RNAi drug for inhibiting PNPLA3 expression, its pharmaceutical composition, and method of use.

Abstract

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

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 000,137, filed on 26 March 2020, which is incorporated herein by reference in its entirety.

[0002] (Sequence Listing) This application includes a sequence listing, which is submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy was created on March 25, 2021, named 103693_002475_PCT_SL.txt, and has a size of 248,025 bytes.

[0003] (Field of Invention) This disclosure relates to a pharmaceutical composition containing an RNA interference (RNAi) agent for inhibiting Patatin-like phospholipase domain-containing protein 3 (PNPLA3), such as a double-stranded RNAi agent or a PNPLA3 RNAi agent, and to a method of using the same.

[0004] (background) Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, affecting an estimated 20% of the global population. In some individuals, ectopic fat accumulation in the liver, known as steatosis, leads to inflammation and liver cell damage, progressing to a more advanced stage of the disease called nonalcoholic steatohepatitis (NASH). Treatment for NAFLD often focuses on weight loss and addressing any secondary conditions, such as insulin resistance or dyslipidemia.

[0005] Patatin-like phospholipase domain-containing protein 3 (PNPLA3), a type II transmembrane protein, is expressed in various cells, including liver cells. In hepatocytes, PNPLA3 is expressed on the endoplasmic reticulum and lipid membranes, and primarily exhibits triacylglycerol hydrolase activity.

[0006] This invention provides a novel approach to treat liver diseases such as NAFLD by reducing PNPLA3 levels.

[0007] (overview) Disclosed herein is an RNAi agent for inhibiting the expression of the PNPLA3 gene, comprising an antisense strand containing at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any one of the sequences of SEQ ID NOs. 46-60, 176, 181, and 188, and a sense strand containing a nucleotide sequence at least partially complementary to the antisense strand.

[0008] In some embodiments, the antisense strand contains nucleotides 2-18 of any one of the sequences of SEQ ID NOs. 46-60, 176, 181, and 188.

[0009] In some embodiments, the sense strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any one of the sense strand sequences of SEQ ID NOs: 2, 3, 4, 9-20, 214, 219, and 220, and the sense strand has a region of at least 85% complementarity with the antisense strand over the 17 consecutive nucleotides.

[0010] In some embodiments, at least one nucleotide of the RNAi drug is a modified nucleotide or contains modified nucleoside linkages.

[0011] According to some embodiments, all or substantially all of the nucleotides in the sense strand and / or antisense strand of the RNAi drug are modified nucleotides.

[0012] In some embodiments, the modified nucleotide is selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, reversed nucleotide, reversed 2'-O-methylnucleotide, reversed 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methylnucleotide.

[0013] In other embodiments, all or substantially all of the modified nucleotides are 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof.

[0014] In some embodiments, the antisense strand consists of, essentially consists of, or includes one of the modified antisense strand sequences of SEQ ID NOs: 90, 95, and 102.

[0015] In some embodiments, the sense strand consists of, essentially consists of, or includes one of the modified sense strand sequences of SEQ ID NOs: 131, 136, and 137.

[0016] In some embodiments, the antisense strand comprises one nucleotide sequence from among the modified sequences of SEQ ID NOs: 90, 95, and 102, and the sense strand comprises one nucleotide sequence from among the modified sequences of SEQ ID NOs: 131, 136, and 137.

[0017] In other embodiments, the RNAi drug 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 further 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.

[0018] In some embodiments, the sense strand is 18 - 30 nucleotides in length and the antisense strand is 18 - 30 nucleotides in length. In other embodiments, the sense strand and the antisense strand for targeting are each 18 - 27 nucleotides in length. In other embodiments, the sense strand and the antisense strand for targeting are each 18 - 24 nucleotides in length. In still other embodiments, the sense strand and the antisense strand are each 21 nucleotides in length.

[0019] In some embodiments, the RNAi drug has two blunt ends.

[0020] In some embodiments, the sense strand comprises one or two terminal caps. In other embodiments, the sense strand comprises one or two inverted deoxy base residues.

[0021] In some embodiments, the RNAi drug consists of a sense strand and an antisense strand that form a double-stranded sequence having SEQ ID NOs: (176 and 214), (90 and 131), (181 and 219), (95 and 136), (188 and 220), and / or (102 and 137).

[0022] In some embodiments, the sense strand further comprises inverted deoxy base residues at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or both.

[0023] In some embodiments, the RNAi drugs provided herein include an antisense strand comprising, consisting of, or essentially consisting of, a modified nucleotide sequence in which one of the nucleotide sequences of SEQ ID NOs. 90, 95, and 102 differs by 0 or 1 nucleotide, where 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 linkage; and all or substantially all of the nucleotides in the sense strand are modified nucleotides.

[0024] In some embodiments, the sense strand of the present disclosure contains, consists of, or essentially consists of, a modified nucleotide sequence in which zero or one nucleotide from one of the nucleotide sequences of SEQ ID NOs: 214, 219, and 256 differs, where 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 linkage; and all or substantially all of the nucleotides of the antisense strand are modified nucleotides. In other embodiments, the sense strand further includes a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both. In other embodiments, the sense strand of the RNAi drug is linked to a targeted ligand.

[0025] In some embodiments, the targeted ligand has affinity for the asialoclycoprotein receptor. In some embodiments, the targeted ligand includes N-acetyl-galactosamine.

[0026] In further embodiments, the targeted ligand is

[0027] [ka] Includes.

[0028] In further embodiments, the antisense strand consists of the modified nucleotide sequences of SEQ ID NOs: 90, 95, and 102, and the sense strand consists of the modified nucleotide sequences of SEQ ID NOs: 131, 136, and 137, 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 linkage; (invAb) is a reverse debasing deoxyribose residue; and (NAG37)s has the following chemical structure.

[0029] [ka]

[0030] Also disclosed are compositions comprising the RNAi drug of the present disclosure, further comprising pharmaceutically acceptable excipients.

[0031] A method for inhibiting the expression of the PNPLA3 gene in cells is also provided herein, comprising introducing an effective amount of the RNAi drug or composition of the disclosure into the cells.

[0032] In some embodiments, the cells are located within the subject. In some embodiments, the subject is a human subject.

[0033] 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.

[0034] A method for treating PNPLA3-related disease or disorder is further provided herein, comprising administering a therapeutically effective amount of the composition of this disclosure to a human subject in need thereof.

[0035] In some embodiments, the disease is NAFLD, NASH, hepatic fibrosis, alcoholic fatty liver disease, or cirrhosis.

[0036] In some embodiments, RNAi drugs are administered in doses of approximately 0.05 mg to 5.0 mg per kg of body weight in human subjects.

[0037] In other embodiments, the RNAi drug is administered in two or three doses.

[0038] The use of the RNAi agents or compositions of the disclosure for the treatment of diseases, disorders, or conditions mediated at least partially by PNPLA3 gene expression is also provided herein.

[0039] In some embodiments, the symptom is cirrhosis of the liver.

[0040] The use of the RNAi agents or compositions of the disclosure for the preparation of pharmaceutical compositions for treating diseases, disorders, or symptoms that are at least partially mediated by PNPLA3 gene expression is further provided herein.

[0041] In some embodiments, the disease is alcoholic or alcoholic liver disease such as NAFLD, NASH, hepatic fibrosis, or cirrhosis. In some embodiments, the RNAi drug is administered in a dose of approximately 0.05 mg to approximately 5.0 mg per kg of body weight in human subjects. [Brief explanation of the drawing]

[0042] [Figure 1] This graph shows the baseline relative whole-liver expression of PNPLA3 by PCR from non-human primates (NHPs) treated with RNAi drugs. Study #1: Data are geometric mean ± SD (n=4). [Figure 2]This graph shows the mean PNPLA3 mRNA knockdown in the cytoplasm of hepatocytes by quantitative ISH in all tested animals (Study #1 and Study #2). Study #2: Data are mean ± SD (n=14).

[0043] (Detailed description of the embodiment) The methods of this disclosure can be more readily understood by referring to the following detailed description made in relation to the accompanying drawings, which form part of this disclosure. It should be understood that the methods of this disclosure are not limited to any particular methods described and / or shown herein, and furthermore, that the terms used herein are intended solely to illustrate particular embodiments by example and are not intended to limit them to the methods described in the claims.

[0044] In this specification, for the sake of clarity, several features of the methods of the disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the methods of the disclosure described as a single embodiment for the sake of brevity may also be provided separately or in any partial combination.

[0045] definition As used herein, “RNAi drug” (also referred to as “RNAi trigger”) means a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting the translation of mRNA transcripts of a target messenger RNA (mRNA) in a sequence-specific manner. As used herein, RNAi drugs may act via an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex, RISC) in mammalian cells) or by any alternative mechanism or pathway. While the term RNAi drug as used herein is primarily understood to act via an RNA interference mechanism, the RNAi drugs of this disclosure are not bound by or limited to any particular pathway or mechanism of action. The RNAi drugs 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 drugs described herein is at least partially complementary to the targeted mRNA (i.e., PNPLA3 mRNA). The RNAi drugs may contain one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0046] As used herein, the terms “silence,” “reduction,” “inhibition,” “downregulate,” or “knockdown,” when relating to the expression of a given gene, mean that the expression of the gene is reduced when the cell, cell population, tissue, organ, or subject is treated with an oligomeric compound such as an RNAi drug described herein, compared to a second cell, cell population, tissue, organ, or subject that is not treated in the same way, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in the cell, cell population, tissue, organ, or subject from which the gene is transcribed.

[0047] As used herein, the terms “sequence” or “nucleotide sequence” mean a sequence or order of nucleic acid bases or nucleotides, and are written as a sequence of letters using standard nomenclature. Nucleic acid molecules may contain unmodified and / or modified nucleotides. Nucleic acid sequences may contain unmodified and / or modified nucleotides.

[0048] As used herein, “base,” “nucleotide base,” “nucleotide,” or “nucleic acid base” refers to heterocyclic pyrimidine or purine compounds that are components of polynucleotides, including primary purine bases adenine and guanine, and primary pyrimidine bases cytosine, thymine, and uracil. Nucleotides may be unmodified. Nucleic acid bases may be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, for example, 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.

[0049] As used herein, and unless otherwise specified, the term “complementary” means that an oligonucleotide or polynucleotide containing the first nucleotide sequence (e.g., the sense strand of an RNAi drug or the mRNA to be targeted) can hybridize (form base-pair hydrogen bonds under mammalian physiological conditions (or otherwise preferred in vivo or in vitro conditions)) with the oligonucleotide or polynucleotide containing the second nucleotide sequence under specific standard conditions to form a double-stranded or double-helical structure. A person skilled in the art will be able to select the set of conditions best suited for hybridization testing. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and also include native nucleotides, modified nucleotides, or nucleotide mimetic compounds, insofar as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modification. For example, a and Af as defined herein are complementary to U (or T) and are identical to A for the purpose of determining identity or complementarity.

[0050] As used herein, “perfectly complementary” or “completely complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, all (100%) of the bases in the sequence of the first oligonucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may consist of all or part of the first or second nucleotide sequence.

[0051] As used herein, “partially complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, at least 70% but not all of the bases in the sequence of the first polynucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may comprise all or part of the first or second nucleotide sequence.

[0052] As used herein, “substantially complementary” means that in a pair of hybridized nucleic acid bases or nucleotide sequence molecules, at least about 85% of, but not all, of the bases in the sequence of the first polynucleotide hybridize to the same number of bases in the sequence of the second polynucleotide. The sequence may comprise all or part of the first or second nucleotide sequence.

[0053] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used in reference to the matching of nucleic acid bases or nucleotides between the sense strand and antisense strand of an RNAi drug, or between the antisense strand of an RNAi drug and the sequence of PNPLA3 mRNA.

[0054] As used herein, the terms “substantially identical” or “substantially identical,” when applied to nucleic acid sequences, mean that a nucleic acid sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity, preferably at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window. This percentage is calculated by determining the number of positions in both sequences where the same type of nucleic acid base occurs, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the window, and multiplying the result by 100 to obtain the percentage of sequence identity (%). The inventions disclosed herein encompass nucleotide sequences that are substantially identical to those disclosed herein.

[0055] As used herein, the terms “individual,” “patient,” and “subject” are used interchangeably to refer to birds, humans, and other primates, as well as members of any animal species, including but not limited to commercially relevant mammals or other mammals, including animal models such as mice, rats, monkeys, cattle, pigs, horses, sheep, cattle, and dogs. Preferably, the subject is human.

[0056] As used herein, the terms “to treat,” “treatment,” etc., mean a method or step taken to provide relief or mitigation of the number, severity, and / or frequency of one or more symptoms of a disease or condition in a subject. As used herein, “to treat” and “treatment” may also include prevention, control, 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.

[0057] As used herein, the phrase “introduced into cells” when referring to RNAi drugs means functionally delivering the RNAi drug into cells. The phrase “functionally delivered” means delivering the RNAi drug to cells in a manner that enables the RNAi drug to have its expected biological activity, such as sequence-specific inhibition of gene expression.

[0058] Unless otherwise specified, symbols used herein

[0059] [ka] The use of means any one of the bases which may be linked in accordance with the scope of the invention as described herein.

[0060] As used herein, the term "isomer" means a compound having the same molecular formula but differing in the nature or arrangement of the bonds between 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 "diastereomers," and stereoisomers that are mirror images that cannot be superimposed are called "enantiomers" or sometimes "optical isomers." A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0061] When used herein, unless otherwise specified in the structure as having a particular conformation, for each structure in which an asymmetric center exists, resulting in an enantiomer, diastereomer, or other stereoisomer configuration, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure form and racemates. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers, as well as individual stereoisomers.

[0062] As used herein, the terms “comprising,” “including,” “containing,” and “characterized by” are interchangeable, comprehensive, open, and do not exclude additional undescribed elements or method steps. Any use of the term “including” herein is understood to encompass compositions and methods that are essentially made up of and comprised of the described elements or components, particularly in descriptions of components of a composition or elements of a device.

[0063] When used in the claims of this application, the phrase "consists of" excludes any elements, steps, or components not specified in the claims. When used in the claims of this application, the phrase "essentially consists of" limits the scope of the claims to the specified materials or steps, and any that do not substantially affect the essential and novel features of the claimed patent.

[0064] It will be readily understood and appreciated by those skilled in the art that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Therefore, when used herein, the structures disclosed herein are assumed to have certain functional groups (e.g., OH, SH, or NH) that can be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of the protonation state based on the environment (e.g., pH), as will be readily understood by those skilled in the art. Accordingly, compounds described herein having an unstable proton or basic atom should be understood as representing a salt form of the corresponding compound. The compounds described herein may be free acids, free bases, or salt forms. pharmaceutically acceptable salts of the compounds described herein should be understood to be within the scope of the invention.

[0065] When used herein, the terms “linked” or “conjugated” mean that the two compounds or molecules are joined by a covalent bond. Unless otherwise stated herein, the terms “linked” and “conjugated” may also mean a connection between a first compound and a second compound, with or without any intervening atoms or atomic groups.

[0066] As used herein, the term “including” means “including, but not limited to” and is used interchangeably. Unless otherwise explicitly stated by the context, the term “or” means “and / or” and is used interchangeably.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly interpreted by those skilled in the art. Methods and materials similar to or equivalent to those described herein may be used to carry out or test the present invention, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In case of any inconsistency, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0068] Other objects, features, embodiments, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims.

[0069] (Detailed explanation) RNAi drugs RNAi drugs for inhibiting the expression of the PNPLA3 gene (hereinafter referred to as PNPLA3 RNAi drugs or PNPLA3 RNAi triggers) are described herein. Each PNPLA3 RNAi drug comprises a sense strand and an antisense strand. The sense strand and antisense strand may each be 16 to 49 nucleotides long. The sense strand and antisense strand may be the same length or of different lengths. In some embodiments, the sense strand and antisense strand are each independently 18 to 27 nucleotides long. In some embodiments, both the sense strand and antisense strand are each 21 to 26 nucleotides long. In some embodiments, the sense strand and antisense strand are each 21 to 24 nucleotides long. In some embodiments, the sense strand and antisense strand are each independently 19 to 21 nucleotides long. In some embodiments, the sense strand is about 19 nucleotides long and the antisense strand is about 21 nucleotides long. In some embodiments, the sense strand is about 21 nucleotides long and the antisense strand is about 23 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long and the antisense strand is 21 nucleotides long. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides long, respectively. In some embodiments, the sense strand and antisense strand of the RNAi drug are 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 long, respectively. In some embodiments, the double-stranded RNAi drug has a double-strand length of approximately 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0070] Examples of nucleotide sequences used to form PNPLA3 RNAi drugs are provided in Tables 2, 3, and 4. Examples of RNAi drug double helices containing the sense and antisense strand sequences from Tables 2, 3, and 4 are shown in Tables 5A and 5B.

[0071] In some embodiments, the region of complete, substantial, or partial complementarity between the sense strand and the antisense strand is 16–26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides long and is located at or near the 5' end of the antisense strand (e.g., this region may be separated from the 5' end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not completely, substantial, or partially complementary).

[0072] The sense strand of the PNPLA3 RNAi drug described herein contains at least 16 consecutive nucleotides having at least 85% identity with the core stretch sequence (also referred herein as the “core stretch” or “core sequence”) of the same number of nucleotides in the PNPLA3 mRNA. In some embodiments, the sense strand core stretch sequence is 100% (completely) complementary to the antisense strand core stretch sequence, or at least about 85% (substantially) complementary, and therefore, the sense strand core stretch sequence is typically completely identical, or at least about 85% identical, to a nucleotide sequence of the same length present in the PNPLA3 mRNA target (which may also be referred to as the target sequence). In some embodiments, this sense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, this sense strand core stretch is 17 nucleotides long. In some embodiments, this sense strand core stretch is 19 nucleotides long.

[0073] The antisense strand of the PNPLA3 RNAi drug described herein comprises a core stretch of the same number of nucleotides in the PNPLA3 mRNA and the same number of nucleotides in the corresponding sense strand, and at least 16 consecutive nucleotides having at least 85% complementarity. In some embodiments, the antisense strand core stretch is 100% (completely) complementary or at least about 85% (substantially) complementary to a nucleotide sequence of the same length present in the PNPLA3 mRNA target (e.g., the target sequence). In some embodiments, this antisense strand core stretch is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, this antisense strand core stretch is 19 nucleotides long. In some embodiments, this antisense strand core stretch is 17 nucleotides long. The sense strand core stretch sequence may be the same length as the corresponding antisense core sequence, or it may be of a different length.

[0074] The sense and antisense strands of a PNPLA3 RNAi drug anneal to form a double helix. The sense and antisense strands of a PNPLA3 RNAi drug may be partially, substantially, or completely complementary to each other. Within the complementary double helix region, the sense strand core stretch sequence is at least about 85% or 100% complementary to the antisensor core stretch sequence. In some embodiments, the sense strand core stretch sequence includes 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 are at least approximately 85% or 100% complementary to the corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequences of the antisense strand core stretch sequence (i.e., the sense and antisense core stretch sequences of a PNPLA3 RNAi drug have regions 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 are at least 85% or 100% base-paired).

[0075] In some embodiments, the antisense strand of the PNPLA3 RNAi drug disclosed herein differs from either the antisense strand sequence in Table 2 or Table 3 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of the PNPLA3 RNAi drug disclosed herein differs from either the sense strand sequence in Table 2 or Table 4 by 0, 1, 2, or 3 nucleotides.

[0076] In some embodiments, the sense strand 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' and 5' ends 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 or may not be identical to the corresponding sequence in the PNPLA3 mRNA. The additional nucleotides of the antisense strand, if present, may or may not be complementary to the corresponding additional nucleotides of the sense strand (if present).

[0077] When used herein, the extensions contain 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' ends of the sense strand core stretch sequence and / or antisense strand core stretch sequence. The sense strand extension nucleotides may or may not be complementary to the corresponding antisense strand nucleotides (either core stretch sequence nucleotides or extension nucleotides). Conversely, the antisense strand extension nucleotides may or may not be complementary to the corresponding sense strand nucleotides (either core stretch sequence nucleotides or extension nucleotides). In some embodiments, both the sense and antisense strands of the RNAi drug contain 3' and 5' extensions. In some embodiments, one or more 3' extension nucleotides on one strand are base-paired with one or more 5' extension nucleotides on the other strand. In other embodiments, one or more 3' extension nucleotides on one strand are not base-paired with one or more 5' extension nucleotides on the other strand. In some embodiments, the PNPLA3 RNAi drug has an antisense strand having a 3' extension and a sense strand having a 5' extension. In some embodiments, the extension nucleotides are unpaired and form an overhang. As used herein, “overhang” refers to a stretch of one or more unpaired nucleotides located at the end of either the sense strand or the antisense strand that does not form part of the hybridized or double-stranded portion of the RNAi drug disclosed herein.

[0078] In some embodiments, the PNPLA3 RNAi drug comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, the PNPLA3 RNAi drug comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more nucleotides in the antisense strand extension contain nucleotides complementary to the corresponding PNPLA3 mRNA sequence. In some embodiments, one or more nucleotides in the antisense strand extension contain nucleotides that are not complementary to the corresponding PNPLA3 mRNA sequence.

[0079] In some embodiments, the PNPLA3 RNAi drug comprises a sense strand having a 3' extension of 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 thymidine nucleotide, AT dinucleotide, or a nucleotide corresponding to or identical to a nucleotide 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 (each listed from 5' to 3').

[0080] The sense strand may have a 3' extension and / or a 5' extension. In some embodiments, the PNPLA3 RNAi drug includes a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more nucleotides in the sense strand extension include nucleotides that correspond to or are identical to nucleotides in the PNPLA3 mRNA sequence.

[0081] Examples of nucleotide sequences used to form PNPLA3 RNAi drugs are provided in Tables 2, 3, and 4. In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains a nucleotide sequence from either Table 2 or Table 3. In certain embodiments, the PNPLA3 RNAi drug antisense strand contains or consists of one of the modified sequences from Table 3. In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains the nucleotide (5' end → 3' end) sequences 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 from either Table 2 or Table 3. In some embodiments, the sense strand of the PNPLA3 RNAi drug contains a sequence from either Table 2 or Table 4. In some embodiments, the PNPLA3 RNAi drug sense strand contains the sequence of nucleotides (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 from any of the sequences in Table 2 or Table 4. In certain embodiments, the PNPLA3 RNAi drug sense strand contains or consists of any one of the modified sequences in Table 4.

[0082] In some embodiments, the sense and antisense strands of the RNAi drug described herein have the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi drug described herein have 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 drug form a blunt end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi drug form a blunt end. In some embodiments, both ends of the RNAi drug form a blunt end. In some embodiments, neither end of the RNAi drug is a blunt end. As used herein, “blunt end” refers to the end of a double-stranded RNAi gene where the terminal nucleotides of the two annealed strands are complementary (forming complementary base pairs).

[0083] In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi drug form a frayed end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi drug form a frayed end. In some embodiments, both ends of the RNAi drug form a frayed end. In some embodiments, neither end of the RNAi drug is a frayed end. As used herein, a frayed end refers to the end of a double-stranded RNAi drug where 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 drug form an overhang. The unpaired nucleotides may be on the sense strand or the antisense strand and form either a 3' or 5' overhang. In some embodiments, RNAi drugs include a blunt end and a frayed end, a blunt end and a 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends. Typically, if present, the overhangs are located at the 3' end of the sense strand, the antisense strand, or both the sense and antisense strands.

[0084] The PNPLA3 RNAi drugs disclosed herein may also consist of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand of the PNPLA3 RNAi drug are modified nucleotides. The PNPLA3 RNAi drugs disclosed herein may further consist of one or more modified nucleoside linkages, for example, one or more phosphorothioate linkages. In some embodiments, the PNPLA3 RNAi drug comprises one or more modified nucleotides and one or more modified nucleoside linkages. In some embodiments, 2'-modified nucleotides are combined with modified nucleoside linkages.

[0085] In some embodiments, the PNPLA3 RNAi drug is prepared or provided as a salt, a mixed salt, or a free acid. In some embodiments, the PNPLA3 RNAi drug is prepared as a sodium salt. Such forms well known in the art are within the scope of the present invention as disclosed herein.

[0086] Modified nucleotides Modified nucleotides, when used in various oligonucleotide constructs, can maintain the activity of the compounds in cells, while simultaneously enhancing the serum stability of these compounds, and can also minimize the potential for activating interferon activity in humans when the oligonucleotide constructs are administered.

[0087] In some embodiments, the PNPLA3 RNAi drug comprises one or more modified nucleotides. As used herein, “modified nucleotide” is a nucleotide other than a ribonucleotide (2'-hydroxylnucleotide). 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. When used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimes, debasalized nucleotides, 2'-modified nucleotides, reversed nucleotides, modified nucleic acid base-containing nucleotides, cross-linked nucleotides, peptide nucleic acids (PNA), 2',3'-seconucleotide mimes (locked nucleic acid base analogs), locked nucleotides, 3'-O-methoxy(2'-nucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholinonucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. Examples of 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a five-membered sugar ring) include, but are not limited to, 2'-O-methylnucleotides, 2'-fluoronucleotides (also referred herein as 2'-deoxy-2'-fluoronucleotides), 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl)) nucleotides (also referred to as 2'-MOEs), 2'-aminonucleotides, and 2'-alkylnucleotides. It is not necessary to uniformly modify all positions in a given compound. Conversely, two or more modifications can be incorporated into a single PNPLA3 RNAi drug, or even into that single nucleotide. The sense and antisense strands of a PNPLA3 RNAi drug can be synthesized and / or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.

[0088] Modified nucleic acid bases 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, adenine, and 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) derivatives of guanine, and other alkyl derivatives, 2-thiouracil Examples include syl, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azouracil, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl, 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.

[0089] In some embodiments, the 5' and / or 3' ends of the antisense strand may contain a debasic residue (Ab), which may also be referred to as a “debasic site” or “debasic nucleotide.” A debasic residue (Ab) is a nucleotide or nucleoside lacking a nucleic acid base at the 1' position of the sugar moiety. (See, for example, U.S. Patent No. 5,998,203). In some embodiments, the debasic residue may be located internally in the nucleotide sequence. In some embodiments, Ab or AbAb may be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand may contain one or more additional debasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab may be added to the 3' end of the sense strand. In some embodiments, a debasic (deoxyribose) residue may be replaced with a ribitol (debasic ribose) residue.

[0090] In some embodiments, all or substantially all of the nucleotides of an RNAi drug are modified nucleotides. As used herein, an RNAi drug in which substantially all of the nucleotides present are modified nucleotides is an RNAi drug having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand, which are ribonucleotides (i.e., unmodified). 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) modified nucleotides in the sense strand, which is a ribonucleotide. 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) modified nucleotides in the sense strand, which is a ribonucleotide. In some embodiments, one or more nucleotides of an RNAi drug are unmodified ribonucleotides.

[0091] Inter-modified nucleoside linking In some embodiments, one or more nucleotides of the PNPLA3 RNAi drug are linked by non-standard linkages or skeletons (i.e., modified nucleoside linkages or modified skeletons). Modified nucleoside linkages or skeletons include 5'-phosphorothioate groups (represented herein by lowercase "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylenephosphonate), chiral phosphonates, phosphinates, and phosphoramidates (e.g., 3'-aminophosphoramidates). Examples include, but are not limited to, aminoalkylphosphoromidates or thionophosphoromidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having the usual 3'-5' linkages, 2'-5' linked analogues of boranophosphates, or boranophosphates having opposite polarity in which adjacent nucleoside pair units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkages or skeletons do not contain phosphorus atoms. Examples of modified internucleoside linkages that do not contain phosphorus atoms include, but are not limited to, short-chain alkyl or cycloalkyl sugar linkages, mixed heteroatom and alkyl or cycloalkyl sugar linkages, or one or more short-chain heteroatom or heterocyclic sugar linkages. In some embodiments, the modified internucleoside skeletons include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 components.

[0092] In some embodiments, the sense strand of a PNPLA3 RNAi drug may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of a PNPLA3 RNAi drug may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand may independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of a PNPLA3 RNAi drug may contain 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of a PNPLA3 RNAi drug may contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand may independently contain 1, 2, 3, or 4 phosphorothioate linkages.

[0093] In some embodiments, the sense strand of the PNPLA3 RNAi drug contains at least two phosphorothioate nucleoside linkages. In some embodiments, the phosphorothioate nucleoside linkages are located between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate nucleoside linkage is located at the 5' end of the sense strand nucleotide sequence, and another phosphorothioate linkage is located at the 3' end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate nucleoside linkages are located at the 5' end of the sense strand, and another phosphorothioate linkage is located at the 3' end of the sense strand. In some embodiments, the sense strand does not contain any phosphorothioate nucleoside linkages between nucleotides, but contains one, two, or three phosphorothioate linkages between both the 5' and 3' terminal nucleotides, and optionally includes a reverse debase residue terminal cap. In some embodiments, the targeted ligand is linked to the sense strand via phosphorothioate linkages.

[0094] In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains four phosphorothioate nucleoside linkages. In some embodiments, the four phosphorothioate nucleoside linkages are located between nucleotides 1-3 from the 5' end of the antisense strand, and between nucleotides 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, three phosphorothioate nucleoside linkages are located between 1-4 from the 5' end of the antisense strand, and a fourth phosphorothioate nucleoside linkage is located between 20-21 from the 5' end of the antisense strand. In some embodiments, the PNPLA3 RNAi drug contains at least three or four phosphorothioate nucleoside linkages in the antisense strand.

[0095] Capping residue or portion In some embodiments, the sense strand may include one or more capping residues or portions, which may also be referred to in the art as “caps,” “terminal caps,” or “capping residues.” As used herein, “capping residues” are non-nucleotide compounds or other portions that can be incorporated into one or more ends of the nucleotide sequence of an RNAi drug disclosed herein. Capping residues can provide an RNAi drug, and in some cases, can provide an RNAi drug having certain beneficial properties, such as protection against exonuclease degradation. In some embodiments, inverted abasic residues (invAb) (also referred to in the art as “inverted base sites”) are added as capping residues. (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 and terminal C3H7 (propyl), C6H 13 (Hexyl), or C 12 H 25The capping residue includes a carbon chain such as a (dodecyl) group. In some embodiments, the capping residue is located at either the 5' end, the 3' end, or both the 5' and 3' ends of the sense chain. In some embodiments, the 5' and / or 3' ends of the sense chain may contain two or more reverse debasing deoxyribose moieties as capping residues.

[0096] In some embodiments, one or more reverse debase residues (invAbs) are added to the 3' end of the sense strand. In some embodiments, one or more reverse debase residues (invAbs) are added to the 5' end of the sense strand. In some embodiments, one or more reverse debase nucleosides may be inserted between the targeting ligand and the nucleic acid base sequence of the sense strand of the RNAi drug. In some embodiments, including one or more reverse debase residues or reverse debase sites at or near the end or both ends of the sense strand of the RNAi drug can enhance the activity of the RNAi drug or enable other desired properties.

[0097] In some embodiments, one or more reverse debasing residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more reverse debasing nucleosides may be inserted between the targeting ligand and the nucleic acid base sequence of the sense strand of the RNAi drug. The reverse debasing residues may be linked via a phosphate, a phosphorothioate (e.g., indicated herein as (invAb)s), or other internucleoside linkages. In some embodiments, including one or more reverse debasing residues at the end or both ends of the sense strand of the RNAi drug, or near the end or both ends, may enable enhanced activity or other desired properties of the RNAi drug. In some embodiments, a reverse debasing (deoxyribose) residue may be replaced with a reverse ribitol (debasing ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence, or the 3' end of the antisense strand sequence, may contain a reverse debasing residue. The chemical structure of the reverse debasic deoxyribose residue is shown in Table 6 below.

[0098] PNPLA3 RNAi drug The PNPLA3 RNAi drugs disclosed herein are designed to target specific locations of the PNPLA3 gene (e.g., SEQ ID NO: 1).

[0099] NM_025225.2 Homo sapiens spatatin-like phospholipase domain 3 (PNPLA3), mRNA (SEQ ID NO: 1):

[0100] As defined herein, an antisense strand sequence is designed to target the PNPLA3 gene at a given position on the gene when the 5' terminal nucleic acid base of the antisense strand aligns with a position 19 nucleotides downstream (towards the 3' end) from the position on the gene during base pairing with the gene. For example, as shown in Tables 1 and 2 herein, an antisense strand sequence designed to target the PNPLA3 gene at position 2180 requires that the 5' terminal nucleic acid base of the antisense strand align with position 2198 on the PNPLA3 gene during base pairing with the gene.

[0101] As provided herein, if there is at least 85% complementarity between the antisense strand and the gene across a core stretch 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), then the PNPLA3 RNAi drug does not require that the nucleic acid base at position 1 (5'→3') of the antisense strand be complementary to the gene. For example, in the case of a PNPLA3 RNAi drug disclosed herein designed to target position 2180 of the PNPLA3 gene, the 5' terminal nucleic acid base of the antisense strand of the PNPLA3 RNAi drug needs to be aligned with position 2198 of the gene; however, the 5' terminal nucleic acid base of the antisense strand may be complementary to position 2200 of the PNPLA3 gene, but this is not necessary if there is at least 85% complementarity between the antisense strand and the gene across a core stretch 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). In particular, as demonstrated in the various examples disclosed herein, the specific binding site of the antisense strand of the PNPLA3 RNAi drug to the gene (e.g., whether the PNPLA3 RNAi drug is designed to target the PNPLA3 gene at position 2180, 1586, 1179, or some other position) is important to the level of inhibition achieved by the PNPLA3 RNAi drug.

[0102] In some embodiments, the PNPLA3 RNAi drugs disclosed herein target the PNPLA3 gene at or near the PNPLA3 location shown in Table 1. In some embodiments, the antisense strand of the PNPLA3 RNAi drug disclosed herein includes a core stretch sequence that is fully, substantially, or at least partially complementary to the target PNPLA3 19mer sequence disclosed in Table 1.

[0103] [Table 1] 1. When referring to gene locations in this specification, 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. The updated gene reference changes the number of “target gene locations” identified in Table 1 above, but this does not affect the nucleotide sequences used in the RNAi drugs disclosed herein.

[0104] In some embodiments, the PNPLA3 RNAi drug includes an antisense strand (5'→3') whose 19th position can form a base pair with position 1 of the 19mer target sequence disclosed in Table 1.

[0105] In some embodiments, the PNPLA3 RNAi drug includes an antisense strand (5'→3') whose 2 position can base-pair with position 18 of the 19mer target sequence disclosed in Table 1. In some embodiments, the PNPLA3 RNAi drug includes an antisense strand (5'→3') whose 2-18 positions can base-pair with each of the complementary bases located at positions 18-2 of the 19mer target sequence disclosed in Table 1.

[0106] In the PNPLA3 RNAi drugs disclosed herein, the nucleotide at position 1 of the antisense strand (5' end → 3' end) may be either perfectly complementary to the PNPLA3 gene or non-complementary to the PNPLA3 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) is U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) forms an A:U or U:A base pair with the sense strand.

[0107] In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains the sequence of nucleotides (5' end to 3' end) 2-18, 2-19, 2-20, or 2-21 from any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the PNPLA3 RNAi sense strand contains the sequence of nucleotides (5' end to 3' end) 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 any of the sense strand sequences in Table 2 or Table 4.

[0108] In some embodiments, the PNPLA3 RNAi drug antisense strand includes a sequence of nucleotides (5' end to 3' end) 2-18, 2-19, 2-20, or 2-21 from any of the antisense strand sequences of SEQ ID NOs. 46-60, 176, 181, and 188. In some embodiments, the PNPLA3 RNAi sense strand includes a sequence of nucleotides (5' end to 3' end) 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 any of the sense strand sequences of SEQ ID NOs. 2, 3, 4, 9-20, 214, 219, and 220.

[0109] In some embodiments, the PNPLA3 RNAi drug comprises (i) an antisense strand containing the sequence of nucleotides (5' end to 3' end) 2-18 or 2-19 from any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand containing the sequence of nucleotides (5' end to 3' end) 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 any of the sense strand sequences in Table 2 or Table 4.

[0110] In some embodiments, the PNPLA3 RNAi drug comprises (i) an antisense strand containing a sequence of nucleotides (5' end to 3' end) 2-18 or 2-19 from any of the antisense strand sequences of SEQ ID NOs. 46-60, 176, 181, and 188, and (ii) a sense strand containing a sequence of nucleotides (5' end to 3' end) 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 any of the sense strand sequences of SEQ ID NOs. 2, 3, 4, 9-20, 214, 219, and 220.

[0111] In some embodiments, the PNPLA3 RNAi drug contains the core 19mer nucleotide sequence shown in Table 2 below.

[0112] [Table 2-1]

[0113] [Table 2-2]

[0114] The sense and antisense strands of a PNPLA3 RNAi drug containing or comprising the sequences in Table 2 may be modified or unmodified nucleotides. In some embodiments, a PNPLA3 RNAi drug having sense and antisense strand sequences containing or comprising the sequences in Table 2 consists of all or substantially all modified nucleotides.

[0115] In some embodiments, the antisense strand of the PNPLA3 RNAi drug disclosed herein differs from any 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 drug disclosed herein differs from any 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 drug disclosed herein differs from any 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 drug disclosed herein differs from any 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.

[0116] When used herein, each N listed in the sequences disclosed in Table 2 may be independently selected from any and all nucleic acid bases (including those found in both modified and unmodified nucleotides). In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have nucleic acid bases that are not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have the same nucleic acid bases as the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have different nucleic acid bases from the N nucleotide at the corresponding position on the other strand.

[0117] Table 3 provides specific modified PNPLA3 RNAi drug antisense strand sequences and their underlying unmodified sequences. Table 4 provides specific modified PNPLA3 RNAi drug sense strands and their underlying unmodified sequences. In the formation of PNPLA3 RNAi drugs, each nucleotide in each underlying unmodified sequence listed in Tables 3 and 4, as well as in Table 2 and above, may be a modified nucleotide.

[0118] The PNPLA3 RNAi drugs described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing the sequences listed in Table 2 or Table 4 can be hybridized with any antisense strand containing the sequences listed in Table 2 or Table 3, provided that it has a region of at least about 85% complementarity across 16, 17, 18, 19, 20, or 21 consecutive nucleotide sequences of the two sequences.

[0119] In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains a nucleotide sequence from either Table 2 or Table 3.

[0120] In some embodiments, the PNPLA3 RNAi drug comprises or consists of a double helix having the nucleic acid base sequences of the sense and antisense strands of any of the sequences in Table 2, Table 3, or Table 4. In some embodiments, the PNPLA3 RNAi drug comprises or consists of a double helix having SEQ ID NOs (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 drug double helix having SEQ ID NOs (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.

[0121] Examples of antisense strands containing modified nucleotides are provided in Table 3. Examples of sense strands containing modified nucleotides are provided in Table 4.

[0122] When used in Tables 3 and 4, the following nomenclature is used to indicate modified nucleotides and linking groups. A = adenosine-3'-phosphate C = cytidine-3'-phosphate G = Guanosine-3'-phosphate U = uridine-3'-phosphate I = Inosine-3'-phosphate a=2'-O-methyladenosine-3'-phosphate as=2'-O-methyladenosine-3'-phosphorothioate c=2'-O-methylcytidine-3'-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate t=2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us=2'-O-methyluridine-3'-phosphorothioate i=2'-O-methylinosine-3'-phosphate is=2'-O-methylinosine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate Tf = 2'-fluoro-5'-methyluridine-3'-phosphate Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate A UNA = 2',3'-seco-adenosine-3'-phosphate, see Table 6. A UNA s = 2',3'-seco-adenosine-3'-phosphorothioate, see Table 6. C UNA = 2',3'-seco-cytidine-3'-phosphate, see Table 6. C UNA s = 2',3'-seco-cytidine-3'-phosphorothioate, see Table 6. G UNA = 2',3'-seco-guanosine-3'-phosphate, see Table 6. G UNA s = 2',3'-seco-guanosine-3'-phosphorothioate, see Table 6. U UNA = 2',3'-seco-uridine-3'-phosphate, see Table 6. U UNA s = 2',3'-seco-uridine-3'-phosphorothioate, see Table 6. a_2N = 2'-O-methyl-2-aminoadenosine-3'-phosphate, see Table 6. a_2Ns = 2'-O-methyl-2-aminoadenosine-3'-phosphorothioate, see Table 6. (invAb) = reverse abasic deoxyribonucleotide, see Table 6. (invAb)s = reverse abasic deoxyribonucleotide-5'-phosphorothioate, see Table 6.

[0123] As will be readily apparent to those skilled in the art, unless otherwise indicated by the sequence (e.g., by a phosphorothioate linkage "s"), nucleotide monomers in oligonucleotides are linked to each other by a 5'-3'-phosphodiester bond. As will be clearly apparent to those skilled in the art, the inclusion of phosphorothioate linkages shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester bond typically present in oligonucleotides. Furthermore, those skilled in the art will readily understand that the terminal nucleotide at the 3' end of a given oligonucleotide sequence typically has a hydroxyl (-OH) group at the 3' position of each given monomer in ex vivo, instead of a phosphate moiety. Furthermore, in the embodiments disclosed herein, when viewing each chain from 5' to 3', the reverse debasing residue is inserted such that the 3' position of the deoxyribose is linked to the 3' end of the preceding monomer on each chain (see, for example, Table 6). Furthermore, as will be readily understood and recognized by those skilled in the art, the phosphorothioate chemical structures shown herein typically represent an anion on a sulfur atom, and the present invention disclosed herein encompasses all phosphorothioate tautomers (e.g., those in which the sulfur atom has a double bond and the anion is on an oxygen atom). Unless otherwise expressly stated herein, such understanding of those skilled in the art is used when describing the PNPLA3 RNAi drugs and compositions of PNPLA3 RNAi drugs disclosed herein.

[0124] Specific examples of targeted ligands, targeting groups, and linking groups used with the PNPLA3 RNAi drugs disclosed herein are provided in Table 6 below. More specifically, the targeting groups and linking groups (which together can form a targeted ligand) include (NAG37) and (NAG37)s, whose chemical structures are provided in Table 6 below. Each sense strand and / or antisense strand may have any of the targeted ligands, targeting groups, or linking groups listed herein, as well as other groups, conjugated at the 5' and / or 3' ends of the sequence.

[0125] [Table 3-1]

[0126] [Table 3-2]

[0127] [Table 4-1]

[0128] [Table 4-2] (A 2N ) = 2-aminoadenine nucleotide, I = hypoxanthine (inosine) nucleotide

[0129] The PNPLA3 RNAi drugs described herein are formed by annealing an antisense strand and a sense strand. A sense strand containing the sequences listed in Table 2 or Table 4 can be hybridized to any antisense strand containing the sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least about 85% complementarity across 16, 17, 18, 19, 20, or 21 consecutive nucleotide sequences.

[0130] In some embodiments, the antisense strand of the PNPLA3 RNAi drug 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 drug disclosed herein differs from any of the sense strand sequences in Table 4 by 0, 1, 2, or 3 nucleotides.

[0131] In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains a nucleotide sequence from either Table 2 or Table 3. In some embodiments, the PNPLA3 RNAi drug antisense strand contains a nucleotide sequence (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 from either Table 2 or Table 3. In some embodiments, the PNPLA3 RNAi drug antisense strand contains or consists of one of the modified sequences from Table 3.

[0132] In some embodiments, the sense strand of the PNPLA3 RNAi drug contains a nucleotide sequence from either Table 2 or Table 4. In some embodiments, the antisense strand of the PNPLA3 RNAi drug contains a nucleotide sequence (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 some embodiments, the sense strand of the PNPLA3 RNAi drug contains or consists of one of the modified sequences from Table 4.

[0133] In the PNPLA3 RNAi drugs disclosed herein, the nucleotide at position 1 of the antisense strand (5' end → 3' end) may be either perfectly complementary to the PNPLA3 gene or non-complementary to the PNPLA3 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) is U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (5' end → 3' end) forms an A:U or U:A base pair with the sense strand.

[0134] A sense strand containing sequences listed in Table 2 or Table 4 can be hybridized with any antisense strand containing sequences listed in Table 2 or Table 3, provided that the two sequences have a region of at least approximately 85% complementarity across 16, 17, 18, 19, 20, or 21 consecutive nucleotide sequences. In some embodiments, a PNPLA3 RNAi drug has a sense strand consisting of one of the modified sequences in Table 4 and an antisense strand consisting of one of the modified sequences in Table 3. Specific representative sequence pairs are exemplified by the double-stranded ID numbers shown in Tables 5A and 5B.

[0135] In some embodiments, the PNPLA3 RNAi drug comprises, consists of, or essentially consists of a double helix represented by any one of the double helix ID numbers presented herein. In some embodiments, the PNPLA3 RNAi drug comprises either the sense strand and antisense strand nucleotide sequence of the double helix represented by any one of the double helix ID numbers presented herein. In some embodiments, the PNPLA3 RNAi drug comprises either the sense strand and antisense strand nucleotide sequence of the double helix represented by any one of the double helix ID numbers presented herein, as well as a targeting group and / or linking group, the targeting group and / or linking group being covalently linked (i.e., conjugated) to the sense strand or antisense strand. In some embodiments, the PNPLA3 RNAi drug comprises either the sense strand and antisense strand modified nucleotide sequence of any one of the double helix ID numbers presented herein. In some embodiments, the PNPLA3 RNAi drug comprises a modified nucleotide sequence of a sense strand and / or antisense strand of any of the dual-stranded ID numbers presented herein, and a targeting group and / or linking group, the targeting group and / or linking group being covalently linked to the sense strand or antisense strand.

[0136] In some embodiments, the PNPLA3 RNAi drug comprises an antisense strand and a sense strand having a nucleotide sequence of either the antisense strand / sense strand duplex of Table 2 or Tables 5A and 5B, and further comprises a targeting group or a targeting ligand. In some embodiments, the PNPLA3 RNAi drug comprises an antisense strand and a sense strand having a nucleotide sequence of either the antisense strand / sense strand duplex of Table 2 or Tables 5A and 5B, and further comprises an asialoclycoprotein receptor ligand targeting group.

[0137] The targeting group may be attached to the 5' or 3' end of any sense chain and / or antisense chain disclosed in Tables 2, 3, and 4, with or without a linker. The linker may be attached to the 5' or 3' end of any sense chain and / or antisense chain disclosed in Tables 2, 3, and 4, with or without a targeting group.

[0138] In some embodiments, the PNPLA3 RNAi drug comprises an antisense strand and a sense strand having one of the nucleotide sequences of the antisense / sense strand duplexes of Table 2 or Tables 5A and 5B, and further comprising a targeted ligand selected from the group consisting of (NAG37) and (NAG37)s, as defined in Table 6.

[0139] In some embodiments, the PNPLA3 RNAi drug comprises an antisense strand and a sense strand having a modified nucleotide sequence in either the antisense strand and / or sense strand nucleotide sequences of Table 3 or Table 4.

[0140] In some embodiments, the PNPLA3 RNAi drug comprises an antisense strand and a sense strand having a modified nucleotide sequence in either of the antisense strands and / or sense strand nucleotide sequences of the double helix shown in Tables 5A and 5B, and further comprising an asialoglycoprotein receptor ligand targeting group.

[0141] In some embodiments, the PNPLA3 RNAi drug comprises, consists of, or essentially consists of, one of the double helixes shown in Tables 5A and 5B.

[0142] [Table 5]

[0143] [Table 6]

[0144] In some embodiments, the PNPLA3 RNAi drug is prepared or provided as a salt, a mixed salt, or a free acid. When delivered to cells expressing PNPLA3, the RNAi drugs described herein inhibit the expression of one or more PNPLA3 genes in vivo and / or in vitro.

[0145] Targeted ligand or targeting group, linking group, and delivery vehicle In some embodiments, the PNPLA3 RNAi drug is conjugated to one or more non-nucleotide groups, including but not limited to targeting groups, linking groups, targeting ligands, delivery polymers, or delivery vehicles. These non-nucleotide groups can enhance the targeting, delivery, or conjugation of the RNAi drug. Table 6 provides examples of targeting groups and linking groups. The non-nucleotide groups may be covalently linked to the 3' and / or 5' ends of either the sense strand and / or antisense strand. In some embodiments, the PNPLA3 RNAi drug includes non-nucleotide groups linked to the 3' and / or 5' ends of the sense strand. In some embodiments, the non-nucleotide groups are linked to the 5' end of the sense strand of the PNPLA3 RNAi drug. The non-nucleotide groups may be linked to the RNAi drug directly or indirectly via linker / linking groups. In some embodiments, the non-nucleotide groups are linked to the RNAi drug via unstable cleavageability, or via invertable linkers or linkers.

[0146] In some embodiments, non-nucleotide groups are attached to improve the pharmacokinetic or in vivo distribution characteristics of RNAi drugs or conjugates, which are intended to enhance cell-specific or tissue-specific distribution and cell-specific uptake of the RNAi drug or conjugate. In some embodiments, non-nucleotide groups improve the endocytosis of RNAi drugs.

[0147] Targeting groups or moieties are attached to improve the pharmacokinetic or in vivo distribution characteristics of a conjugate or RNAi drug, thereby enhancing the cell-specific (and in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugate or RNAi drug. Targeting groups can be monovalent, divalent, trivalent, tetravalent, or have a higher valency for the target they are directed at. Typical targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetic compounds with affinity for cell surface molecules.

[0148] In some embodiments, the targeting group is linked to the RNAi drug using a linker, for example, a PEG linker that can function as a linker in some cases, or one, two, or three debase and / or ribitol (debase-ribose) residues. In some embodiments, the targeting ligand comprises a galactose derivative cluster.

[0149] The PNPLA3 RNAi drugs described herein can be synthesized to have a reactive group, such as an amino group (also referred to herein as an amine), at the 5' and / or 3' ends. This reactive group can then be used to attach a targeting moiety using methods typical in the art.

[0150] In some embodiments, the targeting group comprises an asialoglycoprotein receptor ligand. As used herein, an asialoglycoprotein receptor ligand is a ligand comprising a moiety having 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 both galactose and galactose derivatives having an affinity greater than or equal to that of galactose for the asialoglycoprotein receptor. Examples of galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, Nn-butanoylgalactosamine, and N-isobutanoylgalactosamine (see, for example, STIobst and K. Drickamer, JBC, 1996, 271, 6686). Galactose derivatives and a group of galactose derivatives useful for in vivo targeting of oligonucleotides and other molecules against the liver are a base in this art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

[0151] Galactose derivatives have been used in vivo to target molecules to hepatocytes by binding to asialoglycoprotein receptors expressed on the surface of hepatocytes. Binding of asialoglycoprotein receptor ligands to asialoglycoprotein receptors promotes cell-specific targeting to hepatocytes and endocytosis of molecules to hepatocytes. Asialoglycoprotein receptor ligands 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). Galactose derivatives or galactose derivative clusters can be attached to the 3' or 5' end of the sense or antisense strand of an RNAi drug using methods known in the art. The preparation of targeted ligands, such as galactose derivative clusters, is described, for example, in International Patent Application Publication No. 2018 / 044350 and International Patent Application Publication No. 2017 / 156012 to Arrowhead Pharmaceuticals, Inc., both of which are incorporated herein by reference in their entirety.

[0152] When used herein, a galactose derivative cluster comprises a molecule having 2 to 4 terminal galactose derivatives. The terminal galactose derivatives are attached to the molecule via their C-1 carbon. In some embodiments, the galactose derivative cluster is a galactose derivative trimer (also called a ternary galactose derivative or trivalent galactose derivative). In some embodiments, the galactose derivative cluster contains N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster contains three N-acetyl-galactosamines. In some embodiments, the galactose derivative cluster is a galactose derivative tetramer (also called a tetraary galactose derivative or tetravalent galactose derivative). In some embodiments, the galactose derivative cluster contains four N-acetyl-galactosamines.

[0153] As used herein, a galactose derivative trimer comprises three galactose derivatives linked to a central branch point. As used herein, a galactose derivative tetramer comprises four galactose derivatives linked to a central branch point. The galactose derivatives may be attached to the central branch point via the C-1 carbon of the sugar. In some embodiments, the galactose derivatives are linked to the branch point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer such as a PEG group (see, for example, 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. This branch point can be any small molecule to which three galactose derivatives can be attached, and further, to which the branch point can be attached to an RNAi drug. Examples of branch point groups are dyridine or diglutamate salts. The attachment of the branching point to the RNAi drug can be done via a linker or spacer. In some embodiments, the linker or spacer includes a flexible hydrophilic spacer (e.g., a PEG spacer). In some embodiments, the linker includes a rigid linker (e.g., a cyclic group). In some embodiments, the galactose derivative includes or comprises 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.

[0154] Embodiments of the present disclosure include pharmaceutical compositions for delivering PNPLA3 RNAi drugs to hepatocytes in vivo. Such pharmaceutical compositions may, for example, include PNPLA3 RNAi drugs conjugated to a group of galactose derivatives. 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).

[0155] A targeted ligand or targeting group may be ligated to the 3' or 5' end of the sense or antisense strand of a PNPLA3 RNAi drug disclosed herein.

[0156] Targeted ligands include, but are not limited to, (NAG37) and (NAG37)s as defined in Table 6. Other targeted groups and targeted ligands, including galactose cluster targeted ligands, are known in the art.

[0157] In some embodiments, a linking group is conjugated to the RNAi drug. The linking group facilitates covalent linkage of the drug to a targeting group or a delivery polymer or delivery vehicle. The linking group can be linked to the 3' or 5' end of the sense or antisense strand of the RNAi drug. In some embodiments, the linking group is linked to the sense strand of the RNAi drug. In some embodiments, the linking group is conjugated to the 5' or 3' end of the sense strand of the RNAi drug. In some embodiments, the linking group is conjugated to the 5' end of the sense strand of the RNAi drug. Examples of linking groups include, but are not limited to, reactive groups such as primary amines and alkynes, alkyl groups, debased nucleosides, ribitors (debased ribose), and / or PEG groups.

[0158] In some embodiments, the targeting group is internally linked to nucleotides on the sense and / or antisense strands of the RNAi drug. In some embodiments, the targeting group is linked to the RNAi drug via a linker.

[0159] A linker or linking group is a connection between two atoms that, by one or more covalent bonds, links one chemical group (such as an RNAi drug) or segment of interest to another chemical group (e.g., a targeting group or a delivery polymer) or segment of interest. Unstable linkages include unstable bonds. Linkages may optionally include spacers that increase the distance between the two bonding atoms. Spacers can further add flexibility and / or length to the linkage. Examples of spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkyl groups. Each of these may include one or more heteroatoms, heterocycles, amino acids, nucleotides, and sugars. Spacer groups are well known in the art, and the above list is not intended to limit the scope of this specification.

[0160] In some embodiments, when two or more RNAi drugs are contained in a single composition, each RNAi drug may be linked to the same targeting group or to two different targeting groups (i.e., targeting groups having different chemical structures). In some embodiments, the targeting groups are linked to the PNPLA3 RNAi drugs disclosed herein without the use of additional linkers. In some embodiments, the targeting groups themselves are designed to have linkers or other sites to facilitate the presence of conjugation. In some embodiments, when two or more PNPLA3 RNAi drugs are contained in a single molecule, each RNAi drug may utilize the same linker or different linkers (i.e., linkers having different chemical structures).

[0161] Any of the PNPLA3 RNAi drug nucleotide sequences listed in Table 2, Table 3, or Table 4, whether modified or unmodified, may contain a 3' and / or 5' targeting group or linking group. Alternatively, any of the PNPLA3 RNAi drug sequences listed in Table 3 or Table 4 or described herein that contain a 3' or 5' targeting group or linking group may not contain a 3' or 5' targeting group or linking group, or may contain different 3' or 5' targeting group or linking group, including but not limited to those shown in Table 6. Any of the PNPLA3 RNAi drug duplexes listed in Tables 5A and 5B, whether modified or unmodified, may further contain targeting groups or linking groups, including but not limited to those shown in Table 6, and the targeting group or linking group may be attached to the 3' or 5' end of either the sense strand or the antisense strand of the PNPLA3 RNAi drug duplex.

[0162] Table 6 provides examples of targeting groups and linking groups (which can form targeted ligands when combined). Table 4 provides several embodiments of the sense strand of a PNPLA3 RNAi drug having a targeting group or linking group linked to the 5' or 3' end.

[0163] [Table 7-1]

[0164] [Table 7-2]

[0165] [Table 7-3]

[0166] In each of the structures shown in Table 6, NAG comprises N-acetyl-galactosamine or another galactose derivative, which would be understood by those skilled in the art to be attached in consideration of the above structures and the descriptions provided herein.

[0167] Each (NAGx) may be attached to the PNPLA3 RNAi drug via a phosphate group (as in (NAG37)), a phosphorothioate group (as in (NAG37)s), or another binding group.

[0168] [ka]

[0169] Other linking groups known in the relevant art may be used.

[0170] In some embodiments, a delivery vehicle can be used to deliver RNAi drugs to cells or tissues. The delivery vehicle is a compound that improves the delivery of RNAi drugs to cells or tissues. Examples of delivery vehicles include, but are not limited to, polymers (e.g., amphiphilic polymers, membrane-active polymers), peptides, melittin peptides, melittin-like peptides (MLPs), lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines. In some embodiments, the RNAi drug can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art. RNAi drugs can also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, International Publications 2000 / 053722, 2008 / 0022309, 2011 / 104169, and 2012 / 083185, 2013 / 032829, and 2013 / 158141, each of which is incorporated herein by reference), hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, protein vectors, or other delivery systems known and available in the art that are suitable for the delivery of nucleic acids or oligonucleotides.

[0171] Pharmaceutical compositions and preparations The PNPLA3 RNAi drugs disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as “pharmaceuticals”). In some embodiments, the pharmaceutical composition comprises at least one PNPLA3 RNAi drug. These pharmaceutical compositions are particularly useful for inhibiting the expression of target mRNA in target cells, cell populations, tissues, or in vivo.

[0172] The pharmaceutical composition can be used to treat subjects having a disease, disorder, or condition in which they would benefit from a reduction in the level of a target PNPLA3 mRNA or inhibition of the expression of a target gene. The pharmaceutical composition can be used to treat subjects having a risk of developing a disease, disorder, or condition in which they would benefit from a reduction in the level of a target mRNA or inhibition of the expression of a target gene. In one embodiment, the method comprises administering a PNPLA3 RNAi drug linked to a targeted ligand as described herein to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) are added to the pharmaceutical composition containing the PNPLA3 RNAi drug to form a pharmaceutical formulation or drug suitable for in vivo delivery to subjects including humans.

[0173] Pharmaceutical compositions and methods comprising PNPLA3 RNAi agents disclosed herein involve administering a therapeutically effective amount of the PNPLA3 RNAi agent described herein to a subject, thereby reducing the level of target mRNA in cells, cell populations, tissues, organs, or the subject by inhibiting PNPLA3 expression in the subject. In some embodiments, the subject has been previously identified or diagnosed with pathogenic upregulation of the target gene in the targeted cells or tissues. In some embodiments, the subject has been previously identified or diagnosed with alcoholic or non-alcoholic liver disease such as NAFLD, NASH, hepatic fibrosis, and / or cirrhosis. In some embodiments, the subject suffers from symptoms associated with alcoholic or non-alcoholic liver disease such as NAFLD, NASH, hepatic fibrosis, and / or cirrhosis.

[0174] In some embodiments, the described pharmaceutical compositions comprising a PNPLA3 RNAi agent are used to treat or manage clinical symptoms associated with NAFLD, NASH, hepatic fibrosis, alcoholic or non-alcoholic liver disease, and / or PNPLA3 overexpression, including cirrhosis, in a subject. In some embodiments, one or more therapeutic (including prophylactic) doses of one or more pharmaceutical compositions are administered to a subject requiring such treatment. In some embodiments, administration of any of the PNPLA3 RNAi agents of this disclosure can be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0175] A pharmaceutical composition comprising a PNPLA3 RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder in which reduction or inhibition of PNPLA3 mRNA expression would be beneficial. In some embodiments, the subject is administered one or more therapeutically effective doses of a pharmaceutical composition comprising a PNPLA3 RNAi agent to treat the symptom. In other embodiments, the subject is administered one or more prophylactically effective doses of a PNPLA3 RNAi agent to prevent or inhibit at least one symptom.

[0176] The route of administration is the route through which the PNPLA3 RNAi drug comes into contact with the body. Generally, methods for administering drugs, oligonucleotides, and nucleic acids for the treatment of mammals are well known in the art and can be applied to the administration of the compositions described herein. The PNPLA3 RNAi drugs disclosed herein can be administered via any suitable route in preparations tailored to specific routes. Thus, the pharmaceutical compositions described herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intra-articularly, or intraperitoneally. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[0177] Pharmaceutical compositions comprising the PNPLA3 RNAi drug described herein can be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery techniques known in the art. In general, any preferred 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 may be by parenteral routes including local administration (e.g., direct injection, implantation, local administration), systemic administration, subcutaneous administration, intravenous, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or local (including oral and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0178] 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.

[0179] When used herein, a pharmaceutical composition or drug comprises at least one of the described therapeutic compounds in a pharmacologically effective amount and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product, e.g., PNPLA3 RNAi drug) that is intentionally included in a drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dose. An excipient may act to a) assist in the processing of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance the overall safety, efficacy, or any other attributes of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0180] Excipients include, but are not limited to, absorption enhancers, anti-adhesion agents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, surfactants (detergents), dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, fragrances, lubricants, humectants, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0181] Suitable pharmaceutical compositions for use by injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). Suitable carriers must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained by coating, for example, lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it is preferable that the composition contains, for example, sugars, polyhydric alcohols such as mannitol and sorbitol, and isotonic agents such as sodium chloride. By including substances that slow down absorption, such as aluminum monostearate and gelatin, in the composition, the long-term absorption of the injectable composition can be achieved.

[0182] Sterile injection solutions can be prepared by adding the required amount of the active compound to a suitable solvent, along with one or a combination of the components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a basic dispersion medium and a sterile excipient containing the other necessary components listed above. In the case of sterile powders for the preparation of sterile injection solutions, preparation methods include vacuum drying and freeze-drying, which yield powders of the active ingredient and any further desired components from a pre-sterilized and filtered solution.

[0183] In some embodiments, pharmaceutical formulations containing PNPLA3 RNAi drugs disclosed herein that are suitable for subcutaneous administration can be prepared in aqueous sodium phosphate buffer (e.g., PNPLA3 RNAi drugs formulated in 0.5 mM mononucleotide sodium phosphate or 0.5 mM dibasic sodium phosphate in water).

[0184] Formulations suitable for intra-articular administration may be in the form of sterile aqueous preparations of drugs that may be in microcrystalline form, such as aqueous microcrystalline suspensions. Liposome formulations or biodegradable polymer systems can also be used to administer drugs for both intra-articular and intraocular administration.

[0185] Formulations suitable for oral administration of the PNPLA3 RNAi drugs disclosed herein can also be prepared. In some embodiments, the PNPLA3 RNAi drugs disclosed herein are administered orally. In some embodiments, the PNPLA3 RNAi drugs disclosed herein are formulated in capsules for oral administration.

[0186] The active compound can be prepared with a carrier that prevents the compound from being rapidly eliminated from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The methods for preparing such formulations will be obvious to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared, for example, by methods well known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0187] PNPLA3 RNAi drugs can be formulated in dose unit form for ease of administration and dose uniformity. A dose unit form refers to a physically distinct unit suitable as a unit dose for the target of treatment, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications for dose unit forms in this disclosure are determined and directly depend on the inherent properties of the active compound, the therapeutic effect to be achieved, and the limitations inherent in the art to formulate such an active compound for individual treatments.

[0188] Pharmaceutical compositions may include other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritics, astringents, topical anesthetics, analgesics, antihistamines, or anti-inflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine). It is also conceivable that cells, tissues, or isolated organs expressing or containing an RNAi drug as defined herein may be used as a “pharmaceutical composition.” As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” refers to the amount of RNAi drug that produces a pharmacological, therapeutic, or prophylactic effect.

[0189] In some embodiments, the methods disclosed herein further include the step of administering a second therapeutic agent or treatment in addition to administering the RNAi agent disclosed herein. In some embodiments, the second therapeutic agent is another PNPLA3 RNAi agent (e.g., a PNPLA3 RNAi agent that targets a different sequence within the PNPLA3 target). In other embodiments, the second therapeutic agent may be a small molecule drug, an antibody, an antibody fragment, or an aptamer.

[0190] In some embodiments, the described PNPLA3 RNAi drug is optionally combined with one or more additional therapeutic agents. The PNPLA3 RNAi drug and the additional therapeutic agents may be administered in a single composition, or they may be administered separately. In some embodiments, one or more additional therapeutic agents are administered separately from the RNAi drug in separate dosage forms (for example, the PNPLA3 RNAi drug is administered by subcutaneous injection, and the additional therapeutic agents used in the therapeutic administration regimen are administered orally). In some embodiments, the described PNPLA3 RNAi drug is administered by subcutaneous injection to a subject who needs it, while one or more optional additional therapeutic agents are administered orally, and together they provide a therapeutic regimen for diseases and conditions associated with alcoholic or non-alcoholic liver disease, including NAFLD, NASH, hepatic fibrosis, and / or cirrhosis. In some embodiments, the described PNPLA3 RNAi drug is administered by subcutaneous injection to a subject who needs it, and one or more optional additional therapeutic agents are administered by separate subcutaneous injections. In some embodiments, the PNPLA3 RNAi drug and one or more additional therapeutic agents are combined in a single dosage form (e.g., a “cocktail” formulated in a single composition for subcutaneous injection). The PNPLA3 RNAi drug can be combined with one or more excipients, with or without one or more additional therapeutic agents, to form a pharmaceutical composition.

[0191] Generally, the effective dose of a PNPLA3 RNAi drug is in the range of about 0.1 to about 100 mg / kg body weight / dose, for example, about 1.0 to about 50 mg / kg body weight / dose. In some embodiments, the effective dose of the active compound is in the range of about 0.25 to about 5 mg / kg body weight per single dose. In some embodiments, the effective dose of the active ingredient is in the range of about 0.5 to about 4 mg / kg body weight per single dose. Dosage may be weekly, bi-weekly, monthly, or at any other interval, depending on the dose of the PNPLA3 RNAi drug administered, the activity level of the particular PNPLA3 RNAi drug, and the desired level of inhibition against a particular target. The examples herein show levels suitable for inhibition in specific animal species. The dosage will depend on variables such as the patient's overall health, the relative biological potency of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, and the route of administration. Furthermore, it will be understood that the initial dose administered may be increased beyond the above upper limit to rapidly achieve the desired blood or tissue level, or the initial dose may be less than the optimal dose.

[0192] For the treatment of a disease, or for the formation of a pharmaceutical or composition for the treatment of a disease, the pharmaceutical compositions described herein, comprising a PNPLA3 RNAi drug, may be combined with excipients or a second therapeutic agent or treatment (including, but not limited to, a second or other RNAi drug, small molecule drug, antibody, antibody fragment, peptide and / or aptamer).

[0193] The PNPLA3 RNAi drugs described herein may be packaged in kits, containers, packs, or dispensers when added to pharmaceutically acceptable excipients or adjuvants. The pharmaceutical compositions described herein may be packaged in pre-filled syringes, pen syringes, auto-injectors, infusion bags / devices, or vials.

[0194] Treatment methods and methods for inhibiting expression The PNPLA3 RNAi agents disclosed herein can be used to treat subjects (e.g., humans or mammals) who have a disease or disorder in which they would benefit from administration of the compound. In some embodiments, the RNAi agents disclosed herein can be used to treat subjects (e.g., humans) who would benefit from reduction and / or inhibition of PNPLA3 mRNA expression and / or PNPLA3 protein levels, for example, subjects diagnosed with or suffering from symptoms associated with alcoholic or non-alcoholic liver disease, including NAFLD, NASH, hepatic fibrosis, or cirrhosis.

[0195] In some embodiments, a subject is administered a therapeutically effective dose of any one or more PNPLA3 RNAi agents. Treatment of the subject may include therapeutic measures and / or prophylactic treatment. A subject is administered a therapeutically effective dose of any one or more PNPLA3 RNAi agents described herein. The subject may be a human, a patient, or a human patient. The subject may be an adult, adolescent, child, or infant. Administration of the pharmaceutical compositions described herein may be to humans or animals.

[0196] The PNPLA3 RNAi agents described herein may be used to treat at least one symptom in a subject having a PNPLA3-related disease or disorder, or a disease or disorder at least partially mediated by PNPLA3 gene expression. In some embodiments, the PNPLA3 RNAi agent is used to treat or manage the clinical symptoms of a subject having a disease or disorder that benefits from or is at least partially mediated by a reduction in PNPLA3 mRNA. The subject is administered a therapeutically effective dose of one or more of the PNPLA3 RNAi agents described herein or a PNPLA3 RNAi agent-containing composition. In some embodiments, the method disclosed herein includes administering a composition containing the PNPLA3 RNAi agent described herein to the subject to be treated. In other embodiments, the subject is administered a prophylactically effective dose of one or more of the described PNPLA3 RNAi agents to treat the subject by preventing or inhibiting at least one symptom.

[0197] In certain embodiments, the present disclosure provides a method for treating a disease, disorder, condition, or medical condition at least partially mediated by PNPLA3 gene expression, the method comprising administering to a patient one of the PNPLA3 RNAi agents described herein.

[0198] In some embodiments, the gene expression level and / or mRNA level of the PNPLA3 gene in subjects administered with the described PNPLA3 RNAi drug is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 99% compared to subjects before administration of the PNPLA3 RNAi drug or subjects not receiving the PNPLA3 RNAi drug. The gene expression level and / or mRNA level in the subjects may be reduced in the cells, cell populations, and / or tissues of the subjects. In some embodiments, PNPLA3 gene expression is inhibited by at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, or more than 70% in the cytoplasm of hepatocytes compared to subjects before administration of the PNPLA3 RNAi drug or subjects not receiving the PNPLA3 RNAi drug.

[0199] In some embodiments, PNPLA3 protein levels in subjects administered with the described PNPLA3 RNAi drug are reduced by at least approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% compared to subjects before administration of the PNPLA3 RNAi drug or subjects not receiving the PNPLA3 RNAi drug. Protein levels within the subjects may be reduced in the subjects' cells, cell populations, tissues, blood, and / or other bodily fluids.

[0200] The reduction of PNPLA3 mRNA levels and PNPLA3 protein levels can be evaluated by any method known in the art. As used herein, the reduction or decrease of PNPLA3 mRNA levels and / or protein levels is collectively referred to as the reduction or decrease of PNPLA3 or the inhibition or reduction of PNPLA3 expression. The examples described herein illustrate known methods for evaluating the inhibition of PNPLA3 gene expression. Those skilled in the art will be able to learn of further preferred methods for evaluating the inhibition of PNPLA3 gene expression in vivo and / or in vitro.

[0201] In some embodiments, methods for treating (including prophylactic or preventative treatment) diseases, disorders, or symptoms caused by alcoholic or non-alcoholic liver diseases, including NAFLD, NASH, hepatic fibrosis, and / or cirrhosis, comprising administering to a subject in need a therapeutically effective amount of a PNPLA3 RNAi drug comprising an antisense strand at least partially complementary to a portion of PNPLA3 mRNA having the sequence of Table 1. In some embodiments, methods for treating (including prophylactic or preventative treatment) diseases or symptoms caused by alcoholic or non-alcoholic liver diseases, including NAFLD, NASH, hepatic fibrosis, and / or cirrhosis, comprising administering to a subject in need a therapeutically effective amount of a PNPLA3 RNAi drug comprising an antisense strand having a sequence of either Table 2 or Table 3, and a sense strand having a sequence of either Table 2 or Table 4 that is at least partially complementary to the antisense strand, are disclosed herein. In some embodiments, methods for treating (including prophylactic or preventative treatment) diseases or symptoms caused by alcoholic or non-alcoholic liver diseases, including NAFLD, NASH, hepatic fibrosis, and / or cirrhosis, comprising administering to a subject in need of such treatment a therapeutically effective amount of a PNPLA3 RNAi agent comprising a sense strand comprising one of the sequences in Table 2 or Table 4, and an antisense strand comprising one of the sequences in Table 2 or Table 3 that is at least partially complementary to the sense strand, are disclosed herein.

[0202] In some embodiments, methods for inhibiting the expression of the PNPLA3 gene in cells are disclosed herein, comprising administering the cells a PNPLA3 RNAi drug comprising an antisense strand at least partially complementary to a portion of the PNPLA3 mRNA having the sequence of Table 1. In some embodiments, methods for inhibiting the expression of the PNPLA3 gene in cells are disclosed herein, comprising administering the cells a PNPLA3 RNAi drug comprising an antisense strand containing either the sequence of Table 2 or Table 3, and a sense strand containing either the sequence of Table 2 or Table 4, which is at least partially complementary to the antisense strand. In some embodiments, methods for inhibiting the expression of the PNPLA3 gene in cells are disclosed herein, comprising administering a PNPLA3 RNAi drug comprising a sense strand containing either the sequence of Table 2 or Table 4, and an antisense strand containing either the sequence of Table 2 or Table 3, which is at least partially complementary to the sense strand.

[0203] The use of PNPLA3 RNAi agents provides methods for the therapeutic (including prophylactic) treatment of diseases / disorders associated with enhanced or improved PNPLA3 expression, including alcoholic or non-alcoholic liver diseases such as NAFLD, NASH, hepatic fibrosis, and cirrhosis. The described PNPLA3 RNAi agents inhibit the expression of one or more genes necessary for the production of the PNPLA3 protein by mediating RNA interference. PNPLA3 RNAi agents may also be used to treat or prevent a variety of diseases, disorders, or conditions, including alcoholic or non-alcoholic liver diseases such as NAFLD, NASH, hepatic fibrosis, and / or cirrhosis. Furthermore, compositions for in vivo delivery of PNPLA3 RNAi agents to hepatocytes are described.

[0204] Cells, tissues, organs, and non-human organisms Cells, tissues, organs, and non-human organisms comprising at least one of the PNPLA3 RNAi drugs described herein are intended. These cells, tissues, organs, or non-human organisms are generated by delivering the RNAi drug to cells, tissues, organs, or non-human organisms.

[0205] Exemplary Embodiments Exemplary embodiments of the technology of the present disclosure are provided herein. These embodiments are for illustrative purposes only and do not limit the scope of the present disclosure or the claims appended thereto.

[0206] Embodiment 1. An RNAi drug for inhibiting the expression of the PNPLA3 gene, An RNAi drug comprising an antisense strand containing at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any one of the sequences of sequence numbers 46-87, 174-211, and 257-258, and a sense strand containing a nucleotide sequence that is at least partially complementary to the antisense strand.

[0207] Embodiment 2. The RNAi drug according to Embodiment 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one sequence from sequence numbers 46 to 87, 174 to 211, and 257 to 258.

[0208] Embodiment 3. The RNAi drug according to Embodiment 1 or Embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any one of the sense strand sequences of SEQ ID NOs. 2-45 and 212-256, and the sense strand has a region of at least 85% complementarity with the antisense strand over the 17 consecutive nucleotides.

[0209] Embodiment 4. An RNAi drug according to any one of Embodiments 1 to 3, wherein at least one nucleotide of the RNAi drug is a modified nucleotide or comprises a modified nucleoside linkage.

[0210] Embodiment 5. An RNAi drug according to any one of Embodiments 1 to 3, wherein all or substantially all of the nucleotides of the sense strand and antisense strand of the RNAi drug are modified nucleotides.

[0211] Embodiment 6. The RNAi drug according to Embodiment 4 or 5, wherein the modified nucleotide is selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasic nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methylnucleotide, reverse 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinyl phosphonate-containing nucleotide, cyclopropyl phosphonate-containing nucleotide, and 3'-O-methylnucleotide.

[0212] Embodiment 7. The RNAi drug according to Embodiment 5, wherein all or substantially all of the modified nucleotides are 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof.

[0213] Embodiment 8. An RNAi drug according to any one of Embodiments 1 to 7, wherein the antisense strand comprises one nucleotide sequence from among the modified antisense strand sequences of SEQ ID NOs. 88 to 128.

[0214] Embodiment 9. An RNAi drug according to any one of Embodiments 1 to 8, wherein the sense strand comprises a nucleotide sequence of any of the modified sense strand sequences of SEQ ID NOs. 129 to 173.

[0215] Embodiment 10. The RNAi drug according to Embodiment 1, wherein the antisense strand comprises one nucleotide sequence from among the modified sequences of SEQ ID NOs. 88 to 128, and the sense strand comprises one nucleotide sequence from among the modified sequences of SEQ ID NOs. 129 to 173.

[0216] Embodiment 11. An RNAi drug according to any one of Embodiments 1 to 10, wherein the RNAi drug is linked to a targeted ligand.

[0217] Embodiment 12. The method according to Embodiment 11, wherein the targeted ligand comprises n-acetyl-galactosamine.

[0218] Embodiment 13. The RNAi drug according to Embodiment 11 or 12, wherein the targeted ligand comprises the structure of (NAG37) or (NAG37)s.

[0219] Embodiment 14. An RNAi drug according to any one of Embodiments 11 to 14, wherein the targeted ligand is ligated to the sense strand.

[0220] Embodiment 15. The RNAi drug according to Embodiment 15, wherein the targeted ligand is ligated to the 5' end of the sense strand.

[0221] Embodiment 16. An RNAi drug according to any one of Embodiments 1 to 16, wherein the sense strand is 18 to 30 nucleotides long and the antisense strand is 18 to 30 nucleotides long.

[0222] Embodiment 17. The RNAi drug according to Embodiment 17, wherein the sense strand and the antisense strand are each 18 to 27 nucleotides long.

[0223] Embodiment 18. The RNAi drug according to Embodiment 18, wherein the sense strand and the antisense strand are each 18 to 24 nucleotides long.

[0224] Embodiment 19. The RNAi drug according to Embodiment 19, wherein the sense strand and the antisense strand are each 21 nucleotides long.

[0225] Embodiment 20. An RNAi drug according to any one of Embodiments 17 to 20, wherein the RNAi drug has two blunt ends.

[0226] Embodiment 21. An RNAi drug according to any one of Embodiments 1 to 21, wherein the sense strand comprises one or two terminal caps.

[0227] Embodiment 22. An RNAi drug according to any one of Embodiments 1 to 22, wherein the sense strand comprises one or two reverse debase residues.

[0228] Embodiment 23. The RNAi drug according to Embodiment 1, wherein the RNAi drug comprises a sense strand and an antisense strand that form one of the double helix sequences having the sequence numbers listed in Table 5B.

[0229] Embodiment 24. The RNAi drug according to any one of Embodiments 1 to 23, wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[0230] Embodiment 25. An RNAi drug according to Embodiment 1, comprising, consisting of, or essentially consisting of a modified nucleotide sequence in which one of the nucleotide sequences of SEQ ID NOs. 88 to 128 differs from zero or one 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 linkage; and all or substantially all of the nucleotides of the sense strand are modified nucleotides.

[0231] Embodiment 26. The RNAi drug according to Embodiment 1, wherein the sense strand contains, essentially consists of, or essentially consists of, a modified nucleotide sequence in which one of the nucleotide sequences of SEQ ID NOs. 129-173 differs from zero or one nucleotide, and 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 linkage; and all or substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0232] Embodiment 27. The RNAi drug according to any one of Embodiments 24 to 26, wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[0233] Embodiment 28. An RNAi drug according to any one of Embodiments 24 to 27, wherein the sense strand of the RNAi drug is linked to a targeted ligand.

[0234] Embodiment 29. The RNAi drug according to Embodiment 28, wherein the targeted ligand has affinity for the asialocrypoprotein receptor.

[0235] Embodiment 30. The RNAi drug according to Embodiment 29, wherein the targeted ligand comprises N-acetyl-galactosamine.

[0236] Embodiment 31. The targeted ligand is

[0237] [ka] An RNAi drug according to Embodiment 1, including the above.

[0238] Embodiment 32. The RNAi drug according to Embodiment 1, wherein the antisense strand consists of the modified nucleotide sequences of SEQ ID NOs. 88 to 128, the sense strand consists of the modified nucleotide sequences of SEQ ID NOs. 129 to 173, 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 linkage, (invAb) is a reverse debased deoxyribose residue, and (NAG37)s has the following chemical structure.

[0239] [ka]

[0240] Embodiment 33. A composition comprising any one of the RNAi drugs of Embodiments 1 to 32, further comprising a pharmaceutically acceptable excipient.

[0241] Embodiment 34. A method for inhibiting the expression of the PNPLA3 gene in a cell, comprising introducing into the cell an effective amount of the RNAi drug described in any one of Embodiments 1 to 32 or the composition described in any one of Embodiments 33.

[0242] Embodiment 35. The method according to Embodiment 34, wherein the cell is within a subject.

[0243] Embodiment 36. The method according to Embodiment 35, wherein the subject is a human subject.

[0244] 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%.

[0245] Embodiment​​​​​​​​​​​​​​​Embodiment 42. Use of an RNAi drug 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 at least partially mediated by PNPLA3 gene expression.

[0250] Embodiment 43. The use described in Embodiment 42, wherein the symptom is cirrhosis of the liver.

[0251] Embodiment 44. Use of an RNAi drug according to any one of Embodiments 1 to 32 or the composition according to Embodiment 33 for the preparation of a pharmaceutical composition for treating a disease, disorder, or symptom that is at least partially mediated by PNPLA3 gene expression.

[0252] Embodiment 45. Use of an RNAi agent according to any one of Embodiments 42 to 44, wherein the prior disease is an alcoholic or non-alcoholic liver disease such as NAFLD, NASH, hepatic fibrosis, or cirrhosis.

[0253] Embodiment 46. Use of the composition according to Embodiment 33, wherein the RNAi drug is administered at a dose of approximately 0.05 mg to approximately 5.0 mg per kg of body weight of a human subject.

[0254] Herein, the embodiments and items described above are illustrated in the following non-limiting embodiments. [Examples]

[0255] Example 1. Synthesis of PNPLA3 RNAi drug. The PNPLA3 RNAi drug duplexes shown in Tables 5A and 5B above were synthesized according to the following general procedure:

[0256] A. Synthesis. Based on solid-phase phosphoramidite techniques used for oligonucleotide synthesis, sense and antisense strands of RNAi drugs were synthesized. Such standard synthesis is generally known in the art. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was carried out on solid supports made of controlled porous glass (CPG, 500 Å or 600 Å, available from Prime Synthesis, Aston, PA, USA). Monomers located at the 3' end of each strand were attached to the solid support as starting points for synthesis. All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA) or Hongene Biotech (Shanghai, PRC). Examples of 2'-O-methylphosphoramidites include: (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-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)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. 2'-deoxy-2'-fluoro-phosphoramidite had the same protecting group as 2'-O-methylamidite. 5'-(4,4'-dimethoxytrityl)-2',3'-secor-uridine and 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were 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 debasing (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.

[0257] Phosphoamidites containing the targeting ligand were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), and all other amidites were dissolved in anhydrous acetonitrile (50 mM) or anhydrous dimethylformamide, and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Binding times were 12 minutes (RNA), 15 minutes (targeting ligand), 90 seconds (2'OMe), and 60 seconds (2'F). To introduce phosphorothioate linkage, a 100 mM solution of 3-phenyl 1,2,4-dithiazolin-5-one (POS, PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used. Unless otherwise specified as a “naked” RNAi drug lacking a targeted ligand, each PNPLA3 RNAi drug double helix synthesized and tested in the following examples utilized N-acetyl-galactosamine as “NAG” in the targeted ligand chemical structure shown in Table 6.

[0258] B. Cleavage and deprotection of support-bound oligomers. After completing the solid-phase synthesis, the dried solid support was treated with a 1:1 solution of 40% by weight methylamine in water and 28% ammonium hydroxide solution (Aldrich) at 30°C for 1.5 hours. This solution was evaporated, and the solid residue was reconstituted in water (see below).

[0259] C. Purification. Crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A consisted of 20 mM Tris, 5 mM EDTA, pH 9.0, and contained 20% acetonitrile. Buffer B was the same as Buffer A, with the addition of 1.5 M sodium chloride. UV tracing was recorded at 260 nm. After pooling appropriate fractions, the samples were subjected to size exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 fine, with filtered DI water or 100 mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile used as running buffers.

[0260] D. Annealing. The complementary strands were mixed with equimolar amounts of RNA solution (sense and antisense) in 1× phosphate-buffered saline (Corning, Cellgro) to form RNAi drugs. A portion of the RNAi drugs were lyophilized and stored at -15 to -25°C. The double-strand concentration was determined by measuring the solution absorbance using a UV-Vis spectrometer in 1× phosphate-buffered saline. Next, the double-strand concentration was determined by multiplying the solution absorbance at 260 nm by a conversion factor and a dilution factor. The conversion factor used was either 0.050 mg / (mL·cm) or calculated from experimentally determined extinction coefficients.

[0261] Example 2. PNPLA3-SEAP mouse model. To evaluate specific PNPLA3 RNAi drugs, a PNPLA3-SEAP mouse model was used. Depending on availability, female C57BL / 6 albino or ICR mice aged 6–8 weeks were transiently transfected in vivo with plasmids by hydrodynamic tail vein injection at least 29 days prior to administration of the PNPLA3 RNAi drug or control. Two SEAP plasmids were prepared. The first plasmid contained a 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 recognized instability of the full-length transcript over time, a second plasmid containing the human PNPLA3 cDNA sequence (specifically nucleotides 501–2210 of GenBank NM_025225.2) was synthesized. One of two plasmids was selected and transfected into mice. PNPLA3-SEAP model mice were created by injecting 50 μg of each plasmid in Ringer's solution in a total volume of 10% of the animal's body weight into the mice via the tail vein. The solution was injected over 5–7 seconds using a 27-gauge needle, as previously described (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 a PNPLA3 RNAi drug results in simultaneous inhibition of the measured SEAP expression. Before treatment (7 days to 1 day prior to administration), serum SEAP expression levels were measured using the Phospha-Light® SEAP reporter gene assay system (Invitrogen), and mice were grouped according to their mean SEAP levels.

[0262] Mice were anesthetized with 2 - 3% isoflurane, and blood samples were collected from the submandibular region into serum separation tubes (Sarstedt AG & Co., Numbrecht, Germany). The blood was allowed to clot at ambient temperature for 20 minutes. The tubes were centrifuged at 8,000 × g for 3 minutes to separate the serum, which was stored at 4°C. Serum was collected and measured using the Phospha-Light™ SEAP Reporter Gene Assay System (Invitrogen) according to the manufacturer's instructions. The serum SEAP levels of each animal can be normalized to a group of control mice injected with vehicle control to account for the treatment-unrelated decrease in PNPLA3 expression in this model. To do so, first, the SEAP level of each animal at a given time point was divided by the expression level before treatment (-1 day) in that animal to determine the ratio of expression "normalized to pre-treatment". Then, the "normalized to pre-treatment" ratio of an individual animal was divided by the mean "normalized to pre-treatment" ratio of all mice in the normal vehicle control group to normalize the expression at a specific time point to the control group. Alternatively, the serum SEAP levels of each animal were evaluated by normalizing to only the pre-treatment levels.

[0263] Example 3. In vivo test of PNPLA3 RNAi drug in PNPLA3-SEAP mice. Using a plasmid containing the full-length human PNPLA3 transcript, the PNPLA3-SEAP mouse model was used as described in Example 2 above. On day 1, each mouse was administered a single subcutaneous injection of 200 μl / 20 g animal weight containing either 3.0 mg / kg (mpk) of the PNPLA3 RNAi drug formulated in saline or vehicle control (saline without the RNAi drug) according to Table 7 below.

[0264]

Table 8

[0265] Each PNPLA3 RNAi drug contained a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequence described herein in the double-strand structure. (See Tables 3-5 for specific modifications and structural information related to PNPLA3 RNAi drugs, including the (NAG37)s ligand.) PNPLA3 RNAi drugs J1D00001 (group 2) and J1D00002 (group 3) contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 688 of the gene, PNPLA3 RNAi drug J1D00004 (group 4) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1586 of the gene, PNPLA3 RNAi drug J1D00008 (group 5) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180 of the gene, PNPLA3 RNAi drugs J1D00010 (group 6) and J1D00011 (group 7) contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1179 of the gene, and PNPLA3 RNAi drug J1D00012 (group 8) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 571 of the gene. RNAi drug J1D00016 (group 9) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1745. (For example, see Sequence ID 1 and Table 2 for the referenced PNPLA3 gene.)

[0266] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 8 and 9 below.

[0267] [Table 9] *As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0268] [Table 10]

[0269] Example 4. In vivo testing of PNPLA3 RNAi drugs in PNPLA3-SEAP mice. A PNPLA3-SEAP mouse model was used as described in Example 2 above, using a plasmid containing the full-length human PNPLA3 transcript. On day 1, each mouse received a single subcutaneous dose of 200 μl / 20 g animal weight containing either 3.0 mg / kg (mpk) of the PNPLA3 RNAi drug formulated in physiological saline or a vehicle control (physiological saline without the RNAi drug), as shown in Table 10 below.

[0270] [Table 11]

[0271] Each PNPLA3 RNAi drug contains a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequence described herein for the double-strand structure. (See Tables 3-5 for specific modifications and structural information regarding PNPLA3 RNAi drugs containing the (NAG37)s ligand.) PNPLA3 RNAi drug J1D00008 (Group 2) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180, PNPLA3 RNAi drug J1D00014 (Group 3) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 886, PNPLA3 RNAi drug J1D00015 (Group 4) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1584, PNPLA3 RNAi drug J1D00021 (Group 5) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 553, and PNPLA3 RNAi drug J1D00022 (Group 6) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 680. RNAi drug J1D00005 (group 7) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1182, and PNPLA3 RNAi drug J1D00024 (group 8) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 746. (For example, see Sequence ID 1 and Table 2 for the referenced PNPLA3 gene).

[0272] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 11 and 12 below.

[0273] [Table 12] * As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0274] [Table 13]

[0275] Example 5. In vivo testing of PNPLA3 RNAi drugs in PNPLA3-SEAP mice. A PNPLA3-SEAP mouse model was used with a plasmid containing a truncated human PNPLA3 transcript, as described in Example 2 above. On day 1, each mouse received a single subcutaneous dose of 200 μl / 20 g animal weight containing either 3.0 mg / kg (mpk) of the PNPLA3 RNAi drug formulated in saline or a vehicle control (saline without the RNAi drug), as shown in Table 13 below.

[0276] [Table 14]

[0277] Each PNPLA3 RNAi drug contains a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequence described herein for the double-strand structure. (See Tables 3-5 for specific modifications and structural information related to PNPLA3 RNAi drugs containing the (NAG37)s ligand.) PNPLA3 RNAi drugs J1D00012 (group 2), J1D00027 (group 3), J1D00028 (group 4), and J1D00029 (group 5) each contain nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 571, PNPLA3 RNAi drugs J1D00011 (group 6) and J1D00017 (group 7) each contain nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 1179, PNPLA3 RNAi drugs J1D00004 (group 8), J1D00003 (group 9), and J1D00041 (group 10) each contain nucleotide sequences designed to inhibit the expression of the PNPLA3 gene at position 1586, and PNPLA3 RNAi drug J1D00008 (group 11) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180. (For example, see Sequence ID 1 and Table 2 for the referenced PNPLA3 gene.)

[0278] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 14 and 15 below.

[0279] [Table 15] *As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0280] [Table 16]

[0281] Each PNPLA3 RNAi drug in each treatment group (i.e., groups 2-11) showed a reduction in SEAP at all measurement time points compared to the vehicle control (group 1).

[0282] Example 6. In vivo testing of PNPLA3 RNAi drugs in PNPLA3-SEAP mice. A PNPLA3-SEAP mouse model was used with a plasmid containing a truncated human PNPLA3 transcript, as described in Example 2 above. On day 1, each mouse received a single subcutaneous dose of 200 μl / 20 g animal weight containing either 3.0 mg / kg (mpk) of the PNPLA3 RNAi drug formulated in saline or a vehicle control (saline without the RNAi drug), as shown in Table 16 below.

[0283] [Table 17]

[0284] Each PNPLA3 RNAi drug contains a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequence described herein for the double-strand structure. (See Tables 3-5 for specific modifications and structural information related to PNPLA3 RNAi drugs containing the (NAG37)s ligand.) PNPLA3 RNAi drugs J1D00008 (group 2), J1D00046 (group 3), J1D00047 (group 4), and J1D00048 (group 5) each contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180, PNPLA3 RNAi drugs J1D00011 (group 6), J1D00043 (group 7), J1D00044 (group 8), and J1D00045 (group 9) each contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1179, and PNPLA3 RNAi drug J1D00020 (group 10) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 544. RNAi drug J1D00026 (group 11) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1195. (See Sequence ID 1 and Table 2 for the PNPLA3 gene referenced by SEQ).

[0285] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 17 and 18 below:

[0286] [Table 18] *As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0287] [Table 19]

[0288] Example 7. In vivo testing of PNPLA3 RNAi drugs in PNPLA3-SEAP mice. A PNPLA3-SEAP mouse model was used with a plasmid containing a truncated human PNPLA3 transcript, as described in Example 2 above. On day 1, each mouse received a single subcutaneous dose of 200 μl / 20 g animal weight containing either 1.5 mg / kg (mpk) of the PNPLA3 RNAi drug formulated in saline or a vehicle control (saline without the RNAi drug), as shown in Table 19 below.

[0289] [Table 20]

[0290] Each PNPLA3 RNAi drug contains a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequence described herein for the double-strand structure. (See Tables 3-5 for specific modifications and structural information related to PNPLA3 RNAi drugs containing the (NAG37)s ligand.) PNPLA3 RNAi drug J1D00008 (Group 2) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180, PNPLA3 RNAi drugs J1D000018 (Group 3) and J1D00019 (Group 4) contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 538, PNPLA3 RNAi drug J1D00013 (Group 5) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 687, PNPLA3 RNAi drugs J1D000006 (Group 6) and J1D00007 (Group 7) contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 751, and PNPLA3 RNAi drug J1D00035 (Group 8) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1181. RNAi drug J1D00033 (group 9) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 685 of the gene; PNPLA3 RNAi drug J1D00032 (group 10) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 373 of the gene; and PNPLA3 RNAi drug J1D00040 (group 11) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1837 of the gene. (For example, see Sequence ID 1 and Table 2 for the referenced PNPLA3 gene).

[0291] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 20 and 21 below:

[0292] [Table 21] * As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0293] [Table 22]

[0294] Example 8. In vivo testing of PNPLA3 RNAi drugs in PNPLA3-SEAP mice. A PNPLA3-SEAP mouse model was used with a plasmid containing a truncated human PNPLA3 transcript, as described in Example 2 above. On day 1, each mouse received a single subcutaneous dose of 200 μl / 20 g animal weight containing either 1.5 mg / kg (mpk) of the PNPLA3 RNAi drug formulated in saline or a vehicle control (saline without the RNAi drug), as shown in Table 22 below.

[0295] [Table 23]

[0296] Each PNPLA3 RNAi drug contained a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligand) having the modified sequence described herein in the double-strand structure. (See Tables 3-5 for specific modifications and structural information related to PNPLA3 RNAi drugs containing (NAG37)s ligands). Each group (i.e., groups 2-10) of PNPLA3 RNAi drugs contained a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180. (See, for example, SEQ ID NO: 1 and Table 2 for the referenced PNPLA3 gene.)

[0297] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 23 and 24 below:

[0298] [Table 24] * As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0299] [Table 25]

[0300] Each PNPLA3 RNAi drug in each treatment group (i.e., groups 2-10) showed a reduction in SEAP at all measured time points compared to the vehicle control (group 1).

[0301] Example 9. In vivo testing of PNPLA3 RNAi drugs in PNPLA3-SEAP mice. A PNPLA3-SEAP mouse model was used with a plasmid containing a truncated human PNPLA3 transcript, as described in Example 2 above. On day 1, each mouse received a single subcutaneous dose of 200 μl / 20 g animal weight containing either 1.5 mg / kg (mpk) of the PNPLA3 RNAi drug in saline or a vehicle control (saline without the RNAi drug), according to the dosage groups shown in Table 25 below.

[0302] [Table 26]

[0303] Each PNPLA3 RNAi drug contains a modified nucleotide conjugated at the 5' end of the sense strand to a targeting ligand containing three N-acetyl-galactosamine groups (tridentate ligands) having the modified sequence described herein for the double-strand structure. (See Tables 3-5 for specific modifications and structural information related to PNPLA3 RNAi drugs containing the (NAG37)s ligand.) PNPLA3 RNAi drug J1D00008 (Group 2) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180, PNPLA3 RNAi drug J1D00081 (Group 3) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 887, PNPLA3 RNAi drug J1D00083 (Group 4) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1185, PNPLA3 RNAi drugs J1D00084 (Group 5) and J1D00085 (Group 6) each contain a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1191, and PNPLA3 RNAi drug J1D00087 (Group 7) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1746. The RNAi drug J1D00082 (group 8) contains a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1173. (For example, see Sequence ID 1 and Table 2 for the referenced PNPLA3 gene.)

[0304] The injection was administered subcutaneously (i.e., between the skin and muscle) into loose skin in the neck and shoulder region. Four mice were tested in each group (n=4). Serum was collected on day 1 (pre-treatment), day 8, day 15, day 22, and day 29, and SEAP expression levels were determined according to the procedure described in Example 2 above. The experimental data are shown in Tables 26 and 27 below:

[0305] [Table 27] *As described in Example 2 above, the gradual decrease in SEAP over time in the vehicle control group (Group 1) is due to the loss of the SEAP reporter gene in mouse cells due to the replication of native cells in animals, and not a result of the inhibitory compound.

[0306] [Table 28]

[0307] Example 10. In vivo study of PNPLA3 RNAi drugs in cynomolgus monkeys. The PNPLA3 RNAi drugs J1D00003, J1D00008, and J1D00017 were evaluated in cynomolgus monkeys (cynos). On days 1 and 43, four cynomolgus monkeys in each group (n=4) were administered either 0.4 mL / kg (approximately 1.5 mL volume depending on animal weight) of each PNPLA3 RNAi drug at a concentration of 4.0 mg / kg (10 mg / mL) in physiological saline, or physiological saline vehicle without the RNAi drug to serve as a control (see Table 28 below).

[0308] [Table 29]

[0309] As shown in Tables 3 to 6, the PNPLA3 RNAi drugs contained a modified nucleotide conjugated to the 5' end of the sense strand and a tridentate N-acetyl-galactosamine-containing targeted ligand ((NAG37)s). PNPLA3 RNAi drug J1D00003 (group 2) contained a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1586, PNPLA3 RNAi drug J1D00008 (group 3) contained a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 2180, and PNPLA3 RNAi drug J1D00017 (group 4) contained a nucleotide sequence designed to inhibit the expression of the PNPLA3 gene at position 1179.

[0310] Liver biopsies were collected on days -13 (pre-administration), 15, 29, 57, and 76. On each biopsy collection day, cynomolgus monkeys were anesthetized, and two liver tissue samples of approximately 80 mg to 120 mg each were extracted using laparoscopy. The biopsy samples were then homogenized, and the level of PNPLA3 mRNA in the liver of the cynomolgus monkeys was measured by RT-qPCR. The obtained values ​​were then normalized to the PNPLA3 mRNA measurement before administration (in this case, on day -13). The obtained mRNA data are reflected in Table 29 below.

[0311] [Table 30]

[0312] Example 11. In vivo study of PNPLA3 RNAi drug J1D00008 in cynomolgus monkeys The objective of this study was to evaluate the RNAi drug J1D00008 in terms of both the depth and duration of knockdown of wild-type PNPLA3 mRNA in the liver of cynomolgus monkeys (cynos). Knockdown was normalized for each animal to baseline values ​​obtained from liver biopsies performed before administration.

[0313] Materials and methods Study 1: Cynomolgus monkeys were grouped according to body weight (2-5 kg) into either a saline-treated group (Group 1) or an RNA-treated group (Groups 2-4), with n=4 per group. The monkeys were subcutaneously administered 4 mg / kg in 0.4 ml / kg saline twice, on days 1 and 43. The monkeys were fasted overnight for at least 12 hours prior to biopsy and blood collection. Liver biopsy samples were collected from all animals before administration and on days 15, 29, 57, and 76 of the study. From each animal, liver biopsy samples (two samples, approximately 100 mg each) were collected for quantification of PNPLA3 mRNA by qPCR and in-situ hybridization (ISH). Blood was collected from the femoral vein of each animal before administration and on days 15, 29, 57, and 76 after administration. RNA was isolated from the outer left liver lobe for all animals, groups, and time points, and qRT-PCR was performed. Data were normalized to the housekeeping gene (ARFGAP2), and relative expression was compared to baseline / pre-treatment biopsy. At the end of the study, PNPLA3 expression was measured in the outer left, right, and central liver lobes from groups 1 and 3 to confirm that expression did not vary by liver lobe and that the outer left liver lobe was representative. RNAscope®-based in-situ hybridization assay (ACDbio, Newark, CA) was performed on cynomolgus monkey liver biopsies. Total PNPLA3 mRNA copies in a given liver cell population were quantified using image-based quantitative software analysis (HALO® software system, Indica Labs, Albuquerque, NM).

[0314] 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 into two RNAi treatment groups based on body weight (2-5 kg), with n=10 per group. The animals were subcutaneously administered 4 mg / kg in physiological saline on days 1 and 29, followed by administration for 57 days.

[0315] result Figure 1 shows the measurement of PNPLA3 expression by PCR for the RNAi drug J1D00008 from Study 1. PNPLA3 mRNA was decreased by 51%, 55%, 47%, and 37% at baseline and normalized time points relative to housekeeping genes, respectively. Following these results, in-situ hybridization (ISH) was performed to assess the intracellular (cytoplasm vs. nucleus) distribution of PNPLA3 mRNA. Approximately 50% of total cellular PNPLA3 mRNA was observed to be distributed in the nucleus. This suggests that the RNAi drug J1D00008 significantly reduced PNPLA3 mRNA at the protein translation site (i.e., the cytoplasm of hepatocytes), but the knockdown at the whole-cell level was underestimated by qPCR due to confounding from the residual nuclear PNPLA3 mRNA pool. Therefore, cytoplasmic PNPLA3 mRNA knockdown was quantified.

[0316] ISH performed on liver biopsies from cynomolgus monkeys treated with the RNAi drug J1D00008 revealed significant residual PNPLA3 mRNA in the cell nuclei, supporting the view that this pool represented a confounding knockdown result in the entire liver as evaluated by qPCR.

[0317] Quantification of ISH images showed that treatment with the RNAi drug J1D00008 reduced PNPLA3 mRNA in the cytoplasm by 44–63% over the entire 76 days (Table 30). Since GalNAc-RNAi drugs such as RNAi drug J1D00008 mediate the degradation of target mRNA, particularly in the cytoplasm of hepatocytes, the whole-liver quantification of PNPLA3 knockdown is weakened by the relative contribution of PNPLA3 mRNA from non-targeted cell types (e.g., astrocytes and Kupffer cells). Therefore, model-based analyses were performed to specifically estimate the reduction of PNPLA3 mRNA, particularly in the cytoplasm of hepatocytes, and to correct for excessive hepatocyte PNPLA3 mRNA expression. Table 31 lists the relevant assumptions about liver physiology used in the modeling.

[0318] [Table 31] The data represents the mean relative to baseline ± SD (n=4).

[0319] [Table 32]

[0320] Using this model, PNPLA3 mRNA was reduced by 81% on day 15 and 78% on day 29 in the cytoplasm of hepatocytes after administration of a single SC dose of 4 mg / kg of the RNAi drug J1D00008. Repeating this modeling in Study 2 achieved reductions of 66% and 65% on days 15 and 29, respectively. On average, across all NHPs (n=14) tested with the RNAi drug J1D00008, PNPLA3 mRNA was reduced by 70% in the cytoplasm of hepatocytes on days 15 and 29 (Figure 2).

[0321] Example 12. Safety The non-clinical safety of RNAi drug J1D00008 administration for up to 3 months was evaluated in rats and monkeys at doses of up to 500 mg / kg and 300 mg / kg, respectively. In both species, RNAi drug J1D00008 was well-tolerated, and no adverse signs of toxicity were observed up to the highest dose evaluated.

[0322] Other Embodiments Although the present invention has been described in connection with its detailed description, the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, and the scope of the invention is understood to be defined by the appended claims. Other aspects, advantages, and modifications are within the following claims. The following embodiments may be included. [1] RNAi drugs for inhibiting the expression of the PNPLA3 gene, An antisense strand containing at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any one of the sequences of sequence numbers 46-60, 176, 181, and 188, An RNAi drug comprising a sense strand having a nucleotide sequence that is at least partially complementary to the antisense strand. [2] The RNAi drug according to [1] above, wherein the antisense strand comprises nucleotides 2 to 18 of any one sequence of sequence numbers 46 to 60, 176, 181, and 188. [3] The RNAi drug according to [1] or [2], wherein the sense strand comprises a sequence of at least 17 consecutive nucleotides that differ by 0 or 1 nucleotide from any one of the sense strand sequences of SEQ ID NOs: 2, 3, 4, 9-20, 214, 219, and 220, and the sense strand has a region of at least 85% complementarity with the antisense strand over the 17 consecutive nucleotides. [4] The RNAi drug according to any one of the above [1] to [3], wherein at least one nucleotide of the RNAi drug is a modified nucleotide or includes a modified nucleoside linkage. [5] The RNAi drug according to any one of the above [1] to [3], wherein all or substantially all of the nucleotides of the sense strand and / or antisense strand of the RNAi drug are modified nucleotides. [6] The RNAi drug according to any one of the above [4] to [5], wherein the modified nucleotide is selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methylnucleotide, reverse 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinylphosphonate-containing nucleotide, cyclopropylphosphonate-containing nucleotide, and 3'-O-methylnucleotide. [7] The RNAi drug according to [5] above, wherein all or substantially all of the modified nucleotides are 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof. [8] The RNAi drug according to any one of the above [1] to [7], wherein the antisense strand consists of, essentially consists of, or comprises one nucleotide sequence from among the modified antisense strand sequences of SEQ ID NOs. 90, 95, and 102. [9] The RNAi drug according to any one of the above [1] to [8], wherein the sense strand consists of, essentially consists of, or comprises any of the modified sense strand sequences of SEQ ID NOs: 131, 136, and 137.

[10] The RNAi drug according to [1] above, wherein the antisense strand comprises one nucleotide sequence from among the modified sequences of SEQ ID NOs: 90, 95, and 102, and the sense strand comprises one nucleotide sequence from among the modified sequences of SEQ ID NOs: 131, 136, and 137.

[11] The RNAi drug according to any one of the above [1] to

[10] , wherein the RNAi drug is linked to a targeted ligand.

[12] The RNAi drug according to

[11] above, wherein the targeted ligand comprises N-acetyl-galactosamine.

[13] The RNAi drug according to

[11] or

[12] above, wherein the targeted ligand comprises the structure of (NAG37) or (NAG37)s.

[14] The RNAi drug according to any one of the above

[11] to

[13] , wherein the targeted ligand is linked to the sense strand.

[15] The RNAi drug according to

[14] above, wherein the targeted ligand is ligated to the 5' end of the sense strand.

[16] The RNAi drug according to any one of the above [1] to

[15] , wherein the sense strand is 18 to 30 nucleotides long and the antisense strand is 18 to 30 nucleotides long.

[17] The RNAi drug according to

[16] , wherein the sense strand and the antisense strand are each 18 to 27 nucleotides long.

[18] The RNAi drug according to

[17] , wherein the sense strand and the antisense strand are each 18 to 24 nucleotides long.

[19] The RNAi drug according to

[18] , wherein the sense strand and the antisense strand are each 21 nucleotides long.

[20] The RNAi drug according to any one of the above

[16] to

[19] , wherein the RNAi drug has two blunt ends.

[21] The RNAi drug according to any one of the above [1] to

[20] , wherein the sense strand comprises one or two terminal caps.

[22] The RNAi drug according to any one of the above [1] to

[22] , wherein the sense strand comprises one or two reverse debase residues.

[23] The RNAi drug according to [1] above, wherein the RNAi drug comprises a sense strand and an antisense strand forming a double-stranded sequence having sequence numbers (176 and 214), (90 and 131), (181 and 219), (95 and 136), (188 and 220), and / or (102 and 137).

[24] The RNAi drug according to any one of the above [1] to

[23] , wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[25] An antisense strand comprising, consisting of, or essentially consisting of, a modified nucleotide sequence that differs from one of the nucleotide sequences of sequence numbers 90, 95, and 102 by 0 or 1 nucleotides, The RNAi drug according to [1] above, wherein a, c, g, and u each represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf each represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the sense strand are modified nucleotides.

[26] The sense strand contains, consists of, or is essentially composed of, a modified nucleotide sequence that differs from one of the nucleotide sequences of SEQ ID NOs. 214, 219, and 256 by 0 or 1 nucleotides, The RNAi drug according to [1] above, wherein a, c, g, i, and u each represent 2'-O-methyladenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf each represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the antisense strand are modified nucleotides.

[27] The RNAi drug according to any one of the above

[24] to

[26] , wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

[28] The RNAi drug according to any one of the above

[24] to

[27] , wherein the sense strand of the RNAi drug is linked to a targeted ligand.

[29] The RNAi drug according to

[28] above, wherein the targeted ligand has affinity for an asial glycoprotein receptor.

[30] The RNAi drug according to

[29] above, wherein the targeted ligand comprises N-acetyl-galactosamine.

[31] The targeted ligand is

change

[32] The antisense strand consists of the modified nucleotide sequences of SEQ ID NOs. 90, 95, and 102, and the sense strand consists of the modified nucleotide sequences of SEQ ID NOs. 131, 136, and 137. 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 linkage; (invAb) is a reverse debasing deoxyribose residue; and (NAG37)s has the following chemical structure:

change

[33] A composition comprising an RNAi drug as described in any one of the above items [1] to

[32] , further comprising a pharmaceutically acceptable excipient.

[34] A method for inhibiting the expression of the PNPLA3 gene in cells, comprising introducing an effective amount of the RNAi drug described in any one of the above [1] to

[32] or the composition described in

[33] into the cells.

[35] The method according to

[34] above, wherein the cells are located within the subject.

[36] The method according to

[35] above, wherein the subject is a human subject.

[37] The method according to any one of the above

[34] to

[36] , wherein the PNPLA3 gene expression is inhibited by at least about 30%.

[38] A method for treating a PNPLA3-related disease or disorder, comprising administering a therapeutically effective amount of the composition described in

[33] above to a human subject in need thereof.

[39] The method according to

[38] above, wherein the disease is NAFLD, NASH, hepatic fibrosis, alcoholic fatty liver disease, or cirrhosis.

[40] The method according to any one of the above

[34] to

[39] , wherein the RNAi drug is administered in a dose of approximately 0.05 mg to approximately 5.0 mg per kg of body weight of the human subject.

[41] The method according to any one of the above

[34] to

[40] , wherein the RNAi drug is administered in two or three or more doses.

[42] Use of an RNAi agent or composition described in any one of the above [1] to

[32] for the treatment of a disease, disorder, or condition that is at least partially mediated by PNPLA3 gene expression.

[43] The use described in

[42] above, wherein the symptom is cirrhosis of the liver.

[44] Use of an RNAi drug according to any one of the above [1] to

[32] or the composition according to the above

[33] for the preparation of a pharmaceutical composition for treating a disease, disorder or condition that is at least partially mediated by PNPLA3 gene expression.

[45] The use described in any one of the above paragraphs

[42] to

[44] , wherein the disease is an alcoholic or non-alcoholic liver disease such as NAFLD, NASH, hepatic fibrosis, or cirrhosis.

[46] The use described in any one of the above

[42] to

[45] , wherein the RNAi drug is administered in a dose of approximately 0.05 mg to approximately 5.0 mg per kg of body weight of the human subject.

Claims

1. RNAi drugs for inhibiting the expression of the PNPLA3 gene, below: usAfsgsUfgAfaAfaAfcUfgGfgAfaAfgGfsu (Sequence ID 90), usUfsasGfuUfaGfgUfgAfaAfaAfgGfuGfsu (SEQ ID NO: 95), and usCfsusUfgCfU UNA CfaUfgUfaUfcCfaCfcUfsg (Sequence ID 102) (Here, 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 linkage; and U UNA represents 2',3'-seco-uridine-3'-phosphate.) An antisense strand containing one modified nucleotide sequence from among the antisense strand sequences selected from, An RNAi drug comprising a sense strand having a nucleotide sequence that is at least partially complementary to the antisense strand.

2. The RNAi drug according to claim 1, wherein at least one nucleotide of the sense strand of the RNAi drug is a modified nucleotide or comprises a modified nucleoside linkage.

3. The RNAi drug according to claim 1 or 2, wherein all or substantially all of the nucleotides of the sense strand of the RNAi drug are modified nucleotides.

4. The RNAi drug according to claim 2 or 3, wherein the modified nucleotide is selected from the group consisting of 2'-O-methylnucleotide, 2'-fluoronucleotide, 2'-deoxynucleotide, 2',3'-seconucleotide mimetic, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, debasalized nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methylnucleotide, reverse 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholinonucleotide, vinylphosphonate-containing nucleotide, cyclopropylphosphonate-containing nucleotide, and 3'-O-methylnucleotide.

5. The RNAi drug according to claim 3, wherein all or substantially all of the modified nucleotides are 2'-O-methylnucleotides, 2'-fluoronucleotides, or a combination thereof.

6. The RNAi drug according to any one of claims 1 to 5, wherein the antisense strand comprises, essentially comprises, or includes the nucleotide sequence of SEQ ID NO: 95, and the sense strand comprises the nucleotide sequence of SEQ ID NO:

136.

7. The RNAi drug according to any one of claims 1 to 5, wherein the sense strand consists of, essentially consists of, or comprises any of the modified sense strand sequences of SEQ ID NOs: 131, 136, and 137.

8. The RNAi drug according to any one of claims 1 to 7, wherein the RNAi drug is linked to a targeted ligand.

9. The RNAi agent according to claim 8, wherein the targeted ligand comprises N-acetyl-galactosamine.

10. The RNAi drug according to claim 8 or 9, wherein the targeted ligand comprises the structure of (NAG37) or (NAG37)s.

11. The RNAi drug according to any one of claims 8 to 10, wherein the targeted ligand is linked to the sense strand.

12. The RNAi drug according to claim 11, wherein the targeted ligand is ligated to the 5' end of the sense strand.

13. The RNAi drug according to any one of claims 1 to 12, wherein the sense strand is 18 to 30 nucleotides long.

14. The RNAi drug according to claim 13, wherein the sense strand is 18 to 27 nucleotides long.

15. The RNAi drug according to claim 14, wherein the sense strand is 18 to 24 nucleotides long.

16. The RNAi drug according to claim 15, wherein the sense strand is 21 nucleotides long.

17. The RNAi drug according to any one of claims 13 to 16, wherein the RNAi drug has two blunt ends.

18. The RNAi drug according to any one of claims 1 to 17, wherein the sense strand comprises one or two terminal caps.

19. The RNAi drug according to any one of claims 1 to 18, wherein the sense strand comprises one or two reverse debase residues.

20. The RNAi drug according to claim 1, wherein the RNAi drug comprises a sense strand and an antisense strand forming a double-stranded sequence having sequence numbers (90 and 131), (95 and 136), and / or (102 and 137).

21. The RNAi drug according to any one of claims 1 to 20, wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

22. The sense strand contains, consists of, or is essentially composed of, a modified nucleotide sequence in which zero or one nucleotide differs from the nucleotide sequence of SEQ ID NO:

136. The RNAi drug according to claim 1, wherein a, c, g, and u each represent 2'-O-methyladenosine, cytidine, guanosine, and uridine, respectively; Af, Cf, Gf, and Uf each represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the sense strand are modified nucleotides.

23. The sense strand contains, consists of, or is essentially composed of, a modified nucleotide sequence that differs from one of the nucleotide sequences of SEQ ID NOs. 131, 136, and 137 by 0 or 1 nucleotides. The RNAi drug according to claim 1, wherein a, c, g, i, and u each represent 2'-O-methyladenosine, cytidine, guanosine, inosine, and uridine, respectively; Af, Cf, Gf, and Uf each represent 2'-fluoroadenosine, cytidine, guanosine, and uridine, respectively; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the antisense strand are modified nucleotides.

24. The RNAi drug according to any one of claims 21 to 23, wherein the sense strand further comprises a reverse debase residue at the 3' end of the nucleotide sequence, the 5' end of the nucleotide sequence, or both.

25. The RNAi drug according to any one of claims 22 to 24, wherein the sense strand of the RNAi drug is linked to a targeted ligand.

26. The RNAi drug according to claim 25, wherein the targeted ligand has affinity for an asialoclycoprotein receptor.

27. The RNAi drug according to claim 26, wherein the targeted ligand comprises N-acetyl-galactosamine.

28. The targeted ligand is 【Chemistry 1】 The RNAi drug according to claim 8, comprising:

29. The antisense strand consists of the modified nucleotide sequences of SEQ ID NOs: 90, 95, and 102, and the sense strand consists of the modified nucleotide sequences of SEQ ID NOs: 131, 136, and 137. 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 linkage; (invAb) is a reverse debasing deoxyribose residue; and (NAG37)s has the following chemical structure: 【Chemistry 2】 The RNAi drug according to claim 1, having the following characteristics.

30. A composition comprising an RNAi drug according to any one of claims 1 to 29, further comprising a pharmaceutically acceptable excipient.

31. An ex vivo method for inhibiting the expression of the PNPLA3 gene in cells, comprising introducing an effective amount of an RNAi drug according to any one of claims 1 to 29 or the composition according to claim 30 into the cells.

32. The method according to claim 31, wherein the expression of the PNPLA3 gene is inhibited by at least 30%.

33. The composition according to claim 30 for use in treating PNPLA3-related diseases or disorders.

34. The composition for use according to claim 33, wherein the disease is NAFLD, NASH, hepatic fibrosis, alcoholic fatty liver disease, or cirrhosis.

35. An RNAi agent according to any one of claims 1 to 29 or a composition according to claim 30, for use in treating a disease, disorder, or symptom that is at least partially mediated by PNPLA3 gene expression.

36. The RNAi agent or composition for use according to claim 35, wherein the aforementioned symptom is cirrhosis of the liver.

37. An RNAi agent according to any one of claims 1 to 29 or a composition according to claim 30, for use in preparing a pharmaceutical composition for treating a disease, disorder, or symptom that is at least partially mediated by PNPLA3 gene expression.

38. The RNAi agent or composition for use according to any one of claims 35 to 37, wherein the disease is NAFLD, NASH, hepatic fibrosis, or alcoholic or non-alcoholic cirrhosis.

39. The RNAi drug or composition for use according to any one of claims 35 to 38, wherein the RNAi drug or composition is prepared for administration in a dose of 0.05 mg to 5.0 mg per kg of body weight of a human subject.

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