PNPLA3 gene (e.g., SEQ ID NO:1).

[0108] NM_025225.2 Homo sapiens patatin-like phospholipase domain-containing 3 (PNPLA3), mRNA (SEQ ID NO:1):

[0109]

[0110] As defined herein, the antisense strand sequences are designed to target the PNPLA3 gene at a given position on the gene when the 5'-terminal nucleobase of the antisense strand aligns with a position 19 nucleotides downstream (towards the 3'-end) from the position on the gene when base-paired with the gene bases. For example, as shown in Tables 1 and 2 herein, the antisense strand sequence designed to target the PNPLA3 gene at position 2180 requires that when base-paired with the gene bases, the 5'-terminal nucleobase of the antisense strand aligns with position 2198 of the PNPLA3 gene.

[0111] As provided herein, the PNPLA3 RNAi agent does not require the nucleobase at position 1 (5'→3') of the antisense strand to be complementary to the gene, provided that there is at least 85% complementarity of the antisense strand with the gene on a core extension sequence of at least 16 consecutive nucleotides (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity). For example, for the PNPLA3 RNAi agent disclosed herein that is designed to target position 2180 of the PNPLA3 gene, the 5'-terminal nucleobase of the antisense strand of the PNPLA3 RNAi agent must align with position 2198 of the gene; however, the 5'-terminal nucleobase of the antisense strand may but is not required to be complementary to position 2200 of the PNPLA3 gene, provided that there is at least 85% complementarity of the antisense strand with the gene on a core extension sequence of at least 16 consecutive nucleotides (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity). Among other things, as shown in the various examples disclosed herein, binding of the antisense strand of the PNPLA3 RNAi agent to a specific site on the gene (e.g., whether the PNPLA3 RNAi agent is designed to target the PNPLA3 gene at position 2180, 1586, 1179 or some other position) is important for the level of inhibition achieved by the PNPLA3 RNAi agent.

[0112] In some embodiments, the PNPLA3 RNAi agents disclosed herein target the PNPLA3 gene at or near the PNPLA3 gene sequence positions shown in Table 1. In some embodiments, the antisense strand of the PNPLA3 RNAi agents disclosed herein includes a core stretch sequence that is fully, substantially or at least partially complementary to the target PNPLA3 19-mer sequence disclosed in Table 1.

[0113] Table 1. PNPLA3 19 - mer mRNA Target Sequences (from Homo sapiens containing patatin - like phospholipase domain 3 (PNPLA3), mRNA, GenBank NM_025225.2 (SEQ ID NO:1)) 1

[0114]

[0115] 1 When referring to the gene positions in this text, the Applicant uses Genebank NM_025225.2 as the reference gene for human PNPLA3. On or around February 9, 2020, the gene sequence was updated to NM_025225.3. Although the reference to the updated gene will change the numbers identified as "target gene positions" in Table 1 above, this has no impact on the nucleotide sequences used in the RNAi agents disclosed herein.

[0116] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand, wherein the 19th position (5'→3') of the antisense strand is capable of forming a base pair with the 1st position of the 19-mer target sequence disclosed in Table 1. In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand, wherein the 1st position (5'→3') of the antisense strand is capable of forming a base pair with the 19th position of the 19-mer target sequence disclosed in Table 1.

[0117] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand, wherein the 2nd position (5'→3') of the antisense strand is capable of forming a base pair with the 18th position of the 19-mer target sequence disclosed in Table 1. In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand, wherein positions 2 to 18 (5'→3') of the antisense strand are capable of forming base pairs with each of the corresponding complementary bases located at positions 18 to 2 of the 19-mer target sequence disclosed in Table 1.

[0118] For the RNAi agents disclosed herein, the 1st nucleotide of the antisense strand (from the 5'-end → 3'-end) may be fully complementary to the PNPLA3 gene or may not be complementary to the PNPLA3 gene. In some embodiments, the 1st nucleotide of the antisense strand (from the 5'-end → 3'-end) is U, A or dT. In some embodiments, the 1st nucleotide of the antisense strand (from the 5'-end → 3'-end) forms an A:U or U:A base pair with the sense strand.

[0119] In some embodiments, the antisense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of positions 2-18, 2-19, 2-20 or 2-21 (from the 5'-end → 3'-end) of any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the sense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18 or 1-18 (from the 5'-end → 3'-end) of any of the sense strand sequences in Table 2 or Table 4.

[0120] In some embodiments, the antisense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of 2-18, 2-19, 2-20 or 2-21 (from the 5'-end → 3'-end) of any one of the antisense strand sequences of SEQ ID NOs: 46-60, 176, 181 and 188. In some embodiments, the sense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18 or 1-18 (from the 5'-end → 3'-end) of any one of the sense strand sequences of SEQ ID NOs: 2, 3, 4, 9-20, 214, 219 and 220.

[0121] In some embodiments, the PNPLA3 RNAi agent consists of: (i) an antisense strand that comprises the nucleotide sequence of 2-18 or 2-19 (from the 5'-end → 3'-end) of any one of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand that comprises the nucleotide sequence of 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18 or 1-18 (from the 5'-end → 3'-end) of any one of the sense strand sequences in Table 2 or Table 4.

[0122] In some embodiments, the PNPLA3 RNAi agent consists of: (i) an antisense strand that comprises the nucleotide sequence of 2-18 or 2-19 (from the 5'-end → 3'-end) of any one of the antisense strand sequences of SEQ ID NOs: 46-60, 176, 181 and 188, and (ii) a sense strand that comprises the nucleotide sequence of 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18 or 1-18 (from the 5'-end → 3'-end) of any one of the sense strand sequences of SEQ ID NOs: 2, 3, 4, 9-20, 214, 219 and 220.

[0123] In some embodiments, the PNPLA3 RNAi agent comprises the core 19-mer nucleotide sequence shown in Table 2 below.

[0124]

[0125]

[0126] The sense and antisense strands of the PNPLA3 RNAi agent comprising or consisting of the sequences in Table 2 can be modified nucleotides or unmodified nucleotides. In some embodiments, the PNPLA3 RNAi agent having sense and antisense strand sequences comprising or consisting of the sequences in Table 2 is entirely modified or substantially entirely modified nucleotides.

[0127] In some embodiments, the antisense strand of the PNPLA3 RNAi agent disclosed herein differs from any one of the antisense strand sequences in Table 2 by 0, 1, 2, or 3 nucleotides. In some embodiments, the antisense strand of the PNPLA3 RNAi agent disclosed herein differs from any one of the antisense strand sequences of SEQ ID NOs: 46 - 60, 176, 181, and 188 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the PNPLA3 RNAi agent disclosed herein differs from any one of the sense strand sequences in Table 2 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the PNPLA3 RNAi agent disclosed herein differs from any one of the sense strand sequences of SEQ ID NOs: 2, 3, 4, 9 - 20, 214, 219, and 220 by 0, 1, 2, or 3 nucleotides.

[0128] As used herein, each N listed in the sequences disclosed in Table 2 can independently be selected from any and all nucleobases (including those found on modified and unmodified nucleotides). In some embodiments, the N nucleotides in the sequences disclosed in Table 2 have nucleobases that are complementary to the N nucleotides at the corresponding positions on the other strand. In some embodiments, the N nucleotides in the sequences disclosed in Table 2 have nucleobases that are not complementary to the N nucleotides at the corresponding positions on the other strand. In some embodiments, the N nucleotides in the sequences disclosed in Table 2 have nucleobases that are the same as the N nucleotides at the corresponding positions on the other strand. In some embodiments, the N nucleotides in the sequences disclosed in Table 2 have nucleobases that are different from the N nucleotides at the corresponding positions on the other strand.

[0129] Certain modified antisense strands of the PNPLA3 RNAi agent, as well as their potential unmodified nucleobase sequences, are provided in Table 3. Certain modified sense strands of the PNPLA3 RNAi agent, as well as their potential unmodified nucleobase sequences, are provided in Table 4. When forming the PNPLA3 RNAi agent, each nucleotide in each of the potential base sequences listed in Table 3, Table 4, and Table 2 can be a modified nucleotide.

[0130] The PNPLA3 RNAi agents described herein are formed by annealing an antisense strand with a sense strand. The sense strand containing the sequences listed in Table 2 or Table 4 can hybridize with any antisense strand containing the sequences listed in Table 2 or Table 3, provided that the two sequences have a region that is at least 85% complementary over an adjacent 16, 17, 18, 19, 20 or 21 nucleotide sequence.

[0131] In some embodiments, the PNPLA3 RNAi agent antisense strand comprises the nucleotide sequence of any one of the sequences in Table 2 or Table 3.

[0132] In some embodiments, the PNPLA3 RNAi agent comprises or consists of a duplex having the nucleobase sequences of a sense strand and an antisense strand of any one of the sequences in Table 2, Table 3 or Table 4. In some embodiments, the PNPLA3 RNAi agent comprises or consists of a duplex sequence having SEQ ID NO: (176 and 214); (90 and 131); (181 and 219); (95 and 136); (188 and 220) and / or (102 and 137). In some embodiments, the PNPLA3 RNAi agent duplex sequence comprising SEQ ID NO: (176 and 214); (90 and 131); (181 and 219); (95 and 136); (188 and 220) or (102 and 137) is prepared or provided as a sodium salt, a mixed salt or a free acid.

[0133] Examples of antisense strands containing modified nucleotides are provided in Table 3. Examples of sense strands containing modified nucleotides are provided in Table 4.

[0134] As used in Tables 3 and 4, the following symbols are used to denote modified nucleotides and linking groups.

[0135] A = adenosine-3'-phosphate;

[0136] C = cytidine-3'-phosphate;

[0137] G = guanosine-3'-phosphate;

[0138] U = uridine-3'-phosphate

[0139] I = inosine-3'-phosphate

[0140] a = 2'-O-methyladenosine-3'-phosphate

[0141] as = 2'-O-methyladenosine-3'-thiophosphate

[0142] c = 2'-O-methylcytidine-3'-phosphate

[0143] cs = 2'-O-methylcytidine-3'-thiol phosphate

[0144] g = 2'-O-methylguanosine-3'-phosphate

[0145] gs = 2'-O-methylguanosine-3'-phosphate

[0146] t = 2'-O-methyl-5-methyluridine-3'-phosphate

[0147] ts = 2'-O-methyl-5-methyluridine-3'-thiol phosphate

[0148] u = 2'-O-methyluridine-3'-phosphate

[0149] us = 2'-O-methyluridine-3'-thiol phosphate

[0150] i = 2'-O-methylinosine-3'-phosphate

[0151] is = 2'-O-methylinosine-3'-thiol phosphate

[0152] Af = 2'-fluoroadenosine-3'-phosphate

[0153] Afs = 2'-fluoroadenosine-3'-thiol phosphate

[0154] Cf = 2'-fluorocytidine-3'-phosphate

[0155] Cfs = 2'-fluorocytidine-3'-thiol phosphate

[0156] Gf = 2'-fluoroguanosine-3'-phosphate

[0157] Gfs = 2'-fluoroguanosine-3'-thiol phosphate

[0158] Tf = 2'-fluoro-5'-methyluridine-3'-phosphate

[0159] Tfs = 2'-fluoro-5'-methyluridine-3'-thiol phosphate

[0160] Uf = 2'-fluorouridine-3'-phosphate

[0161] Ufs = 2'-fluorouridine-3'-thiol phosphate

[0162] A UNA = 2',3'-anhydro-adenosine-3'-phosphate, see Table 6

[0163] A UNA s = 2',3'-anhydro-adenosine-3'-thiol phosphate, see Table 6

[0164] CUNA = 2',3'-anhydro-cytidine-3'-phosphate, see Table 6

[0165] C UNA s = 2',3'-anhydro-cytidine-3'-thiophosphate, see Table 6

[0166] G UNA = 2',3'-anhydro-guanosine-3'-phosphate, see Table 6

[0167] G UNA s = 2',3'-anhydro-guanosine-3'-thiophosphate, see Table 6

[0168] U UNA = 2',3'-anhydro-uridine-3'-phosphate, see Table 6

[0169] U UNA s = 2',3'-anhydro-uridine-3'-thiophosphate, see Table 6

[0170] a_2N = 2'-O-methyl-2-aminoadenosine-3'-phosphate, see Table 6

[0171] a_2Ns = 2'-O-methyl-2-aminoadenosine-3'-thiophosphate, see Table 6

[0172] (invAb) = inverted abasic deoxyribonucleotide, see Table 6

[0173] (invAb)s = inverted abasic deoxyribonucleotide-5'-thiophosphate, see Table 6

[0174] Unless otherwise specified for a sequence (such as phosphorothioate bond “s”), as will be readily understood by one of ordinary skill in the art, when present in an oligonucleotide, nucleotide monomers are linked to each other by 5'-3'-phosphodiester bonds. As will be clearly understood by one of ordinary skill in the art, as shown in the modified nucleotide sequences disclosed herein, phosphorothioate bonds are included in place of the phosphodiester bonds that are normally present in oligonucleotides. In addition, one of ordinary skill in the art will readily understand that the terminal nucleotide at the 3'-end of a given oligonucleotide sequence will typically have a hydroxyl (-OH) group at the corresponding 3'-position of a given monomer, rather than a phosphate moiety in vitro. Additionally, for the embodiments disclosed herein, when observing the corresponding strand 5'→3', inverted abasic residues are inserted such that the 3'-position of the deoxyribose is linked at the 3'-end of the previous monomer on the corresponding strand (see, for example, Table 6). Further, as will be readily understood and appreciated by one of ordinary skill in the art, while the phosphorothioate chemical structures depicted herein typically show an anion on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers (e.g., where the sulfur atom has a double bond and the anion is on an oxygen atom). Such understanding of one of ordinary skill in the art is used when describing the PNPLA3 RNAi agents and compositions of PNPLA3 RNAi agents disclosed herein, unless otherwise explicitly specified herein.

[0175] Certain examples of targeting ligands, targeting groups, and linking groups for use with the PNPLA3 RNAi agents disclosed herein are provided in Table 6 below. More specifically, the targeting groups and linking groups (which together can form a targeting ligand) include (NAG37) and (NAG37)s, the chemical structures of which are provided in Table 6 below. Each sense strand and / or antisense strand can have any of the targeting ligands, targeting groups, or linking groups listed herein, as well as other groups conjugated to the 5'-end and / or 3'-end of the sequence.

[0176]

[0177]

[0178]

[0179]

[0180] The PNPLA3 RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2 or Table 4 can hybridize with any antisense strand containing a sequence listed in Table 2 or Table 3, provided that the two sequences have a region that is at least 85% complementary over an adjacent 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0181] In some embodiments, the antisense strand of the PNPLA3 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 3 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the PNPLA3 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 4 by 0, 1, 2, or 3 nucleotides.

[0182] In some embodiments, the antisense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 3. In some embodiments, the antisense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 3 from (5' end → 3' end) 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21. In certain embodiments, the antisense strand of the PNPLA3 RNAi agent comprises or consists of the modified sequence of any of the modified sequences in Table 3.

[0183] In some embodiments, the sense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 4. In some embodiments, the sense strand of the PNPLA3 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 4 from (5' end → 3' end) 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, or 4-21. In certain embodiments, the sense strand of the PNPLA3 RNAi agent comprises or consists of the modified sequence of any of the modified sequences in Table 4.

[0184] For the PNPLA3 RNAi agent disclosed herein, the nucleotide at position 1 of the antisense strand (from 5' end → 3') may be fully complementary to the PNPLA3 gene or may not be complementary to the PNPLA3 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end → 3') is U, A, or dT (or a modified form thereof). In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end → 3') forms an A:U or U:A base pair with the sense strand.

[0185] The sense strand containing the sequences listed in Table 2 or Table 4 can hybridize with any antisense strand containing the sequences listed in Table 2 or Table 3, provided that the two sequences have a region that is at least 85% complementary over an adjacent 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some embodiments, the PNPLA3 RNAi agent has a sense strand consisting of a modified sequence of any of the modified sequences in Table 4, and an antisense strand consisting of a modified sequence of any of the modified sequences in Table 3. Certain representative sequence pairings are illustrated by the duplex ID numbers shown in Table 5A and Table 5B.

[0186] In some embodiments, the PNPLA3 RNAi agent comprises, consists of, or consists essentially of a duplex represented by any of the duplex ID numbers presented herein. In some embodiments, the PNPLA3 RNAi agent comprises the nucleotide sequences of the sense and antisense strands of any of the duplexes represented by any of the duplex ID numbers presented herein. In some embodiments, the PNPLA3 RNAi agent comprises the nucleotide sequences of the sense and antisense strands of any of the duplexes represented by any of the duplex ID numbers presented herein, and a targeting group and / or a linking group, wherein the targeting group and / or the linking group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, the PNPLA3 RNAi agent comprises the modified nucleotide sequences of the sense and antisense strands of any of the duplex ID numbers presented herein. In some embodiments, the PNPLA3 RNAi agent comprises the modified nucleotide sequences of the sense and antisense strands of any of the duplex ID numbers presented herein, and a targeting group and / or a linking group, wherein the targeting group and / or the linking group is covalently linked to the sense strand or the antisense strand.

[0187] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense / sense strand duplexes of Table 2 or Table 5A and Table 5B, and further comprises a targeting group or a targeting ligand. In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense / sense strand duplexes of Table 2 or Table 5A and Table 5B, and further comprises an asialoglycoprotein receptor ligand targeting group.

[0188] With or without a linker, the targeting group can be attached to the 3' end or the 5' end of any sense strand and / or antisense strand disclosed in Table 2, Table 3, and Table 4. With or without a targeting group, the linker can be attached to the 3' end or the 5' end of any sense strand and / or antisense strand disclosed in Table 2, Table 3, and Table 4.

[0189] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand and a sense strand having a nucleotide sequence of any of the antisense / sense strand duplexes of Table 2 or Tables 5A and 5B, and further comprises a targeting ligand selected from the group consisting of (NAG37) and (NAG37)s, each as defined in Table 6.

[0190] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense and / or sense strand nucleotide sequences of Table 3 or Table 4.

[0191] In some embodiments, the PNPLA3 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense and / or sense strand nucleotide sequences of any of the duplexes of Tables 5A and 5B, and further comprises an asialoglycoprotein receptor ligand targeting moiety.

[0192] In some embodiments, the PNPLA3 RNAi agent comprises, consists of, or consists essentially of any of the duplexes of Tables 5A and 5B.

[0193] Table 5A. PNPLA3 RNAi Agent Duplexes with Corresponding Sense and Antisense Strand ID Numbers

[0194]

[0195] Table 5B. PNPLA3 RNAi Agent Duplexes with Corresponding Sense and Antisense Strand ID Numbers and Sequence ID Numbers of Modified and Unmodified Nucleotide Sequences Targeting Ligand or Group, Linker Group, and Delivery Vehicle

[0196]

[0197]

[0198] In some embodiments, the PNPLA3 RNAi agent is prepared or provided as a salt, mixed salt, or free acid. The RNAi agents described herein, when delivered to cells expressing the PNPLA3 gene, inhibit or knockdown the expression of one or more PNPLA3 genes in vivo and / or in vitro.

[0199] Table 6. Structures Representing Various Modified Nucleotides, Targeting Ligands or Targeting Groups, Blocking Residues, and Linker Groups

[0200] In some embodiments, the PNPLA3 RNAi agent is conjugated to one or more non-nucleotide groups, including but not limited to targeting groups, linking groups, targeting ligands, delivery polymers, or delivery vectors. The non-nucleotide groups can enhance the targeting, delivery, or attachment of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6. The non-nucleotide groups can be covalently linked to the 3'-end and / or 5'-end of the sense strand and / or the antisense strand. In some embodiments, the PNPLA3 RNAi agent contains a non-nucleotide group linked to the 3'-end and / or 5'-end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5'-end of the sense strand of the PNPLA3 RNAi agent. The non-nucleotide group can be directly or indirectly linked to the RNAi agent via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0201] In some embodiments, the non-nucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached to improve the cellular or tissue-specific distribution and cellular-specific uptake of the RNAi agent or conjugate. In some embodiments, the non-nucleotide group enhances the endocytosis of the RNAi agent.

[0202] The targeting group or targeting moiety enhances the pharmacokinetic or biodistribution properties of the conjugate or RNAi agent to which it is attached to improve the cellular-specific (including organ-specific in some cases) distribution and cellular-specific (or organ-specific) uptake of the conjugate or RNAi agent. The targeting group can be monovalent, divalent, trivalent, tetravalent, or of a higher valence for the target to which it is directed. Representative targeting groups include but are not limited to: compounds having an affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics having an affinity for cell surface molecules.

[0203] In some embodiments, the targeting group is linked to the RNAi agent using a linker (such as a PEG linker) or one, two, or three abasic groups and / or ribitol (abasic ribose) residues (which can be used as a linker in some cases). In some embodiments, the targeting ligand comprises a cluster of galactose derivatives.

[0204] The PNPLA3 RNAi agents described herein can be synthesized to have reactive groups, such as amino groups (also referred to herein as amines), at the 5'-end and / or 3'-end. The reactive groups can be used to subsequently attach targeting moieties using methods typical in the art.

[0205] In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. As used herein, an asialoglycoprotein receptor ligand is a ligand that contains a moiety having an affinity for the asialoglycoprotein receptor. As described herein, the asialoglycoprotein receptor is highly expressed on hepatocytes. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl galactosamine (see, for example, S.T. Iobst and K. Drickamer, J.B.C., 1996, 271, 6686). Galactose derivatives and clusters of galactose derivatives useful for targeting the liver in vivo for oligonucleotides and other molecules are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

[0206] Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. The binding of the asialoglycoprotein receptor ligand to the asialoglycoprotein receptor facilitates cell-specific targeting of hepatocytes and the endocytosis of molecules into hepatocytes. The asialoglycoprotein receptor ligand can be monomeric (e.g., having a single galactose derivative, also referred to as monovalent or monodentate) or polymeric (e.g., having multiple galactose derivatives). Methods known in the art can be used to attach galactose derivatives or clusters of galactose derivatives to the 3' or 5' end of the sense or antisense strand of an RNAi agent. The preparation of targeting ligands such as clusters of galactose derivatives is described in, for example, International Patent Application Publication No. WO 2018 / 044350 to Arrowhead Pharmaceuticals, Inc. and International Patent Application Publication No. WO 2017 / 156012 to Arrowhead Pharmaceuticals, Inc., the entire contents of both applications being incorporated herein by reference.

[0207] As used herein, a galactose derivative cluster comprises a molecule having two to four terminal galactose derivatives. The terminal galactose derivatives are attached to the molecule through their C-1 carbon. In some embodiments, the galactose derivative cluster is a galactose derivative trimer (also referred to as a triantennary galactose derivative or trivalent galactose derivative). In some embodiments, the galactose derivative cluster comprises N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster comprises three N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster is a galactose derivative tetramer (also referred to as a tetraantennary galactose derivative or tetravalent galactose derivative). In some embodiments, the galactose derivative cluster comprises four N-acetyl-galactosamine.

[0208] As used herein, a galactose derivative trimer contains three galactose derivatives, each galactose derivative being attached to a central branching point. As used herein, a galactose derivative tetramer comprises four galactose derivatives, each galactose derivative being attached to a central branching point. The galactose derivative may be attached to the central branching point through the C-1 carbon of the sugar. In some embodiments, the galactose derivative is attached to the branching point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer such as a PEG group (see, e.g., U.S. Patent No. 5,885,968; Biessen et al., J. Med. Chem. 1995, Vol. 39, pp. 1538-1546). In some embodiments, the PEG spacer is a PEG3 spacer. The branching point may be any small molecule that allows attachment of three galactose derivatives and also allows attachment of the branching point to an RNAi agent. Examples of branching point groups are dilysine or diglutamic acid. Attachment of the branching point to the RNAi agent may be through a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker such as a cyclic group. In some embodiments, the galactose derivative comprises or consists of N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster consists of a galactose derivative tetramer, which may be, for example, an N-acetyl-galactosamine tetramer.

[0209] Embodiments of the present disclosure include pharmaceutical compositions for in vivo delivery of a PNPLA3 RNAi agent to hepatocytes. Such pharmaceutical compositions may comprise, for example, a PNPLA3 RNAi agent conjugated to a galactose derivative cluster. In some embodiments, the galactose derivative cluster consists of a galactose derivative trimer, which may be, for example, an N-acetyl-galactosamine trimer or a galactose derivative tetramer, which may be, for example, an N-acetyl-galactosamine tetramer.

[0210] A targeting ligand or targeting group can be linked to the 3'-end or 5'-end of the sense strand or the antisense strand of the PNPLA3 RNAi agent disclosed herein.

[0211] Targeting ligands include, but are not limited to, (NAG37) and (NAG37)s as defined in Table 6. Other targeting groups and targeting ligands (including galactose cluster targeting ligands) are known in the art.

[0212] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent attachment of the agent to a targeting group, a delivery polymer, or a delivery vehicle. The linking group can be linked to the 3'-end and / or 5'-end of the sense strand or the antisense strand of the RNAi agent. In some embodiments, the linking group is linked to the sense strand of the RNAi agent. In some embodiments, the linking group is conjugated to the 3'-end or 5'-end of the sense strand of the RNAi agent. In some embodiments, the linking group is conjugated to the 5'-end of the sense strand of the RNAi agent. Examples of linking groups can include, but are not limited to: reactive groups (such as primary amines and alkynes), alkyl groups, abasic nucleotides, ribitol (abasic ribose), and / or PEG groups.

[0213] In some embodiments, the targeting group is internally linked to a nucleotide on the sense strand and / or the antisense strand of the RNAi agent. In some embodiments, the targeting group is linked to the RNAi agent via a linker.

[0214] A linker or linking group is a connection between two atoms that connects one chemical group (such as an RNAi agent) or a segment of interest to another chemical group (such as a targeting group or a delivery polymer) or a segment of interest via one or more covalent bonds. A labile connection contains labile bonds. The connection can optionally include a spacer that increases the distance between the two joined atoms. The spacer can further increase the flexibility and / or length of the connection. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the foregoing list is not intended to limit the scope of the specification.

[0215] In some embodiments, when two or more RNAi agents are included in a single composition, each of the RNAi agents can be linked to the same targeting group or two different targeting groups (i.e., targeting groups having different chemical structures). In some embodiments, the targeting group is linked to the PNPLA3 RNAi agents disclosed herein without the use of an additional linker. In some embodiments, the targeting group itself is designed to have a linker or other site to facilitate conjugation that is readily available. In some embodiments, when two or more PNPLA3 RNAi agents are included in a single molecule, each of the RNAi agents can utilize the same linker or different linkers (i.e., linkers having different chemical structures).

[0216] Any of the PNPLA3 RNAi agent nucleotide sequences listed in Tables 2, 3, and 4, whether modified or unmodified, can contain 3' and / or 5' targeting groups or linking groups. Any of the PNPLA3 RNAi agent sequences listed in Table 3 or 4 or otherwise described herein that contain 3' or 5' targeting groups or linking groups can alternatively not contain 3' or 5' targeting groups or linking groups, or can contain different 3' or 5' targeting groups or linking groups, including but not limited to those shown in Table 6. Any of the PNPLA3 RNAi agent duplexes listed in Tables 5A and 5B, whether modified or unmodified, can also contain targeting groups or linking groups, including but not limited to those shown in Table 6, and the targeting group or linking group can be attached to the 3' end or 5' end of the sense or antisense strand of the PNPLA3 RNAi agent duplex.

[0217] Table 6 provides examples of targeting groups and linking groups (which can form a targeting ligand when combined). Table 4 provides several embodiments of the sense strand of a PNPLA3 RNAi agent having a targeting group or linking group attached to the 5' end or 3' end.

[0218] Pharmaceutical Compositions and Formulations Methods of Treatment and Inhibition of Expression

[0219]

[0220]

[0221]

[0222]

[0223] In each of the above structures in Table 6, NAG contains N-acetyl-galactosamine or another galactose derivative, which is attached as would be understood by one of ordinary skill in the art based on the above structures and the description provided herein.

[0224] Each (NAGx) can be attached to the PNPLA3 RNAi agent via a phosphate group (such as (NAG37)) or a phosphorothioate group (such as (NAG37)s) or another linking group.

[0225]

[0226] Other linking groups known in the art can be used.

[0227] In some embodiments, a delivery vehicle can be used to deliver the RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves the delivery of the RNAi agent to a cell or tissue. The delivery vehicle can include, but is not limited to: polymers (such as amphiphilic polymers), membrane-active polymers, peptides, melittin, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides or reversibly modified membrane-active polyamines, or combinations thereof. In some embodiments, the RNAi agent can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPC, or other delivery systems available in the art. The RNAi agent can also be chemically conjugated to a targeting group, lipids (including but not limited to cholesterol and cholesterol derivatives), nanoparticles, polymers, liposomes, micelles, DPC (see, for example, WO2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169, and WO 2012 / 083185, WO 2013 / 032829, WO2013 / 158141, each of which is incorporated herein by reference), hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, protein carriers, or other delivery systems known and available in the art suitable for nucleic acid or oligonucleotide delivery.

[0228] Cells, Tissues, Organs, and Non - Human Organisms

[0229] The PNPLA3 RNAi agents disclosed herein can be formulated into a pharmaceutical composition or formulation (also referred to herein as an "agent"). In some embodiments, the pharmaceutical composition comprises at least one PNPLA3 RNAi agent. These pharmaceutical compositions are particularly suitable for inhibiting the expression of a target mRNA in a target cell, cell population, tissue, or organism.

[0230] The pharmaceutical composition can be used to treat a subject having a disease, disorder, or condition that would benefit from a reduction in the level of target PNPLA3 mRNA or inhibition of the expression of a target gene. The pharmaceutical composition can be used to treat a subject at risk of developing a disease, disorder, or condition that would benefit from a reduction in the level of target mRNA or inhibition of the expression of a target gene. In one embodiment, the method comprises administering to a subject in need of treatment a PNPLA3 RNAi agent linked to a targeting ligand as described herein. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to a pharmaceutical composition comprising a PNPLA3 RNAi agent to form a pharmaceutical formulation or medicament suitable for delivery into a subject, including a human subject.

[0231] The pharmaceutical composition comprising a PNPLA3 RNAi agent and the methods disclosed herein reduce the level of target mRNA in a cell, cell population, tissue, organ, or subject, including by administering to the subject a therapeutically effective amount of the PNPLA3 RNAi agent described herein, thereby inhibiting the expression of PNPLA3 mRNA in the subject. In some embodiments, the subject has been previously identified or diagnosed as having a pathogenic upregulation of the target gene in the target cell or tissue. In some embodiments, the subject has been previously identified or diagnosed as having NAFLD, NASH, hepatic fibrosis, and / or alcoholic or non-alcoholic liver disease, such as cirrhosis. In some embodiments, the subject has symptoms associated with NAFLD, NASH, hepatic fibrosis, and / or alcoholic or non-alcoholic liver disease, such as cirrhosis.

[0232] In some embodiments, the described pharmaceutical composition comprising a PNPLA3 RNAi agent is used to treat or manage clinical manifestations in a subject associated with NAFLD, NASH, hepatic fibrosis, alcoholic or non-alcoholic liver disease, including cirrhosis, and / or overexpression of PNPLA3. In some embodiments, a therapeutically effective amount of one or more pharmaceutical compositions is administered to a subject in need of such treatment. In some embodiments, administration of any of the disclosed PNPLA3 RNAi agents can be used to reduce the number, severity, and / or frequency of the subject's disease symptoms.

[0233] The described pharmaceutical compositions comprising a PNPLA3 RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from a reduction or inhibition of PNPLA3 mRNA expression. In some embodiments, a therapeutically effective amount of one or more pharmaceutical compositions comprising a PNPLA3 RNAi agent is administered to the subject to treat the symptom. In other embodiments, a prophylactically effective amount of one or more PNPLA3 RNAi agents is administered to the subject to prevent or inhibit at least one symptom.

[0234] The route of administration is the path by which the PNPLA3 RNAi agent comes into contact with the body. Generally, methods of administering drugs, oligonucleotides, and nucleic acids for treating mammals are well known in the art and can be applied to the administration of the compositions described herein. The PNPLA3 RNAi agents disclosed herein can be administered via any suitable route in a formulation suitable for the particular route. The pharmaceutical compositions described herein can be administered by injection, such as intravenous, intramuscular, intradermal, subcutaneous, intra-articular, or intraperitoneal administration. In some embodiments, the pharmaceutical compositions described herein are administered via subcutaneous injection.

[0235] The pharmaceutical compositions comprising a PNPLA3 RNAi agent described herein can be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery techniques known in the art. Generally, any suitable method recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery can be by topical administration (e.g., direct injection, implantation, or topical application), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, parenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or local (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0236] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.

[0237] As used herein, a pharmaceutical composition or a medicament comprises a pharmaceutically effective amount of at least one of the therapeutic compounds and one or more pharmaceutically acceptable excipients. One or more pharmaceutically acceptable excipients are substances other than the active pharmaceutical ingredient (API, therapeutic product, such as a PNPLA3 RNAi agent) that are intentionally included in the drug delivery system. The excipients do not or are not intended to have an effect on the therapeutic effect at the intended dosage. Excipients can be used to a) assist in the processing of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other property of the overall safety and effectiveness of the API during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0238] Excipients include, but are not limited to: absorption enhancers, anti-adhesives, anti-foaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextrans, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweetening agents, thickening agents, tonicity agents, vehicles, water repellents, and wetting agents.

[0239] A pharmaceutical composition suitable for injection comprises a sterile aqueous solution (which is water-soluble herein) or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The suitable carrier should be stable under the conditions of manufacture and storage and should be able to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersions, to maintain the desired particle size, and by the use of surfactants. In many cases, it will be preferable to include in the composition an isotonic agent such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption such as aluminum monostearate and gelatin.

[0240] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent having one or a combination (as required) of the ingredients listed above, followed by filtration sterilization as needed. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods include vacuum drying and freeze drying of the powder, which contains the active ingredient and any additional desired ingredients from the sterile filtered solutions of these ingredients described above.

[0241] In some embodiments, a pharmaceutical formulation comprising a PNPLA3 RNAi agent disclosed herein suitable for subcutaneous administration can be prepared in an aqueous sodium phosphate buffer (e.g., a PNPLA3 RNAi agent formulated in 0.5 mM sodium dihydrogen phosphate, 0.5 mM disodium hydrogen phosphate in water).

[0242] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug, which can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to provide drugs for intra-articular and ophthalmic administration.

[0243] Formulations suitable for oral administration of the PNPLA3 RNAi agent disclosed herein can also be prepared. In some embodiments, the PNPLA3 RNAi agent disclosed herein is administered orally. In some embodiments, the PNPLA3 RNAi agent disclosed herein is formulated in a capsule for oral administration.

[0244] The active compound can be prepared with a carrier that protects the compound from rapid elimination by the body, such as a controlled release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0245] The PNPLA3 RNAi agent can be formulated in a composition in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form refers to physically discrete units suitable as unit doses for the subject to be treated; each unit contains a predetermined quantity of the active compound in association with the required pharmaceutically acceptable carrier, the predetermined quantity being calculated to produce the desired therapeutic effect. The specification of the unit dosage forms of the present disclosure is indicated by the following and directly depends on the unique characteristics of the active compound and the therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such active compounds for individual therapy.

[0246] The pharmaceutical composition may contain other additional components common in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics, analgesics, antihistamines or anti-inflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine, etc.). Cells, tissues or isolated organs expressing or containing the RNAi agent as defined herein are also contemplated as being useful as a "pharmaceutical composition". As used herein, "pharmacologically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of the RNAi agent that produces a pharmacological, therapeutic or prophylactic effect.

[0247] In some embodiments, the methods disclosed herein further comprise the step of administering a second therapeutic agent or treatment in addition to the RNAi agent disclosed herein. In some embodiments, the second therapeutic agent is another PNPLA3 RNAi agent (e.g., a PNPLA3 RNAi agent targeting a different sequence within the PNPLA3 target). In other embodiments, the second therapeutic agent can be a small molecule drug, an antibody, an antibody fragment or an aptamer.

[0248] In some embodiments, the described PNPLA3 RNAi agent is optionally combined with one or more additional therapeutic agents. The PNPLA3 RNAi agent and the additional therapeutic agent(s) can be administered in a single composition, or can be administered separately. In some embodiments, one or more additional therapeutic agents are administered separately in a dosage form separate from the RNAi agent (e.g., the PNPLA3 RNAi agent is administered by subcutaneous injection, while the additional therapeutic agent(s) involved in the treatment dosing regimen are administered orally). In some embodiments, the described PNPLA3 RNAi agent is administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutic agents are administered orally, which together provide a treatment regimen for diseases and disorders associated with NAFLD, NASH, liver fibrosis and / or alcoholic or non-alcoholic liver diseases including cirrhosis. In some embodiments, the described PNPLA3 RNAi agent is administered to a subject in need thereof by subcutaneous injection, and one or more optional additional therapeutic agents are administered by a separate subcutaneous injection. In some embodiments, the PNPLA3 RNAi agent and one or more additional therapeutic agents are combined into a single dosage form (e.g., a "mixture" formulated as a single composition for subcutaneous injection). With or without one or more additional therapeutic agents, the PNPLA3 RNAi agent can be combined with one or more excipients to form a pharmaceutical composition.

[0249] Generally speaking, the effective amount range of the PNPLA3 RNAi agent will be from about 0.1 mg / kg body weight / dose to about 100 mg / kg body weight / dose, such as from about 1.0 mg / kg body weight / dose to about 50 mg / kg body weight / dose. In some embodiments, the effective amount range of the active compound will be from about 0.25 mg / kg body weight / dose to about 5 mg / kg body weight / dose. In some embodiments, the effective amount range of the active ingredient will be from about 0.5 mg / kg body weight / dose to about 4 mg / kg body weight / dose. Depending on the dose of the PNPLA3 RNAi agent administered, the activity level of the specific PNPLA3 RNAi agent, and the desired level of inhibition in the specific subject, administration can be weekly, bi-weekly, monthly, or at any other interval. The examples herein illustrate suitable levels of inhibition in certain animal species. The dosage will depend on variables such as the overall health of the patient, the relative biological efficacy of the compound being delivered, the pharmaceutical formulation, the presence and type of excipients in the formulation, and the route of administration. Additionally, it should be understood that the initial dose administered can be increased to exceed the upper limit described above to rapidly achieve the desired blood level or tissue level, or the initial dose can be less than the optimal dose.

[0250] For treating a disease or forming a drug or composition for treating a disease, the pharmaceutical composition comprising a PNPLA3 RNAi agent described herein can be combined with an excipient or with a second therapeutic agent or treatment combination, and the therapeutic agent or treatment includes, but is not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, a peptide, and / or an aptamer.

[0251] When the PNPLA3 RNAi agent is added to a pharmaceutically acceptable excipient or adjuvant, it can be packaged into a kit, container, package, or dispenser. The pharmaceutical compositions described herein can be packaged in pre-filled syringes, pen injectors, auto-injectors, infusion bags / devices, or vials.

[0252] Exemplary Embodiments

[0253] The PNPLA3 RNAi agents disclosed herein can be used to treat a subject (e.g., a human or other mammal) suffering from a disease or disorder that would benefit from the administration of an RNAi agent. In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from a reduction and / or inhibition in the expression of PNPLA3 mRNA and / or PNPLA3 protein levels, such as a subject that has been diagnosed with or is suffering from symptoms associated with NAFLD, NASH, liver fibrosis, or alcoholic or non-alcoholic liver disease (including cirrhosis).

[0254] In some embodiments, a therapeutically effective amount of any one or more of the PNPLA3 RNAi agents is administered to a subject. Treatment of the subject can include therapeutic and / or prophylactic treatment. A therapeutically effective amount of any one or more of the PNPLA3 RNAi agents described herein is administered to the subject. The subject can be a human, a patient, or a human patient. The subject can be an adult, an adolescent, a child, or an infant. The pharmaceutical compositions described herein can be administered to a human or an animal.

[0255] The PNPLA3 RNAi agents described herein can be used to treat at least one symptom in a subject having a PNPLA3-related disease or disorder, or a disease or disorder that is at least partially mediated by PNPLA3 gene expression. In some embodiments, the PNPLA3 RNAi agent is used to treat or manage the clinical manifestations in a subject having a disease or disorder that would benefit from a reduction in PNPLA3 mRNA or that is at least partially mediated by a reduction in PNPLA3 mRNA. A therapeutically effective amount of any one or more of the PNPLA3 RNAi agents described herein, or a composition comprising a PNPLA3 RNAi agent, is administered to the subject. In some embodiments, the methods disclosed herein include administering to a subject in need of treatment a composition comprising a PNPLA3 RNAi agent described herein. In some embodiments, a prophylactically effective amount of any one or more of the PNPLA3 RNAi agents is administered to the subject, thereby treating the subject by preventing or inhibiting the at least one symptom.

[0256] In certain embodiments, the present disclosure provides a method for treating a disease, disorder, condition, or pathological state that is at least partially mediated by PNPLA3 gene expression in a patient in need thereof, wherein the method comprises administering to the patient any one of the PNPLA3 RNAi agents described herein.

[0257] In some embodiments, the gene expression level and / or mRNA level of the PNPLA3 gene in a subject administered the PNPLA3 RNAi agent is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or greater than 99% relative to a subject before administration of the PNPLA3 RNAi agent or a subject not receiving the PNPLA3 RNAi agent. The gene expression level and / or mRNA level of the subject can be reduced in the cells, cell populations, and / or tissues of the subject. In some embodiments, PNPLA3 gene expression is inhibited by at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70% or greater than 70% in the cytoplasm of hepatocytes relative to a subject before administration of the PNPLA3 RNAi agent or a subject not receiving the PNPLA3 RNAi agent.

[0258] In some embodiments, the PNPLA3 protein level in a subject administered the PNPLA3 RNAi agent is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% relative to a subject before administration of the PNPLA3 RNAi agent or a subject not receiving the PNPLA3 RNAi agent. The protein level of the subject can be reduced in the cells, cell populations, tissues, blood, and / or other fluids of the subject.

[0259] The reduction in PNPLA3 mRNA level and PNPLA3 protein level can be evaluated by any method known in the art. As used herein, a reduction or decrease in the PNPLA3 mRNA level and / or protein level is collectively referred to herein as a reduction or decrease of PNPLA3 or inhibition or reduction of PNPLA3 expression. The examples set forth herein illustrate known methods for evaluating inhibition of PNPLA3 gene expression. One of ordinary skill in the art will further appreciate suitable methods for evaluating inhibition of PNPLA3 gene expression in vivo and / or in vitro.

[0260] In some embodiments, disclosed herein are methods of treating (including prophylactic or preventive treatment) diseases, disorders or symptoms caused by NAFLD, NASH, hepatic fibrosis, and / or alcoholic or non-alcoholic liver diseases (including cirrhosis), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a PNPLA3 RNAi agent, the PNPLA3 RNAi agent comprising an antisense strand that is at least partially complementary to a portion of the PNPLA3 mRNA having the sequence in Table 1. In some embodiments, disclosed herein are methods of treating (including prophylactic or preventive treatment) diseases or symptoms caused by NAFLD, NASH, hepatic fibrosis, and / or alcoholic or non-alcoholic liver diseases (including cirrhosis), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a PNPLA3 RNAi agent, the PNPLA3 RNAi agent comprising an antisense strand comprising the sequence of any one of the sequences in Table 2 or Table 3, and a sense strand comprising the sequence of any one of the sequences in Table 2 or Table 4, the sense strand being at least partially complementary to the antisense strand. In some embodiments, disclosed herein are methods of treating (including prophylactic or preventive treatment) diseases or symptoms caused by NAFLD, NASH, hepatic fibrosis, and / or alcoholic or non-alcoholic liver diseases (including cirrhosis), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a PNPLA3 RNAi agent, the PNPLA3 RNAi agent comprising a sense strand comprising the sequence of any one of the sequences in Table 2 or Table 4, and an antisense strand comprising the sequence of any one of the sequences in Table 2 or Table 3, the antisense strand being at least partially complementary to the sense strand.

[0261] In some embodiments, disclosed herein are methods for inhibiting the expression of the PNPLA3 gene in a cell, wherein the method comprises administering to the cell a PNPLA3 RNAi agent, the PNPLA3 RNAi agent comprising an antisense strand that is at least partially complementary to a portion of the PNPLA3 mRNA having the sequence in Table 1. In some embodiments, disclosed herein are methods for inhibiting the expression of the PNPLA3 gene in a cell, wherein the method comprises administering to the cell a PNPLA3 RNAi agent, the PNPLA3 RNAi agent comprising an antisense strand comprising the sequence of any one of the sequences in Table 2 or Table 3, and a sense strand comprising the sequence of any one of the sequences in Table 2 or Table 4, the sense strand being at least partially complementary to the antisense strand. In some embodiments, disclosed herein are methods for inhibiting the expression of the PNPLA3 gene in a cell, wherein the method comprises administering a PNPLA3 RNAi agent, the PNPLA3 RNAi agent comprising a sense strand comprising the sequence of any one of the sequences in Table 2 or Table 4, and an antisense strand comprising the sequence of any one of the sequences in Table 2 or Table 3, the antisense strand being at least partially complementary to the sense strand.

[0262] The use of a PNPLA3 RNAi agent provides a method for therapeutic (including prophylactic) treatment of diseases / disorders associated with NAFLD, NASH, liver fibrosis, alcoholic or non-alcoholic liver diseases (including cirrhosis) and / or enhanced or elevated PNPLA3 expression. The PNPLA3 RNAi agent mediates RNA interference to inhibit the expression of one or more genes necessary for the production of PNPLA3 protein. The PNPLA3 RNAi agent can also be used to treat or prevent various diseases, disorders or conditions, including NAFLD, NASH, liver fibrosis and / or alcoholic or non-alcoholic liver diseases (including cirrhosis). In addition, compositions for in vivo delivery of PNPLA3 RNAi agents to liver cells are described.

[0263] Examples

[0264] Cells, tissues, organs and non-human organisms comprising at least one of the PNPLA3 RNAi agents described herein are envisioned. The cell, tissue, organ or non-human organism is prepared by delivering the RNAi agent to the cell, tissue, organ or non-human organism.

[0265] Example 1. Synthesis of PNPLA3 RNAi Agent

[0266] Illustrative embodiments of the disclosed technology are provided herein. These embodiments are merely exemplary and do not limit the scope of the disclosure or the appended claims.

[0267] Embodiment 1. An RNAi agent for inhibiting the expression of the PNPLA3 gene, the RNAi agent comprising:

[0268] An antisense strand comprising at least 17 consecutive nucleotides that differ from any of the sequences of SEQ ID NOs: 46-87, 174-211, and 257-258 by 0 or 1 nucleotide; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

[0269] Embodiment 2. The RNAi agent according to Embodiment 1, wherein the antisense strand comprises nucleotides 2-18 of any of the sequences of SEQ ID NOs: 46-87, 174-211, and 257-258.

[0270] Embodiment 3. The RNAi agent according to Embodiment 1 or Embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differ from any of the sense strand sequences of SEQ ID NOs: 2-45 and 212-256 by 0 or 1 nucleotide, and wherein the sense strand has a region that is at least 85% complementary to the antisense strand over 17 consecutive nucleotides.

[0271] Embodiment 4. The RNAi agent according to any one of Embodiments 1 to 3, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or comprises a modified internucleoside bond.

[0272] Embodiment 5. The RNAi agent according to any one of Embodiments 1 to 3, wherein all or substantially all nucleotides of the sense strand and / or the antisense strand of the RNAi agent are modified nucleotides.

[0273] Embodiment 6. The RNAi agent according to any one of Embodiments 4 to 5, wherein the modified nucleotides are selected from the group consisting of: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-acyclic nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxy nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, cyclopropyl phosphonate-containing nucleotides, and 3'-O-methyl nucleotides.

[0274] Embodiment 7. The RNAi agent according to Embodiment 5, wherein all or substantially all of the modified nucleotides are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

[0275] Embodiment 8. The RNAi agent according to any one of Embodiments 1 to 7, wherein the antisense strand comprises the nucleotide sequence of any one of the modified antisense strand sequences of SEQ ID NOs: 88 - 128.

[0276] Embodiment 9. The RNAi agent according to any one of Embodiments 1 to 8, wherein the sense strand comprises the nucleotide sequence of any one of the modified sense strand sequences of SEQ ID NOs: 129 - 173.

[0277] Embodiment 10. The RNAi agent according to Embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences of SEQ ID NOs: 88 - 128, and the sense strand comprises the nucleotide sequence of any one of the modified sequences of SEQ ID NOs: 129 - 173.

[0278] Embodiment 11. The RNAi agent according to any one of Embodiments 1 to 10, wherein the RNAi agent is linked to a targeting ligand.

[0279] Embodiment 12. The RNAi agent according to Embodiment 11, wherein the targeting ligand comprises N-acetyl-galactosamine.

[0280] Embodiment 13. The RNAi agent according to Embodiment 11 or 12, wherein the targeting ligand comprises a structure of (NAG37) or (NAG37)s.

[0281] Embodiment 14. The RNAi agent according to any one of Embodiments 11 to 14, wherein the targeting ligand is linked to the sense strand.

[0282] Embodiment 15. The RNAi agent according to Embodiment 15, wherein the targeting ligand is linked to the 5'-end of the sense strand.

[0283] Embodiment 16. The RNAi agent according to any one of Embodiments 1 to 16, wherein the length of the sense strand is between 18 and 30 nucleotides, and the length of the antisense strand is between 18 and 30 nucleotides.

[0284] Embodiment 17. The RNAi agent according to Embodiment 17, wherein the length of each of the sense strand and the antisense strand is between 18 and 27 nucleotides.

[0285] Embodiment 18. The RNAi agent according to Embodiment 18, wherein the length of each of the sense strand and the antisense strand is between 18 and 24 nucleotides.

[0286] Embodiment 19. The RNAi agent according to Embodiment 19, wherein the length of each of the sense strand and the antisense strand is 21 nucleotides.

[0287] Embodiment 20. The RNAi agent according to any one of Embodiments 17 to 20, wherein the RNAi agent has two blunt ends.

[0288] Embodiment 21. The RNAi agent according to any one of Embodiments 1 to 21, wherein the sense strand comprises one or two terminal caps.

[0289] Embodiment 22. The RNAi agent according to any one of Embodiments 1 to 22, wherein the sense strand contains one or two inverted abasic residues.

[0290] Embodiment 23. The RNAi agent according to Embodiment 1, wherein the RNAi agent is composed of a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex sequence of any one of the duplexes having the SEQ ID NOs listed in Table 5B.

[0291] Embodiment 24. The RNAi agent according to any one of Embodiments 1 to 23, wherein the sense strand further contains an inverted abasic residue at the 3'-end of the nucleotide sequence, at the 5'-end of the nucleotide sequence, or at both.

[0292] Embodiment 25. The RNAi agent according to Embodiment 1, the RNAi agent comprising an antisense strand comprising, consisting of, or consisting essentially of a modified nucleotide sequence that differs from one of the nucleotide sequences of SEQ ID NOs: 88-128 by 0 or 1 nucleotide, wherein a, c, g, and u represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate bond; and all or substantially all of the nucleotides on the sense strand are modified nucleotides.

[0293] Embodiment 26. The RNAi agent according to Embodiment 1, wherein the sense strand comprises, consists of, or consists essentially of a modified nucleotide sequence that differs from one of the nucleotide sequences of SEQ ID NOs: 129-173 by 0 or 1 nucleotide, wherein a, c, g, i, and u represent 2'-O-methyladenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate bond; and all or substantially all of the nucleotides on the antisense strand are modified nucleotides.

[0294] Embodiment 27. The RNAi agent according to any one of Embodiments 24 to 26, wherein the sense strand further comprises a reverse abasic residue at the 3'-end, at the 5'-end, or at both ends of the nucleotide sequence.

[0295] Embodiment 28. The RNAi agent according to any one of Embodiments 24 to 27, wherein the sense strand of the RNAi agent is linked to a targeting ligand.

[0296] Embodiment 29. The RNAi agent according to Embodiment 28, wherein the targeting ligand has an affinity for the asialoglycoprotein receptor.

[0297] Embodiment 30. The RNAi agent according to Embodiment 29, wherein the targeting ligand comprises N-acetyl-galactosamine.

[0298] Embodiment 31. The RNAi agent according to Embodiment 1, wherein the targeting ligand comprises:

[0299]

[0300] Embodiment 32. The RNAi agent according to Embodiment 1, wherein the antisense strand consists of a modified nucleotide sequence of SEQ ID NOs: 88 - 128, and the sense strand consists of a modified nucleotide sequence of SEQ ID NOs: 129 - 173, where a, c, g, and u are 2'-O-methyladenosine, cytidine, guanosine, and uridine respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine, cytidine, guanosine, and uridine respectively; s is a phosphorothioate bond; (invAb) is a reverse abasic deoxyribose residue; and (NAG37)s has the following chemical structure:

[0301]

[0302] Embodiment 33. A composition comprising the RNAi agent according to any one of Embodiments 1 to 32, wherein the composition further comprises a pharmaceutically acceptable excipient.

[0303] Embodiment 34. A method for inhibiting the expression of the PNPLA3 gene in a cell, the method comprising introducing an effective amount of the RNAi agent according to any one of Embodiments 1 to 32 or the composition according to any one of Embodiments 33 into the cell.

[0304] Embodiment 35. The method according to Embodiment 34, wherein the cell is in a subject.

[0305] Embodiment 36. The method according to Embodiment 35, wherein the subject is a human subject.

[0306] Embodiment 37. The method according to any one of Embodiments 34 to 36, wherein the PNPLA3 gene expression is inhibited by at least about 30%.

[0307] Embodiment 38. A method for treating a PNPLA3-related disease or disorder, the method comprising administering a therapeutically effective amount of the composition according to Embodiment 33 to a human subject in need thereof.

[0308] Embodiment 39. The method according to Embodiment 38, wherein the disease is NAFLD, NASH, liver fibrosis, alcoholic fatty liver disease, or cirrhosis.

[0309] Embodiment 40. The method according to any one of Embodiments 34 to 39, wherein the RNAi agent is administered at a dose of about 0.05 mg / kg to about 5.0 mg / kg of the human subject's body weight.

[0310] Embodiment 41. The method according to any one of Embodiments 34 to 40, wherein the RNAi agent is administered in two or more doses.

[0311] Use of an RNAi agent according to any one of embodiments 1 to 32 or a composition according to embodiment 33 for the treatment of a disease, disorder or symptom that is at least partially mediated by PNPLA3 gene expression.

[0312] Embodiment 43. The use according to embodiment 42, wherein the symptom is liver cirrhosis.

[0313] Use of an RNAi agent according to any one of embodiments 1 to 32 or a composition according to embodiment 33 for the preparation of a pharmaceutical composition for the treatment of a disease, disorder or symptom that is at least partially mediated by PNPLA3 gene expression.

[0314] Embodiment 45. The use of an RNAi agent according to any one of embodiments 42 to 44, wherein the disease is NAFLD, NASH, liver fibrosis, or alcoholic or non-alcoholic liver disease such as cirrhosis.

[0315] Embodiment 46. The use of the composition according to embodiment 33, wherein the RNAi agent is administered at a dose of about 0.05 mg / kg to about 5.0 mg / kg of the body weight of a human subject.

[0316] The above-described embodiments and items are now illustrated by the following non-limiting examples.

[0317] Example 2. PNPLA3 - SEAP Mouse Model

[0318] Example 3. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice 。

[0319] Synthesize the PNPLA3 RNAi agent duplexes shown in Table 5A and Table 5B above according to the following general procedure:

[0320] A. Synthesis.

[0321] Synthesize the sense strand and the antisense strand of the RNAi agent according to the solid-phase phosphoramidite technique used in oligonucleotide synthesis. Such standard syntheses are generally known in the art. According to the ratio, use (Bioautomation), (Bioautomation) or OP Pilot 100 (GE Healthcare). On controlled pore glass (CPG, or Synthesis was carried out on a solid support obtained from Prime Synthesis, Aston, PA, USA. Monomers positioned at the 3'-end of the respective strand were attached to the solid support as the starting point of synthesis. All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific, Milwaukee, WI, USA or Hongene Biotech, Shanghai, PRC. The 2'-O-methyl phosphoramidites include the following: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxytrityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5'-O-dimethoxytrityl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidite bears the same protecting groups as the 2'-O-methyl imide. 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was also purchased from Thermo Fisher Scientific or Hongene Biotech. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Glen Research, Virginia or Hongene Biotech. Reverse deoxybase (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes, Wilmington, MA, USA or SAFC, St Louis, MO, USA. 5'-O-dimethoxytrityl-N 2 ,N 6 -(phenoxyacetate)-2'-O-methyl-diaminopurine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was obtained from ChemGenes or Hongene Biotech.

[0322] Dissolve the phosphoramidite-containing targeting ligand in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while dissolve all other amidites in anhydrous acetonitrile (50 mM), or add anhydrous dimethylformamide and molecular sieves. Use 5-benzylthio-1H-tetrazole (BTT, 250 mM, dissolved in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM, dissolved in acetonitrile) as the activator solution. The coupling times are 12 minutes (RNA), 15 minutes (targeting ligand), 90 seconds (2'OMe), and 60 seconds (2'F). To introduce phosphorothioate linkages, use 100 mM 3-phenyl-1,2,4-dithiazolidin-5-one (POS, available from PolyOrg, Inc., Leominster, MA, USA) dissolved in anhydrous acetonitrile. Unless specifically identified as a "naked" RNAi agent without a targeting ligand, each of the PNPLA3 RNAi agent duplexes synthesized and tested in the following examples uses N-acetyl-galactosamine as "NAG" in the targeting ligand chemical structure shown in Table 6.

[0323] B. Cleavage and deprotection of the carrier-bonded oligomers.

[0324] After completion of solid-phase synthesis, treat the dried solid support with a 1:1 volume aqueous solution of 40 wt% methylamine and 28% ammonium hydroxide solution (Aldrich) at 30 °C for 1.5 hours. Evaporate the solution and reconstitute the solid residue in water (see below).

[0325] C. Purification.

[0326] Purify the crude oligomers by anion-exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A is 20 mM Tris, 5 mM EDTA at pH 9.0 and contains 20% acetonitrile. Buffer B is the same as Buffer A except that 1.5 M sodium chloride is added. Record the UV trace at 260 nm. Combine the appropriate fractions and then run on size-exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 gel with a running buffer of filtered DI water or 100 mM ammonium bicarbonate at pH 6.7 and 20% acetonitrile.

[0327] D. Annealing.

[0328] Complementary strands (sense and antisense strands) were mixed by combining equimolar RNA solutions in 1× phosphate buffered saline (Corning, Cellgro) to form RNAi agents. Some RNAi agents were lyophilized and stored at -15 °C to -25 °C. The duplex concentration was determined by measuring the solution absorbance in 1× phosphate buffered saline using a UV-Vis spectrophotometer. The solution absorbance at 260 nm was then multiplied by a conversion factor and a dilution factor to determine the duplex concentration. The conversion factor used was 0.050 mg / (mL·cm), or calculated from the extinction coefficient determined experimentally.

[0329] Table 7. Targeting Positions and Administration Groups of Example 3 。

[0330] To evaluate certain PNPLA3 RNAi agents, the PNPLA3-SEAP mouse model was used. Depending on availability, six- to eight-week-old female C57BL / 6 or ICR mice were transiently transfected in vivo by hydrodynamic tail vein injection with plasmids, at least 29 days before the administration of the PNPLA3 RNAi agent or control. Two types of SEAP plasmids were prepared. The first plasmid contained the human PNPLA3 cDNA sequence (GenBank NM_025225.2 (SEQ ID NO:1)) inserted into the 3'UTR of the SEAP (secreted human placental alkaline phosphatase) reporter gene. Due to the perceived instability of the full-length transcript over time, a second plasmid was synthesized containing a truncated form of the human PNPLA3 cDNA sequence (specifically nucleotides 501 - 2210 of GenBank NM_025225.2). One of the two plasmids was selected for transfection into the mice. 50 μg of the corresponding plasmid in Ringer's solution at a total volume of 10% of the animal's body weight was injected via the tail vein into the mice to generate PNPLA3-SEAP model mice. As previously described, the solution was injected within 5 - 7 seconds through a 27-gauge needle (Zhang G et al., “High levels of foreign gene expression in hepatocytes after tail vein injection of naked plasmid DNA.” Human Gene Therapy 1999, Vol. 10, pp. 1735 - 1737). Inhibition of PNPLA3 expression by the PNPLA3 RNAi agent led to a concomitant inhibition of SEAP expression, which was measured. Before the administration of the treatment (between day -7 and day 1 before dosing), the SEAP expression level in the serum was measured by the Phospha-Light TM SEAP reporter gene assay system (Invitrogen), and the mice were grouped according to the mean SEAP level.

[0331] Mice were anesthetized with 2%-3% isoflurane and blood samples were collected from the submandibular region into serum separator tubes (Sarstedt AG&Co., Nümbrecht, Germany). The blood was allowed to clot for 20 minutes at ambient temperature. The tubes were centrifuged at 8,000×g for 3 minutes to separate the serum and stored at 4°C. Serum was collected and measured by Phospha-Light TM SEAP reporter gene assay system (Invitrogen). The serum SEAP levels of each animal could be normalized to a control group of mice injected with vehicle control to account for non-treatment-related decreases in PNPLA3 expression with this model. To do this, first the SEAP level of each animal at one time point was divided by the expression level of that animal before treatment (day 1) to determine the ratio of expression "normalized to pre-treatment". Then the expression at a particular time point was normalized to the control group by dividing the "normalized to pre-treatment" ratio of a single animal by the mean "normalized to pre-treatment" ratio of all mice in the normal vehicle control group. Alternatively, the serum SEAP level of each animal was evaluated only by normalizing to the pre-treatment level.

[0332] Example 4. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice 。

[0333] Using the PNPLA3-SEAP mouse model described in Example 2 above, a plasmid containing the full-length human PNPLA3 transcript was used. On day 1, according to Table 7 below, each mouse was given a single subcutaneous administration of 200 μl / 20 g of animal body weight, which contained 3.0 mg / kg (mpk) of a PNPLA3 RNAi agent formulated in saline, or vehicle control (saline without the RNAi agent).

[0334] Table 10. Targeting Positions and Administration Groups of Example 4

[0335]

[0336] Each of the PNPLA3 RNAi agents contains a modified nucleotide conjugated at the 5'-end of the sense strand to a targeting ligand that includes three N-acetyl-galactosamine groups (tridentate ligand) having a modified sequence as described in the duplex structures herein. (For specific modification and structural information on PNPLA3 RNAi agents, including the (NAG37)s ligand, see Tables 3 - 5). The PNPLA3 RNAi agents J1D00001 (Group 2) and J1D00002 (Group 3) include nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 688 of the gene; the PNPLA3 RNAi agent J1D00004 (Group 4) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1586 of the gene; the PNPLA3 RNAi agent J1D00008 (Group 5) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene; the PNPLA3 RNAi agents J1D00010 (Group 6) and J1D00011 (Group 7) include nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 1179 of the gene; the PNPLA3 RNAi agent J1D00012 (Group 8) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 571 of the gene; the PNPLA3 RNAi agent J1D00016 (Group 9) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1745 of the gene; (see, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0337] Injections were made into the loose skin of the neck and shoulder regions between the skin and muscle (i.e., subcutaneous injection). Four (4) mice per group (n = 4) were tested. Serum was collected on Day - 1 (before dosing), Day 8, Day 15, Day 22, and Day 29, and SEAP expression levels were determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 8 and 9 below:

[0338]

[0339]

[0340] Example 5. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice Table 13. Targeting Positions and Administration Groups of Example 5 。

[0341] Using the PNPLA3-SEAP mouse model described in Example 2 above, a plasmid containing the full-length human PNPLA3 transcript was used. On day 1, according to Table 10 below, each mouse was administered a single subcutaneous injection of 200 μl / 20 g of animal body weight, which contained 3.0 mg / kg (mpk) of a PNPLA3 RNAi agent formulated in saline, or a vehicle control (saline without the RNAi agent).

[0342] Example 6. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice

[0343]

[0344] Each of the PNPLA3 RNAi agents contains a modified nucleotide conjugated at the 5'-end of the sense strand to a targeting ligand that contains three N-acetyl-galactosamine groups (trident ligand) with a modified sequence as described in the duplex structure herein. (For specific modification and structural information on the PNPLA3 RNAi agents (including the (NAG37)s ligand), see Tables 3 - 5). The PNPLA3 RNAi agent J1D00008 (Group 2) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene; the PNPLA3 RNAi agent J1D00014 (Group 3) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 886 of the gene; the PNPLA3 RNAi agent J1D00015 (Group 4) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1584 of the gene; the PNPLA3 RNAi agent J1D00021 (Group 5) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 553 of the gene; the PNPLA3 RNAi agent J1D00022 (Group 6) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 680 of the gene; the PNPLA3 RNAi agent J1D00005 (Group 7) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1182 of the gene; and the PNPLA3 RNAi agent J1D00024 (Group 8) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 746 of the gene. (See, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0345] The injection was performed subcutaneously (i.e., between the skin and muscle) into the loose skin of the neck and shoulder regions. Four (4) mice per group (n = 4) were tested. Serum was collected on Day -1 (before dosing), Day 8, Day 15, Day 22, and Day 29, and the SEAP expression level was determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 11 and 12 below:

[0346]

[0347]

[0348] Table 16. Targeting Positions and Administration Groups of Example 6 。

[0349] The PNPLA3-SEAP mouse model was used as described in Example 2 above, using a plasmid containing a truncated form of the human PNPLA3 transcript. On Day 1, according to Table 13 below, each mouse was given a single subcutaneous administration of 200 μl / 20 g of animal body weight, which contained 3.0 mg / kg (mpk) of the PNPLA3 RNAi agent formulated in saline, or a vehicle control (saline without the RNAi agent).

[0350] Example 7. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice

[0351]

[0352] Each of the PNPLA3 RNAi agents comprises a modified nucleotide conjugated at the 5'-end of the sense strand to a targeting ligand, the targeting ligand comprising three N-acetyl-galactosamine groups (tridentate ligand) having a modified sequence as described in the duplex structures herein. (For specific modification and structural information on PNPLA3 RNAi agents (including (NAG37)s ligands), see Tables 3 - 5). The PNPLA3 RNAi agents J1D00012 (Group 2), J1D00027 (Group 3), J1D00028 (Group 4), J1D00029 (Group 5) each comprise a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 571 of the gene; the PNPLA3 RNAi agents J1D00011 (Group 6) and J1D00017 (Group 7) comprise a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1179 of the gene; the PNPLA3 RNAi agents J1D00004 (Group 8), J1D00003 (Group 9) and J1D00041 (Group 10) comprise a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1586 of the gene; and the PNPLA3 RNAi agent J1D00008 (Group 11) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene. (See, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0353] Injections were made into the loose skin of the neck and shoulder regions between the skin and muscle (i.e., subcutaneous injection). Four (4) mice per group (n = 4) were tested. Serum was collected on Day -1 (before dosing), Day 8, Day 15, Day 22, and Day 29, and SEAP expression levels were determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 14 and 15 below:

[0354]

[0355]

[0356] Each of the PNPLA3 RNAi agents in each of the dosing groups (i.e., Groups 2 to 11) showed a decrease in SEAP compared to the vehicle control (Group 1) at all measured time points.

[0357] Table 19. Targeting Positions and Administration Groups of Example 7 。

[0358] The PNPLA3-SEAP mouse model was used as described in Example 2 above, using a plasmid containing a truncated form of the human PNPLA3 transcript. On Day 1, according to Table 16 below, each mouse was administered a single subcutaneous injection of 200 μl / 20 g of animal body weight, which contained 3.0 mg / kg (mpk) of the PNPLA3 RNAi agent formulated in saline, or a vehicle control (saline without the RNAi agent).

[0359] Example 8. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice

[0360]

[0361] Each of the PNPLA3 RNAi agents contains a modified nucleotide that is conjugated at the 5'-end of the sense strand to a targeting ligand that contains three N-acetyl-galactosamine groups (tridentate ligand) with a modified sequence as described in the duplex structure herein. (For specific modification and structural information on the PNPLA3 RNAi agents, including the (NAG37)s ligand, see Tables 3 - 5). The PNPLA3 RNAi agents J1D00008 (Group 2), J1D00046 (Group 3), J1D00047 (Group 4), and J1D00048 (Group 5) each include a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene; the PNPLA3 RNAi agents J1D00011 (Group 6), J1D00043 (Group 7), J1D00044 (Group 8), and J1D00045 (Group 9) include a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1179 of the gene; the PNPLA3 RNAi agent J1D00020 (Group 10) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 544 of the gene; and the PNPLA3 RNAi agent J1D00026 (Group 11) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1195 of the gene. (See, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0362] The injection was made (i.e., subcutaneous injection) between the skin and muscle into the loose skin of the neck and shoulder regions. Four (4) mice (n = 4) in each group were tested. Serum was collected on Day -1 (before dosing), Day 8, Day 15, Day 22, and Day 29, and the SEAP expression level was determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 17 and 18 below:

[0363]

[0364]

[0365] Table 22. Targeting Positions and Administration Groups of Example 8 Example 9. In Vivo Testing of PNPLA3 RNAi Agent in PNPLA3 - SEAP Mice 。

[0366] The PNPLA3-SEAP mouse model was used as described in Example 2 above, using a plasmid containing a truncated form of the human PNPLA3 transcript. On day 1, according to Table 19 below, each mouse was given a single subcutaneous administration of 200 μl / 20 g of animal body weight, which contained 1.5 mg / kg (mpk) of the PNPLA3 RNAi agent formulated in saline, or a vehicle control (saline without the RNAi agent).

[0367] Table 25. Targeting Positions and Administration Groups of Example 9

[0368]

[0369] Each of the PNPLA3 RNAi agents comprises a modified nucleotide conjugated at the 5'-end of the sense strand to a targeting ligand that comprises three N-acetyl-galactosamine groups (tridentate ligand) having a modified sequence as described in the duplex structure herein. (For specific modification and structural information on the PNPLA3 RNAi agents (including the (NAG37)s ligand), see Tables 3 - 5). The PNPLA3 RNAi agent J1D00008 (Group 2) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene; the PNPLA3 RNAi agents J1D00018 (Group 3) and J1D00019 (Group 4) comprise nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 538 of the gene; the PNPLA3 RNAi agent J1D00013 (Group 5) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 687 of the gene; the PNPLA3 RNAi agents J1D000006 (Group 6) and J1D00007 (Group 7) comprise nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 751 of the gene; the PNPLA3 RNAi agent J1D00035 (Group 8) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1181 of the gene; the PNPLA3 RNAi agent J1D00033 (Group 9) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 685 of the gene; the PNPLA3 RNAi agent J1D00032 (Group 10) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 373 of the gene; and the PNPLA3 RNAi agent J1D00040 (Group 11) comprises a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1837 of the gene. (See, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0370] Injections were made between the skin and muscle (i.e., subcutaneous injection) into the loose skin of the neck and shoulder regions. Four (4) mice per group (n = 4) were tested. Serum was collected on Day - 1 (before dosing), Day 8, Day 15, Day 22, and Day 29, and SEAP expression levels were determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 20 and 21 below:

[0371]

[0372]

[0373] Example 10. In Vivo Testing of PNPLA3 RNAi Agent in Cynomolgus Monkeys 。

[0374] The PNPLA3-SEAP mouse model was used as described in Example 2 above, using a plasmid containing a truncated form of the human PNPLA3 transcript. On day 1, according to Table 22 below, each mouse was administered a single subcutaneous injection of 200 μl / 20 g of animal body weight, which contained 1.5 mg / kg (mpk) of a PNPLA3 RNAi agent formulated in saline, or a vehicle control (saline without the RNAi agent).

[0375] Table 28. Targeting Positions and Administration Groups of Example 10

[0376]

[0377] Each of the PNPLA3 RNAi agents contains a modified nucleotide conjugated at the 5'-end of the sense strand to a targeting ligand, which targeting ligand contains three N-acetyl-galactosamine groups (trident ligand) with a modified sequence as described in the duplex structure herein. (For specific modification and structural information on the PNPLA3 RNAi agents (including the (NAG37)s ligand), see Tables 3 - 5). Each of the PNPLA3 RNAi agents in each group (i.e., Group 2 - Group 10) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene. (See, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0378] The injection was made (i.e., subcutaneous injection) between the skin and muscle into the loose skin of the neck and shoulder regions. Four (4) mice (n = 4) in each group were tested. Serum was collected on day -1 (before dosing), day 8, day 15, day 22, and day 29, and the SEAP expression level was determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 23 and 24 below:

[0379]

[0380]

[0381] Each of the PNPLA3 RNAi agents in each of the dosing groups (i.e., Group 2 to Group 10) showed a decrease in SEAP compared to the vehicle control (Group 1) at all measured time points.

[0382] Table 29. For Each Group (n = 4), PNPLA3 mRNA Levels Normalized to Pre - Administration (Day - 13) According to Example 10 。

[0383] The PNPLA3-SEAP mouse model was used as described in Example 2 above, using a plasmid containing a truncated form of the human PNPLA3 transcript. On Day 1, according to Table 25 below, each mouse was administered a single subcutaneous injection of 200 μl / 20 g of animal body weight, which contained 1.5 mg / kg (mpk) of the PNPLA3 RNAi agent formulated in saline, or a vehicle control (saline without the RNAi agent), and the vehicle control included the dosing groups.

[0384] Example 11. In Vivo Testing of PNPLA3 RNAi Agent J1D00008 in Cynomolgus Monkeys

[0385]

[0386] Each of the PNPLA3 RNAi agents contains a modified nucleotide that is conjugated at the 5'-end of the sense strand to a targeting ligand, and the targeting ligand contains three N-acetyl-galactosamine groups (trident ligand) having a modified sequence as described in the duplex structure herein. (For specific modification and structural information on the PNPLA3 RNAi agents (including the (NAG37)s ligand), see Tables 3 - 5). The PNPLA3 RNAi agent J1D00008 (Group 2) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene; the PNPLA3 RNAi agent J1D00081 (Group 3) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 887 of the gene; the PNPLA3 RNAi agent J1D00083 (Group 4) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1185 of the gene; the PNPLA3 RNAi agents J1D00084 (Group 5) and J1D00085 (Group 6) each include a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1191 of the gene; the PNPLA3 RNAi agent J1D00087 (Group 7) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1746 of the gene; and the PNPLA3 RNAi agent J1D00082 (Group 8) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1173 of the gene. (See, for example, the PNPLA3 gene cited in SEQ ID NO:1 and Table 2).

[0387] Injections were made between the skin and muscle (i.e., subcutaneous injection) into the loose skin of the neck and shoulder regions. Four (4) mice (n = 4) in each group were tested. Serum was collected on Day -1 (before dosing), Day 8, Day 15, Day 22, and Day 29, and the SEAP expression levels were determined according to the protocol described in Example 2 above. The experimental data are shown in Tables 26 and 27 below:

[0388]

[0389]

[0390] Materials and Methods 。

[0391] The PNPLA3 RNAi agents J1D00003, J1D00008, and J1D00017 were evaluated in cynomolgus monkeys (cynos). On Day 1 and Day 43, four cynomolgus monkeys per group (n = 4) were administered subcutaneously with 0.4 mL / kg (approx. 1.5 mL volume, depending on animal mass) containing the corresponding PNPLA3 RNAi agent formulated at 4.0 mg / kg (10 mg / mL) in saline, or saline vehicle alone (without RNAi agent) as a control (see Table 28 below).

[0392] Results

[0393]

[0394] The PNPLA3 RNAi agents comprise modified nucleotides and a targeting ligand ((NAG37)s) containing tridentate N-acetyl-galactosamine conjugated to the 5'-end of the sense strand, as shown in Tables 3 - 6. The PNPLA3 RNAi agent J1D00003 (Group 2) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1586 of the gene; the PNPLA3 RNAi agent J1D00008 (Group 3) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene; and the PNPLA3 RNAi agent J1D00017 (Group 4) includes a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1179 of the gene.

[0395] On Day - 13 (before dosing), Day 15, Day 29, Day 57, and Day 76, liver biopsies were performed. On the date of each biopsy collection, the cynomolgus monkeys were anesthetized and two liver tissue samples, each approximately 80 mg to 120 mg, were extracted using laparoscopy. The biopsy samples were then homogenized and the levels of PNPLA3 mRNA in the cynomolgus monkey liver were measured by RT-qPCR. The resulting values were then normalized to the PNPLA3 mRNA measurement before dosing (in this case, on Day - 13). The resulting mRNA data are reflected in Table 29 below:

[0396] Figure 1 Table 30. Quantification of ISH Images of Total Hepatocyte Cytoplasmic and Nuclear PNPLA3 Expression Relative to Baseline (Day - 13) in Study #1

[0397]

[0398] Table 31. Parameters Related to Liver Physiology Employed in Modeling

[0399] The objective of this study was to evaluate the depth and duration of knockdown of WT PNPLA3 mRNA in the liver of cynomolgus monkeys (cynos) by the RNAi agent J1D00008. The knockdown in each animal was normalized to baseline measurements obtained from liver biopsies performed prior to dosing.

[0400] Figure 2

[0401] Study 1: Cynomolgus monkeys were grouped by body weight (2 kg - 5 kg) into a saline treatment group (Group 1) or RNA treatment groups (Groups 2 - 4), with n = 4 per group. Cynomolgus monkeys were dosed subcutaneously twice at 4 mg / kg in 0.4 ml / kg saline on Day 1 and Day 43. Cynomolgus monkeys were fasted overnight for at least 12 hours prior to biopsy and blood collection. Liver biopsy samples were collected from all animals before dosing and on Test Days 15, 29, 57, and 76. For each animal, liver biopsy samples (2-fold, approximately 100 mg each) were collected for PNPLA3 mRNA quantification by qPCR and in situ hybridization (ISH). Blood was collected from the femoral vein of each animal before dosing and after dosing on Days 15, 29, 57, and 76. For all animals, groups, and time points, RNA was isolated from the left hepatic lobe to perform qRT-PCR, the data were normalized to a housekeeping gene (ARFGAP2), and the relative expression was compared to the baseline / pre-dose biopsy. At the end of the study, PNPLA3 expression was measured in the left lateral, right lateral, and middle hepatic lobes from Groups 1 and 3 to confirm that there was no variation in expression between lobes and that the left lateral lobe was representative. An in situ hybridization assay (ACDbio, Newark, CA) based on was performed on cynomolgus monkey liver biopsies. Total PNPLA3 mRNA copies in a given hepatocyte population were quantified using image-based quantitative software analysis (HALO TM software system, Indica Labs, Albuquerque, NM).

[0402] Study 2: A second non-human primate study was conducted as described in Study 1, with the following modifications or additions. Cynomolgus monkeys were grouped by body weight (2 kg - 5 kg) into two RNAi treatment groups, with n = 10 per group. Animals were dosed subcutaneously at 4 mg / kg in saline on Day 1 and Day 29, and then continuously for 57 days.

[0403] Example 12. Safety

[0404] From Study 1, PNPLA3 expression was measured by PCR in Other EmbodimentsShown for the RNAi agent J1D00008. At the corresponding time points normalized to baseline and housekeeping genes, PNPLA3 mRNA was reduced by 51%, 55%, 47%, and 37%. Following these results, in situ hybridization (ISH) was performed to evaluate the intracellular (cytoplasmic versus nuclear) distribution of PNPLA3 mRNA. Approximately 50% of the total cellular PNPLA3 mRNA was observed to be distributed within the nucleus. This indicates that the RNAi agent J1D00008 significantly reduces PNPLA3 mRNA at the site of protein translation (i.e., hepatocyte cytoplasm), but qPCR underestimated the knockdown at the whole cell level due to the confounding residual nuclear PNPLA3 mRNA pool. Thus, cytoplasmic PNPLA3 mRNA knockdown was quantified.

[0405] ISH was performed on liver biopsies from cynomolgus monkeys treated with the RNAi agent J1D00008, revealing significant residual PNPLA3 mRNA retained in the nucleus, thus supporting the assertion that this pool is the result of confounding knockdown in the whole liver, as assessed by qPCR.

[0406] Quantification of the ISH images showed that treatment with the RNAi agent J1D00008 resulted in a 44% - 63% reduction in cytoplasmic PNPLA3mRNA over all 76 days (Table 30). Since GalNAc-RNAi agents such as the RNAi agent J1D00008 specifically mediate the degradation of target mRNA in the hepatocyte cytoplasm, the whole liver quantification of PNPLA3 knockdown is diluted by the relative contribution of PNPLA3 mRNA from non-target cell types (e.g., stellate cells and Kupffer cells). Thus, a model-based analysis was performed to specifically evaluate the reduction of PNPLA3 mRNA in the cytoplasm of hepatocytes while correcting for extrahepatic PNPLA3 mRNA expression. Table 31 lists the relevant assumptions of liver physiology employed in the modeling.

[0407] ​ ​ 。

[0408]

[0409] Data are mean ± SD relative to baseline (n = 4)

[0410] ​ 。

[0411]

[0412] Using this model, after administration of a single 4 mg / kg SC dose of the RNAi agent J1D00008, PNPLA3 mRNA was reduced by 81% in the cytoplasm of hepatocytes on day 15 and by 78% on day 29. When this modeling was repeated in Study 2, reductions of 66% and 65% in PNPLA3 mRNA were achieved on days 15 and 29, respectively. In all NHPs (n = 14) tested with the RNAi agent J1D00008, PNPLA3 mRNA was reduced by an average of 70% in the cytoplasm of hepatocytes on days 15 and 29 ( ​ ).

[0413] ​

[0414] The non-clinical safety of the RNAi agent J1D00008 was evaluated in rats and monkeys at doses up to 500 mg / kg and 300 mg / kg, respectively, for up to 3 months. In both species, the RNAi agent J1D00008 was well tolerated and no adverse signs of toxicity were observed up to the highest dose evaluated.

[0415] ​

[0416] It should be understood that although the present invention has been described in connection with specific embodiments thereof, the foregoing description is intended to be illustrative and not restrictive of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. <110> ARROWHEAD PHARMACEUTICALS, INC. <120> RNAi Agent for Inhibiting PNPLA3 Expression, Pharmaceutical Composition Thereof, and Method of Use <130> 103693.002475 PCT <140> <141> <150> 63 / 000,137 <151> 2020-03-26 <160> 258 <170> PatentIn version 3.5 <210> 1 <211> 2805 <212> DNA <213> Homo sapiens <400> 1 atggtccgag gggggcgggg ctgacgtcgc gctgggaatg ccctggccga gacactgagg 60 cagggtagag agcgcttgcg ggcgccgggc ggagctgctg cggatcagga cccgagccga 120 ttcccgatcc cgacccagat cctaacccgc gcccccgccc cgccgccgcc gccatgtacg 180 acgcagagcg cggctggagc ttgtccttcg cgggctgcgg cttcctgggc ttctaccacg 240 tcggggcgac ccgctgcctg agcgagcacg ccccgcacct cctccgcgac gcgcgcatgt 300 tgttcggcgc ttcggccggg gcgttgcact gcgtcggcgt cctctccggt atcccgctgg 360 agcagactct gcaggtcctc tcagatcttg tgcggaaggc caggagtcgg aacattggca 420 tcttccatcc atccttcaac ttaagcaagt tcctccgaca gggtctctgc aaatgcctcc 480 cggccaatgt ccaccagctc atctccggca aaataggcat ctctcttacc agagtgtctg 540 atggggaaaa cgttctggtg tctgactttc ggtccaaaga cgaagtcgtg gatgccttgg 600 tatgttcctg cttcatcccc ttctacagtg gccttatccc tccttccttc agaggcgtgc 660 gatatgtgga tggaggagtg agtgacaacg tacccttcat tgatgccaaa acaaccatca 720 ccgtgtcccc cttctatggg gagtacgaca tctgccctaa agtcaagtcc acgaactttc 780 ttcatgtgga catcaccaag ctcagtctac gcctctgcac agggaacctc taccttctct 840 cgagagcttt tgtccccccg gatctcaagg tgctgggaga gatatgcctt cgaggatatt 900 tggatgcatt caggttcttg gaagagaagg gcatctgcaa caggccccag ccaggcctga 960 agtcatcctc agaagggatg gatcctgagg tcgccatgcc cagctgggca aacatgagtc 1020 tggattcttc cccggagtcg gctgccttgg ctgtgaggct ggagggagat gagctgctag 1080 accacctgcg tctcagcatc ctgccctggg atgagagcat cctggacacc ctctcgccca 1140 ggctcgctac agcactgagt gaagaaatga aagacaaagg tggatacatg agcaagattt 1200 gcaacttgct acccattagg ataatgtctt atgtaatgct gccctgtacc ctgcctgtgg 1260 aatctgccat tgcgattgtc cagagactgg tgacatggct tccagatatg cccgacgatg 1320 tcctgtggtt gcagtgggtg acctcacagg tgttcactcg agtgctgatg tgtctgctcc 1380 ccgcctccag gtcccaaatg ccagtgagca gccaacaggc ctccccatgc acacctgagc 1440 aggactggcc ctgctggact ccctgctccc ccaagggctg tccagcagag accaaagcag 1500 aggccacccc gcggtccatc ctcaggtcca gcctgaactt cttcttgggc aataaagtac 1560 ctgctggtgc tgaggggctc tccacctttc ccagtttttc actagagaag agtctgtgag 1620 tcacttgagg aggcgagtct agcagattct ttcagaggtg ctaaagtttc ccatctttgt 1680 gcagctacct ccgcattgct gtgtagtgac ccctgcctgt gacgtggagg atcccagcct 1740 ctgagctgag ttggttttat gaaaagctag gaagcaacct ttcgcctgtg cagcggtcca 1800 gcacttaact ctaatacatc agcatgcgtt aattcagctg gttgggaaat gacaccagga 1860 agcccagtgc agagggtccc ttactgactg tttcgtggcc ctattaatgg tcagactgtt 1920 ccagcatgag gttcttagaa tgacaggtgt ttggatgggt gggggccttg tgatgggggg 1980 taggctggcc catgtgtgat cttgtggggt ggagggaaga gaatagcatg atcccacttc 2040 cccatgctgt gggaaggggt gcagttcgtc cccaagaacg acactgcctg tcaggtggtc 2100 tgcaaagatg ataaccttga ctactaaaaa cgtctccatg gcgggggtaa caagatgata 2160 atctacttaa ttttagaaca cctttttcac ctaactaaaa taatgtttaa agagttttgt 2220 ataaaaatgt aaggaagcgt tgttacctgt tgaattttgt attatgtgaa tcagtgagat 2280 gttagtagaa taagccttaa aaaaaaaaaa atcggttggg tgcagtggca cacggctgta 2340 atcccagcac tttgggaggc caaggttggc agatcacctg aggtcaggag ttcaagacca 2400 gtctggccaa catagcaaaa ccctgtctct actaaaaata caaaaattat ctgggcatgg 2460 tggtgcatgc ctgtaatccc agctattcgg aaggctgagg caggagaatc acttgaaccc 2520 aggaggcgga ggttgcggtg agctgagatt gcaccatttc attccagcct gggcaacatg 2580 agtgaaagtc tgactcaaaa aaaaaaaatt taaaaaacaa aataatctag tgtgcagggc 2640 attcacctca gccccccagg caggagccaa gcacagcagg agcttccgcc tcctctccac 2700 tggagcacac aacttgaacc tggcttattt tctgcaggga ccagccccac atggtcagtg 2760 agtttctccc catgtgtggc gatgagagag tgtagaaata aagac 2805 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <400> 2 accuuuuuca ccuaacuaa 19 <210> 3 <211> 19 <212> RNA <213> Homo sapiens <400> 3 cuuucccagu uuuucacua 19 <210> 4 <211> 19 <212> RNA <213> Homo sapiens <400> 4 gguggauaca ugagcaaga 19 <210> 5 <211> 19 <212> RNA <213> Homo sapiens <400> 5 gguccaaaga cgaagucgu 19 <210> 6 <211> 19 <212> RNA <213> Homo sapiens <400> 6 aacguacccu ucauugaug 19 <210> 7 <211> 19 <212> RNA <213> Homo sapiens <400> 7 cugaguuggu uuuaugaaa 19 <210> 8 <211> 19 <212> RNA <213> Homo sapiens <400> 8 agcaagauuu gcaacuugc 19 <210> 9 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 9 accuuuuuca ccuaacuan 19 <210> 10 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 10 nccuuuuuca ccuaacuan 19 <210> 11 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (18)..(18) <223> 2-Aminoadenine nucleotide <400> 11 accuuuuuca ccuaacuaa 19 <210> 12 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (18)..(18) <223> 2 - aminoadenine nucleotide <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 12 accuuuuuca ccuaacuan 19 <210> 13 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (18)..(18) <223> 2 - aminoadenine nucleotide <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 13 nccuuuuuca ccuaacuan 19 <210> 14 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 14 cuuucccagu uuuucacun 19 <210> 15 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 15 nuuucccagu uuuucacun 19 <210> 16 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 16 gguggauaca ugagcaagn 19 <210> 17 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 17 nguggauaca ugagcaagn 19 <210> 18 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (14)..(14) <223> Hypoxanthine <400> 18 gguggauaca ugancaaga 19 <210> 19 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (14)..(14) <223> Hypoxanthine <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 19 gguggauaca ugancaagn 19 <210> 20 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (14)..(14) <223> Hypoxanthine <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 20 nguggauaca ugancaagn 19 <210> 21 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <400> 21 gguccaaaga cgaagucga 19 <210> 22 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 22 gguccaaaga cgaagucgn 19 <210> 23 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 23 nguccaaaga cgaagucgn 19 <210> 24 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (18)..(18) <223> Hypoxanthine <400> 24 gguccaaaga cgaagucnu 19 <210> 25 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (18)..(18) <223> Hypoxanthine <400> 25 gguccaaaga cgaagucna 19 <210> 26 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (18)..(18) <223> Hypoxanthine <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 26 gguccaaaga cgaagucnn 19 <210> 27 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (18)..(18) <223> Hypoxanthine <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 27 nguccaaaga cgaagucnn 19 <210> 28 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (12)..(12) <223> Hypoxanthine <220> <221> Modified base <222> (18)..(18) <223> Hypoxanthine <400> 28 gguccaaaga cnaagucnu 19 <210> 29 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (12)..(12) <223> Hypoxanthine <220> <221> Modified base <222> (18)..(18) <223> Hypoxanthine <400> 29 gguccaaaga cnaagucna 19 <210> 30 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified Base <222> (12)..(12) <223> Hypoxanthine <220> <221> Modified Base <222> (18)..(18) <223> Hypoxanthine <220> <221> Modified Base <222> (19)..(19) <223> Any Nucleobase <400> 30 gguccaaaga cnaagucnn 19 <210> 31 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified Base <222> (1)..(1) <223> Any Nucleobase <220> <221> Modified Base <222> (12)..(12) <223> Hypoxanthine <220> <221> Modified Base <222> (18)..(18) <223> Hypoxanthine <220> <221> Modified Base <222> (19)..(19) <223> Any Nucleobase <400> 31 nguccaaaga cnaagucnn 19 <210> 32 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (15)..(15) <223> Hypoxanthine <400> 32 gguccaaaga cgaanucgu 19 <210> 33 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (15)..(15) <223> Hypoxanthine <400> 33 gguccaaaga cgaanucga 19 <210> 34 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (15)..(15) <223> Hypoxanthine <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 34 gguccaaaga cgaanucgn 19 <210> 35 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (15)..(15) <223> Hypoxanthine <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 35 nguccaaaga cgaanucgn 19 <210> 36 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <400> 36 aacguacccu ucauugaua 19 <210> 37 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 37 aacguacccu ucauugaun 19 <210> 38 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 38 nacguacccu ucauugaun 19 <210> 39 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 39 cugaguuggu uuuaugaan 19 <210> 40 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 40 nugaguuggu uuuaugaan 19 <210> 41 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 41 agcaagauuu gcaacuuga 19 <210> 42 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 42 cgcaagauuu gcaacuuga 19 <210> 43 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 43 agcaagauuu gcaacuugn 19 <210> 44 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 44 cgcaagauuu gcaacuugn 19 <210> 45 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 45 ngcaagauuu gcaacuugn 19 <210> 46 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <400> 46 uuaguuaggu gaaaaaggu 19 <210> 47 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 47 nuaguuaggu gaaaaaggu 19 <210> 48 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 48 nuaguuaggu gaaaaaggn 19 <210> 49 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 49 uuaguuaggu gaaaaaggu 19 <210> 50 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 50 nuaguuaggu gaaaaaggu 19 <210> 51 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 51 nuaguuaggu gaaaaaggn 19 <210> 52 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 52 uagugaaaaa cugggaaag 19 <210> 53 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 53 nagugaaaaa cugggaaag 19 <210> 54 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 54 nagugaaaaa cugggaaan 19 <210> 55 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment = "Description of artificial sequence: synthetic oligonucleotide" <400> 55 ucuugcucau guauccacc 19 <210> 56 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 56 ncuugcucau guauccacc 19 <210> 57 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 57 ncuugcucau guauccacn 19 <210> 58 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <400> 58 ucuugcucau guauccacc 19 <210> 59 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 59 ncuugcucau guauccacc 19 <210> 60 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 60 ncuugcucau guauccacn 19 <210> 61 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <400> 61 acgacuucgu cuuuggacc 19 <210> 62 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <400> 62 ucgacuucgu cuuuggacc 19 <210> 63 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified Base <222> (1)..(1) <223> Any nucleobase <400> 63 ncgacuucgu cuuuggacc 19 <210> 64 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified Base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified Base <222> (19)..(19) <223> Any nucleobase <400> 64 ncgacuucgu cuuuggacn 19 <210> 65 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 65 acgacuucgu cuuuggacc 19 <210> 66 <211> 19 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 66 ucgacuucgu cuuuggacc 19 <210> 67 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 67 ncgacuucgu cuuuggacc 19 <210> 68 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 68 ncgacuucgu cuuuggacn 19 <210> 69 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 69 acgacuucgu cuuuggacc 19 <210> 70 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic oligonucleotide" <400> 70 ucgacuucgu cuuuggacc 19 <210> 71 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 71 ncgacuucgu cuuuggacc 19 <210> 72 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 72 ncgacuucgu cuuuggacn 19 <210> 73 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of the artificial sequence: synthetic oligonucleotide” <400> 73 acgacuucgu cuuuggacc 19 <210> 74 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of the artificial sequence: synthetic oligonucleotide” <400> 74 ucgacuucgu cuuuggacc 19 <210> 75 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of the artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 75 ncgacuucgu cuuuggacc 19 <210> 76 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of the artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 76 ncgacuucgu cuuuggacn 19 <210> 77 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 77 naucaaugaa ggguacguu 19 <210> 78 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <400> 78 uaucaaugaa ggguacguu 19 <210> 79 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 79 naucaaugaa ggguacgun 19 <210> 80 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 80 nuucauaaaa ccaacucag 19 <210> 81 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 81 nuucauaaaa ccaacucan 19 <210> 82 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <400> 82 gcaaguugca aaucuugcu 19 <210> 83 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <400> 83 ucaaguugca aaucuugcu 19 <210> 84 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <400> 84 ucaaguugca aaucuugcg 19 <210> 85 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 85 ncaaguugca aaucuugcu 19 <210> 86 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <400> 86 ncaaguugca aaucuugcg 19 <210> 87 <211> 19 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> Any nucleobase <220> <221> Modified base <222> (19)..(19) <223> Any nucleobase <400> 87 ncaaguugca aaucuugcn 19 <210> 88 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiolate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorocytidine-3'-thiolate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methyladenosine-3’-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylguanosine-3'-phosphate <400> 88 ucaucaauga aggguacguu g 21 <210> 89 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorocytidine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2’-fluorocytidine-3’-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methylguanosine-3’-phosphate <400> 89 ucaucaauga aggguacguc g 21 <210> 90 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2’-O-methyluridine-3’-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2’-fluoroadenosine-3’-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methylguanosine-3’-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2’-fluoroguanosine-3’-thionophosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methyluridine-3’-phosphate <400> 90 uagugaaaaa cugggaaagg u 21 <210> 91 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroadenosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methylguanosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoro-guanosine-3'-thio-phosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyl-guanosine-3'-phosphate <400> 91 uagugaaaaa cugggaaagg g 21 <210> 92 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: Synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyl-adenosine-3'-thio-phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoro-adenosine-3'-thio-phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyl-adenosine-3'-thio-phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyl-cytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoroadenosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylcytidine-3'-phosphate <400> 92 aaaucuugcu cauguaucca c 21 <210> 93 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoroadenosine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methylcytidine-3’-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylguanosine-3'-phosphate <400> 93 uacuugacuu uagggcagau g 21 <210> 94 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroadenosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methylcytidine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoro-cytidine-3'-thio-phosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyl-guanosine-3'-phosphate <400> 94 uacuugacuu uagggcagac g 21 <210> 95 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyl-uridine-3'-thio-phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoro-uridine-3'-thio-phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyl-adenosine-3'-thio-phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyl-uridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoro-adenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoroguanosine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyluridine-3'-phosphate <400> 95 uuaguuaggu gaaaaaggug u 21 <210> 96 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorocytidine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyluridine-3'-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiol phosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylguanosine-3'-phosphate <400> 96 ucuugcucau guauccaccu g 21 <210> 97 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: Synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorocytidine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methylguanosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoro guanosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyl adenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoro cytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyl cytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoro guanosine-3'-thio phosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyl adenosine-3'-phosphate <400> 97 acgacuucgu cuuuggaccg a 21 <210> 98 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyl uridine-3'-thio phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoroadenosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoro-cytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyl-uridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoro-guanosine-3'-thio-phosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methylguanosine-3’-phosphate <400> 98 uaucaaugaa ggguacguug g 21 <210> 99 <211> 21 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified Base <222> (1)..(1) <223> 2’-O-methyluridine-3’-thiophosphate <220> <221> Modified Base <222> (2)..(2) <223> 2’-fluorocytidine-3’-thiophosphate <220> <221> Modified Base <222> (3)..(3) <223> 2’-O-methylcytidine-3’-thiophosphate <220> <221> Modified Base <222> (4)..(4) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified Base <222> (5)..(5) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified Base <222> (6)..(6) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified Base <222> (7)..(7) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluoroadenosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylguanosine-3'-phosphate <400> 99 uccaaauauc cucgaaggca g 21 <210> 100 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methylguanosine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2’-fluoroguanosine-3’-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methylguanosine-3’-phosphate <400> 100 uugaaaaacu gggaaaggug g 21 <210> 101 <211> 21 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified Base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiolate <220> <221> Modified Base <222> (2)..(2) <223> 2'-fluorouridine-3'-thiolate <220> <221> Modified Base <222> (3)..(3) <223> 2'-O-methylcytidine-3'-thiolate <220> <221> Modified Base <222> (4)..(4) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified Base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified Base <222> (6)..(6) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified Base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified Base <222> (8)..(8) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified Base <222> (9)..(9) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluorouridine-3'-thiolphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylcytidine-3'-phosphate <400> 101 uucauaaaac caacucagcu c 21 <210> 102 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiolphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorocytidine-3'-thiolphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyluridine-3'-thiolphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’,3’-seco-uridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluorouridine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylguanosine-3'-phosphate <400> 102 ucuugcucau guauccaccu g 21 <210> 103 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroguanosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2',3'-seco-uridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoroadenosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylcytidine-3'-phosphate <400> 103 agaacguuuu ccccaucaga c 21 <210> 104 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyladenosine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoroguanosine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyladenosine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’,3’-Cyclic uridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-Fluorouridine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-Methyluridine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-Fluorouridine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-Methylcytidine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-Fluorocytidine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-Methylcytidine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-Fluorocytidine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-Methyladenosine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-O-Methyluridine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyluridine-3'-phosphate <400> 104 agaacguuuu ccccaucagu u 21 <210> 105 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoroadenosine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methylcytidine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-phosphorothioate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyladenosine-3'-phosphate <400> 105 uacaccagaa cguuuucccc a 21 <210> 106 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroguanosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2',3'-open-ring uridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylguanosine-3'-phosphate <400> 106 ugaaagucag acaccagaac g 21 <210> 107 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoroadenosine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyladenosine-3'-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-Fluoro-guanosine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-Methyl-guanosine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-Fluoro-guanosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’,3’-Open-ring-uridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-Fluoro-adenosine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-Methyl-cytidine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-Fluoro-guanosine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-Methyl-uridine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-Fluoro-uridine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-Methyl-guanosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoroadenosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylcytidine-3'-phosphate <400> 107 uaaggguacg uugucacuca c 21 <210> 108 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroadenosine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methylcytidine-3'-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylguanosine-3'-phosphate <400> 108 uacuuuaggg cagaugucgu g 21 <210> 109 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroguanosine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluorocytidine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylguanosine-3'-phosphate <400> 109 uguagcaagu ugcaaaucuc g 21 <210> 110 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyladenosine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorocytidine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methylguanosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2',3'-Open-ring uridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluoroguanosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methyladenosine-3’-phosphate <400> 110 acgacuucgu cuuuggaccg a 21 <210> 111 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2’-O-methyladenosine-3’-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2’-fluorocytidine-3’-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methyladenosine-3’-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluoroguanosine-3'-thiolphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylcytidine-3'-phosphate <400> 111 acaagaucug agaggaccug c 21 <210> 112 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiolphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorocytidine-3'-thiolphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluorocytidine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylguanosine-3'-phosphate <400> 112 ucaaugaagg guacguuguc g 21 <210> 113 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroadenosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylcytidine-3'-phosphate <400> 113 aaucuugcuc auguauccac c 21 <210> 114 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorouridine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methylguanosine-3'-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylguanosine-3'-phosphate <400> 114 uugucauuuc ccaaccagcu g 21 <210> 115 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluoroadenosine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methylguanosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoro-guanosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyl-cytidine-3'-phosphate <400> 115 uagugaaaaa cugggaaagg c 21 <210> 116 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyl-uridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluoro-cytidine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyl-uridine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyluridine-3'-phosphate <400> 116 ucuugcucau guauccaccu u 21 <210> 117 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorocytidine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methylcytidine-3'-phosphate <400> 117 ucuugcucau guauccaccu c 21 <210> 118 <211> 21 <212> RNA <213> Artificial Sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified Base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiophosphate <220> <221> Modified Base <222> (2)..(2) <223> 2'-fluorouridine-3'-thiophosphate <220> <221> Modified Base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiophosphate <220> <221> Modified Base <222> (4)..(4) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified Base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified Base <222> (6)..(6) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified Base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoro-adenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoro-adenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoro-adenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoroguanosine-3'-thiolphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylcytidine-3'-phosphate <400> 118 uuaguuaggu gaaaaaggug c 21 <210> 119 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 5'-Cyclopropylphosphonate-2'-O-methyluridine-3'-thiolphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorouridine-3'-thiolphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyladenosine-3'-thiolphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyl-uridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyl-adenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoro-guanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoro-uridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyl-guanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoro-adenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2’-fluoroguanosine-3’-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methyluridine-3’-phosphate <400> 119 uuaguuaggu gaaaaaggug u 21 <210> 120 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2’-O-methyluridine-3’-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2’-fluorouridine-3’-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methyladenosine-3’-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’,3’-seco-uridine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluoroguanosine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methyluridine-3'-phosphate <400> 120 uuaguuaggu gaaaaaggug u 21 <210> 121 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorouridine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2',3'-seco-adenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-fluoroguanosine-3'-thiolphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-methyluridine-3'-phosphate <400> 121 uuaguuaggu gaaaaaggug u 21 <210> 122 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-methyluridine-3'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-fluorouridine-3'-thiol phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2',3'-seco-guanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluoro-guanosine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyluridine-3'-phosphate <400> 122 uuaguuaggu gaaaaaggug u 21 <210> 123 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyladenosine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methylcytidine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyladenosine-3'-phosphate <400> 123 auccaaauau ccucgaaggc a 21 <210> 124 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: Synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorocytidine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methyladenosine-3’-thiol phosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyluridine-3'-phosphate <400> 124 ucauguaucc accuuugucu u 21 <210> 125 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2’-O-methyluridine-3’-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2’-fluoroguanosine-3’-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methylcytidine-3’-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorouridine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylcytidine-3'-phosphate <400> 125 ugcaaaucuu gcucauguau c 21 <210> 126 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note = "Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorocytidine-3'-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyladenosine-3'-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoroguanosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-thiol phosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyladenosine-3'-phosphate <400> 126 ucaaguugca aaucuugcuc a 21 <210> 127 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Comment=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Modified base <222> (1)..(1) <223> 2’-O-methyluridine-3’-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2’-fluorocytidine-3’-thiophosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methyladenosine-3’-thiophosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-fluoroadenosine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-fluoroguanosine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-thiotriphosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methylguanosine-3'-phosphate <400> 127 ucaaguugca aaucuugcuc g 21 <210> 128 <211> 21 <212> RNA <213> Artificial sequence <220> <221> Source <223> / note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Modified base <222> (1)..(1) <223> 2'-O-Methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-Fluorouridine-3'-thiotriphosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-Methyluridine-3'-thiotriphosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-Fluoroadenosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2'-Fluorocytidine-3'-thiophosphate <220> <221> Modified base <222> (21)..(21) <223> 2'-O-Methyluridine-3'-phosphate <400> 128 uuucauaaaa ccaacucagc u 21 <210> 129 <211> 23 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note="Description of artificial sequence: synthetic oligonucleotide" <220> <221> Source <223> / Note="5'-NAG37-thiophosphate" <220> <221> Modified base <222> (1)..(1) <223> Reverse abasic deoxyribonucleotide-5'-thiol phosphate <220> <221> Modified base <222> (2)..(2) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (3)..(3) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (4)..(4) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2'-fluorocytidine-3'-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2'-Fluorocytidine-3'-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2'-Fluorouridine-3'-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-O-Methylcytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-Methyluridine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-O-Methylguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-Methyladenosine-3'-phosphate <220> <221> Modified base <222> (20)..(20) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (21)..(21) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (22)..(22) <223> 2’-O-methyladenosine-3’-thiophosphate <220> <221> Modified base <222> (23)..(23) <223> Reverse abasic deoxyribonucleotide <400> 129 ncaacguacc cuucauugau gan 23 <210> 130 <211> 23 <212> RNA <213> Artificial sequence <220> <221> Source <223> / Note=“Description of artificial sequence: synthetic oligonucleotide” <220> <221> Source <223> / Note=“5’-NAG37-thiophosphate” <220> <221> Modified base <222> (1)..(1) <223> Reverse abasic deoxyribonucleotide-5’-thiophosphate <220> <221> Modified base <222> (2)..(2) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (3)..(3) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (4)..(4) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (5)..(5) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (6)..(6) <223> 2’-O-methylguanosine-3’-phosphate <220> <221> Modified base <222> (7)..(7) <223> 2’-O-methyluridine-3’-phosphate <220> <221> Modified base <222> (8)..(8) <223> 2’-O-methyladenosine-3’-phosphate <220> <221> Modified base <222> (9)..(9) <223> 2’-O-methylcytidine-3’-phosphate <220> <221> Modified base <222> (10)..(10) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (11)..(11) <223> 2’-fluorocytidine-3’-phosphate <220> <221> Modified base <222> (12)..(12) <223> 2’-fluorouridine-3’-phosphate <220> <221> Modified base <222> (13)..(13) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (14)..(14) <223> 2'-O-methylcytidine-3'-phosphate <220> <221> Modified base <222> (15)..(15) <223> 2'-O-methyladenosine-3'-phosphate <220> <221> Modified base <222> (16)..(16) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (17)..(17) <223> 2'-O-methyluridine-3'-phosphate <220> <221> Modified base <222> (18)..(18) <223> 2'-O-methylguanosine-3'-phosphate <220> <221> Modified base <222> (19)..(19) <223> 2'-O-methyladenosine-3'-phosphorothioate <220> <...

Claims

1. An RNAi agent for inhibiting the expression of the PNPLA3 gene, the RNAi agent comprising: An antisense strand having a nucleotide sequence of a modified antisense strand sequence usUfsasGfuUfaGfgUfgAfaAfaAfgGfuGfsu (SEQ ID NO:95); and A sense strand having a nucleotide sequence selected from acaccuuuUfUfCfaccuaacuaa (SEQ ID NO:136), a_2NcaccuuuUfUfCfaccuaacuaa (SEQ ID NO:164), acaccuuuUfUfCfaccuaacua_2Na (SEQ ID NO:165), a_2NcaccuuuUfUfCfaccuaacua_2Na (SEQ ID NO:166) and acAfcCfuUfuUfUfCfaccuaacuaa (SEQ ID NO:167); Wherein a, c, g and u represent 2'-O-methyladenosine, cytidine, guanosine and uridine respectively; Af, Cf, Gf and Uf represent 2'-fluoroadenosine, cytidine, guanosine and uridine respectively; s represents a phosphorothioate bond, and a_2N represents 2′-O-methyl-2-aminoadenosine-3′-phosphate.

2. The RNAi agent according to claim 1, wherein the nucleotide sequence of the sense strand is the nucleotide sequence of acaccuuuUfUfCfaccuaacuaa of SEQ ID NO:

136.

3. The RNAi agent according to claim 1, wherein the RNAi agent is linked to a targeting ligand.

4. The RNAi agent according to claim 3, wherein the targeting ligand comprises N-acetyl-galactosamine.

5. The RNAi agent according to claim 3, wherein the targeting ligand comprises a structure of (NAG37) or (NAG37)s.

6. The RNAi agent according to claim 3, wherein the targeting ligand is linked to the sense strand.

7. The RNAi agent according to claim 6, wherein the targeting ligand is linked to the 5' end of the sense strand.

8. The RNAi agent according to claim 1, wherein the RNAi agent has two blunt ends.

9. The RNAi agent according to claim 1, wherein the sense strand comprises one or two terminal caps.

10. The RNAi agent according to claim 1, wherein the RNAi agent is composed of a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex sequence having SEQ ID NO:95 and SEQ ID NO:

136.

11. The RNAi agent according to claim 1, wherein the sense strand further comprises a reverse abasic residue at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or at both.

12. The RNAi agent according to claim 1 or 11, wherein the sense strand of the RNAi agent is linked to a targeting ligand.

13. The RNAi agent according to claim 12, wherein the targeting ligand has an affinity for the asialoglycoprotein receptor.

14. The RNAi agent according to claim 13, wherein the targeting ligand comprises N-acetyl-galactosamine.

15. The RNAi agent according to claim 12, wherein the targeting ligand comprises:

16. The RNAi agent according to claim 1, wherein the antisense strand consists of the modified nucleotide sequence of usUfsasGfuUfaGfgUfgAfaAfaAfgGfuGfsu (SEQ ID NO:95), and the sense strand consists of the modified nucleotide sequence of (NAG37)s(invAb)sacaccuuuUfUfCfaccuaacuaas(invAb) (SEQ ID NO:136); wherein a, c, g, and u are 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s is a phosphorothioate bond; (invAb) is a reverse abasic deoxyribose residue; and (NAG37)s has the following chemical structure:

17. A composition comprising the RNAi agent according to any one of claims 1 to 16, wherein the composition further comprises a pharmaceutically acceptable excipient.

18. Use of a therapeutically effective amount of the composition according to claim 17 in the preparation of a drug for treating a PNPLA3-related disease or disorder, wherein the disease or disorder is NAFLD, liver fibrosis, alcoholic fatty liver disease, or cirrhosis.

19. The use according to claim 18, wherein the RNAi agent is administered at a dose of 0.05 mg / kg to 5.0 mg / kg of the body weight of a human subject.

20. The use according to any one of claim 19, wherein the RNAi agent is administered in two or more doses.

21. Use of the RNAi agent according to any one of claims 1 to 16 or the composition according to claim 17 in the preparation of a drug for treating a disease, disorder, or symptom that is at least partially mediated by PNPLA3 gene expression, wherein the disease, disorder, or symptom is liver fibrosis, alcoholic or non-alcoholic liver disease, or cirrhosis.

22. The use according to claim 21, wherein the symptom is cirrhosis.

23. The use according to claim 21, wherein the disease, disorder, or symptom is NAFLD.

24. The use according to claim 21, wherein the disease, disorder, or symptom is NASH.

25. The use according to any one of claims 21 to 24, wherein the RNAi agent is administered at a dose of 0.05 mg / kg to 5.0 mg / kg of the body weight of a human subject.

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