Dsrna, use thereof and preparation method therefor

Abstract

The present invention relates to a modulator capable of modulating, for example inhibiting, the expression and / or activity of diacylglycerol O-acyltransferase 2 (DGAT2), such as a double-stranded RNA (dsRNA) active agent or an antisense polynucleotide preparation. The present invention further relates to a method for inhibiting the expression and / or activity of DGAT2 using such modulator, and a method for preventing and treating DGAT2-related diseases (such as chronic inflammatory diseases) in a subject.

Description

A dsRNA, uses thereof and methods of making

[0001] Cross-reference to Related Applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202411292200.9, filed on September 14, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to modulators, such as double-stranded RNA (dsRNA) active agents or antisense polynucleotide formulations, that can modulate, e.g., inhibit, the expression and / or activity of diacylglycerol O-acyltransferase 2 (DGAT2). The present invention also relates to methods of using such modulators to inhibit the expression and / or activity of DGAT2, as well as methods of preventing and treating DGAT2-associated diseases, such as chronic inflammation, in a subject.

[0004] BACKGROUND

[0005] DGAT2 (diacylglycerol O-acyltransferase 2) is an enzyme that is widely present in eukaryotes, belonging to the evolutionarily conserved acyltransferase gene family. DGAT2 is a member of the DGAT family, together with DGAT1, which catalyzes the formation of triacylglycerol from diacylglycerol plus fatty acyl-CoA, which is an important form of energy storage in eukaryotic cells. DGAT2 has different localization in different organelles, which can interact with different pools of fatty acids, thereby playing different functions in lipid metabolism. DGAT2 is highly expressed in tissues with strong triglyceride synthesis and storage capacity (adipose tissue, liver, small intestine, and mammary gland), and activation of DGAT2 leads to accumulation of lipid droplets in cells, and accumulation of lipid droplets in the liver, thereby causing inflammation.

[0006] Therefore, DGAT2 can be an effective target for treating metabolic dysfunction-related steatohepatitis. DGAT2 inhibitors have shown consistent efficacy in preclinical studies and good tolerability in early clinical studies. In the treatment of non-alcoholic fatty liver disease (MAFLD) and non-alcoholic steatohepatitis (MASH), DGAT2 inhibitors such as Ervogastat (PF-06865571) show potential for reducing liver fat and have good safety and tolerability. Studies have shown that DGAT2 inhibitors can reduce triglyceride synthesis in the liver and may play a role by reducing the expression of genes associated with lipogenesis. In addition, the role of DGAT2 in tumor progression has also been of interest, as it can promote tumor development through fatty acid metabolism.

[0007] There is still a need for inhibitors that have better activity to effectively modulate the expression and / or activity of DGAT2 for treating metabolic dysfunction-related inflammation, such as fatty liver disease, etc. SUMMARY

[0008] The present application provides specific RNA interference (RNAi) active agents that can effectively reduce the expression and / or activity of DGAT2.

[0009] The RNAi active agents of the present application can affect the cleavage of the mRNA of the DGAT2 gene mediated by the RNA-induced silencing complex (RISC), thereby for inhibiting the expression of DGAT2 in liver cells. By designing the dsRNA against DGAT2, the present application can specifically inhibit the expression of the DGAT2 gene (e.g., in liver tissue or cells), which helps to treat the diseases related to DAGAT2. In some embodiments, the dsRNA against DGAT2 of the present application can more effectively knock out DGAT2 (e.g., in liver tissue or cells), thereby specifically inhibiting the gene expression of DGAT2.

[0010] In some embodiments, the present application provides a double-stranded ribonucleic acid (dsRNA) active agent for inhibiting the expression of a DGAT2 target gene in a cell, such as a fat cell and / or a liver cell; wherein the dsRNA active agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19 contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the sense nucleotide sequences in Table 1, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21 contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in Table 1. In some embodiments, these dsRNA active agents further comprise one or more ligands conjugated to at least one strand of the dsRNA, e.g., via a linker, which ligand(s) is / are capable of targeted delivery of the dsRNA to liver tissue or liver cells, e.g., one or more ligands conjugated to at least one strand of the dsRNA active agent, e.g., via a linker, e.g., a bivalent or trivalent branched linker, such as a ligand targeting ASPGR, e.g., a GalNAc ligand comprising GalNAc or a derivative thereof.

[0011] The present application also provides a cell comprising a dsRNA active agent described herein. In some embodiments, the cell is a liver cell. In some embodiments, the cell is located in an individual, e.g., a human.

[0012] The present invention provides a pharmaceutical composition comprising the dsRNA activator described herein and optionally a pharmaceutically acceptable carrier.

[0013] This invention provides a pharmaceutical combination comprising the dsRNA activator described herein and one or more other therapeutic agents, which are any therapeutic agents effective, for example, in preventing or treating DGAT2-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the DGAT2 gene), covering a wide range of therapeutic agents for treating chronic inflammatory diseases.

[0014] This invention provides the use of the dsRNA activators and / or pharmaceutical compositions and / or drug combinations described herein in the preparation of medicaments for treating DGAT2-related diseases and / or conditions, such as diseases and / or conditions caused by abnormal expression of the DGAT2 gene.

[0015] This invention provides the use of the dsRNA activator and / or pharmaceutical composition and / or pharmaceutical combination described herein in the preparation of a medicament for inhibiting DGAT2 gene expression in tissues or cells, preferably inhibiting DGAT2 expression in liver tissues or hepatocytes.

[0016] The present invention provides a method for preventing or treating DGAT2-related diseases and / or conditions (e.g., diseases and / or conditions caused by abnormal expression of the DGAT2 gene), the method comprising administering an effective amount of the dsRNA activator and / or pharmaceutical composition and / or combination of drugs described herein to a subject in need.

[0017] On the other hand, the present invention provides a method for inhibiting DGAT2 gene expression, the method comprising contacting cells, preferably hepatocytes, with an effective amount of the dsRNA activator and / or pharmaceutical composition and / or drug combination described herein, optionally maintaining the cells produced in this step for a period of time sufficient to degrade the mRNA transcript of the DGAT2 gene, thereby inhibiting the expression of the DGAT2 gene in cells such as hepatocytes. Attached Figure Description

[0018] Figure 1 shows the results of repeated single-dose screening of the dsRNA drug targeting DGAT2 in Hep3B cells, i.e., single-point inhibition of DGAT2 mRNA after transfection of the siRNA molecule of the present invention in Hep3B cells.

[0019] Figure 2 shows the results of multi-dose screening of dsRNA drugs targeting DGAT2 in Hep3B cells, i.e., the normalized inhibition of DGAT2 mRNA after transfection of the siRNA molecule of the present invention in Hep3B cells.

[0020] Figure 3 shows the inhibition of DGAT2 mRNA after transfection of siRNA molecules of the application in human primary hepatocytes.

[0021] Figure 4 shows the inhibition of DGAT2 mRNA after transfection of siRNA molecules of the application in monkey primary hepatocytes.

[0022] Figure 5 shows the results of a multiple dose screening of a dsRNA drug against DGAT2, i.e. the normalized inhibition of DGAT2 mRNA after transfection of siRNA molecules of the application in Hepa-1c1c7 cells.

[0023] Figure 6 shows the single dose validation of a GalNac-siRNA drug against DGAT2 in C57BL / 6N mice, i.e. the inhibition of DGAT2 mRNA after administration of siRNA molecules of the application in C57BL / 6N mice.

[0024] Figure 7 shows the inhibition of DGAT2 mRNA after administration of siRNA molecules of the application in C57BL / 6N mice.

[0025] Figure 8 shows the inhibition of DGAT2 mRNA after administration of siRNA molecules of the application in C57 mice (top panel); and the results of modeled liver Oil red stain (bottom panel).

[0026] Figure 9 shows the inhibition of DGAT2 mRNA after administration of siRNA molecules of the application in ob / ob mice.

[0027] Figure 10 shows the liver triglyceride levels after administration of siRNA molecules of the application in ob / ob mice at 6 and 12 weeks.

[0028] Figure 11 shows the blood biochemistry data AST / ALT / TC expression after administration of siRNA molecules of the application in ob / ob mice at 12 weeks.

[0029] Figure 12 shows the inhibition of DGAT2 mRNA after administration of siRNA molecules of the application in DGAT2 KI mice.

[0030] DETAILED DESCRIPTION

[0031] Before the present application is described in detail below, it is to be understood that the application is not limited to the particular methodology, protocols and reagents described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims.

[0032] I. DEFINITIONS

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0034] To interpret the description, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] The term "about" or "approximately," when used in connection with a numerical value, means within a range of plus or minus 1%, 2%, 3%, 4%, or 5% of the specified numerical value. It is to be understood that the specific value to which the term "about" or "approximately" is directed is itself also specifically and preferably disclosed.

[0036] As used herein, the term "and / or" means any one or any combination of the items in the list of items.

[0037] As used herein, the term "comprises" or "comprising" means including, but not excluding the elements, integers or steps encompassed within the compositions, integers or steps of the specific sequences. In this connection, the term "comprises" or "comprising" is to be construed as meaning "consisting at least in part of the stated elements, integers or steps.

[0038] As used herein, a "modulator" is a molecule that reduces or increases the expression and / or activity of DGAT2. Preferably, the modulator is a molecule that reduces or inhibits the expression and / or activity of DGAT2, such as an RNAi agent, such as a dsRNA agent.

[0039] The term "DGAT2" or "diacylglycerol O-acyltransferase 2" also known as "acyl-CoA retinol O-fatty-acyltransferase" or "Retinol O-fatty-acyltransferase" refers to a known gene encoding a DGAT2 protein from any vertebrate or mammalian source, including but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless otherwise indicated. The term also refers to fragments and variants of native DGAT2 that maintain at least one in vivo or in vitro activity of the native DGAT2. DGAT2, together with DGAT1, catalyzes the formation of triacylglycerol from diacylglycerol plus fatty acyl-CoA, an important form of energy storage in eukaryotic cells. The role of DGAT2 in lipid metabolism is not limited to TAG synthesis, but can also be involved in lipoprotein assembly and regulation of plasma triacylglycerol concentration. As used herein, the term "DGAT2" also refers to a particular polypeptide expressed in a cell by a naturally occurring DNA sequence variation of the DGAT2 gene, such as a single nucleotide polymorphism in the DGAT2 gene. Numerous single nucleotide polymorphisms (SNPs) within the DGAT2 gene have been identified and can be found, for example, at NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov / snp).

[0040] An exemplary nucleotide sequence and amino acid sequence of DGAT2 can be found, for example, in GenBank Accession No. NM_032564.5 for Homo sapiens (SEQ ID NO:342; complement SEQ ID NO:343). Additional examples of DGAT2 mRNA sequences can be readily obtained using publicly available databases (e.g., GenBank, UniProt, and OMIM). Further information on DGAT2 is provided, for example, in the NCBI Gene database at http: / / www.ncbi.nlm.nih.gov / gene. In some embodiments, a dsRNA that is substantially complementary to a region of mouse or rat DGAT2 mRNA cross-reacts with human DGAT2 mRNA and is a potential candidate for human targeting.

[0041] NM_032564.5 (SEQ ID NO:342)

[0042] NM_032564.5 (complement SEQ ID NO:343)

[0043] As used herein, a "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a DGAT2 gene, including mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for directed cleavage by a dsRNA at or near that portion of the nucleotide sequence of an mRNA molecule formed during transcription of a DGAT2 gene. For example, the target sequence can be, e.g., 15-36 nucleotides ("nt") in length, or any sub-length therebetween. By way of non-limiting example, the target sequence can be 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 18 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19 nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30 nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, or 21-22 nt, 21 nt, 22 nt, or 23 nt in length. In some embodiments according to the application, the target sequence is preferably at least 18, 19, 20, or 21 nucleotides in length. In some embodiments according to the application, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments according to the application, the target sequence is about 20 or 21 nucleotides in length.

[0044] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as the base, respectively, and encompass both natural nucleotides and modified nucleotides, unless otherwise specified. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, or surrogate replacement moieties having alternative moieties. It is well understood by those skilled in the art that guanine, cytosine, adenine, and uracil can be replaced with other moieties without materially altering the base pairing properties of an oligonucleotide comprising nucleotides containing such surrogate moieties. For example, and without limitation, a nucleotide comprising inosine (a nucleoside compound formed by the combination of hypoxanthine with ribose) as its base can base pair with a nucleotide comprising adenine, cytosine, or uracil. Thus, in the nucleotide sequences of the dsRNA features of the present application, nucleotides comprising uracil, guanine, or adenine can be replaced with nucleotides comprising, for example, inosine. In another example, adenine and cytosine at any position in an oligonucleotide can be replaced with guanine and uracil, respectively, to form a G-U wobble base pair with a target mRNA. Sequences comprising such surrogate moieties are suitable for use in the compositions and methods of the features of the present application.

[0045] When reference is made herein to a "nucleotide sequence," it refers to a contiguous stretch of nucleotides, wherein the nucleotides can be natural nucleotides or modified nucleotides.

[0046] The terms "dsRNA," "dsRNA active agent," "double-stranded RNA," and "double-stranded RNA molecule," as used interchangeably herein, refer to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientation with respect to a target RNA (i.e., a DGAT2 gene). In some embodiments of the present application, double-stranded RNA (dsRNA) triggers the degradation of a target RNA (e.g., an mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. In some embodiments, the dsRNA of the present application is a small interfering RNA (siRNA). In some embodiments, reference to a dsRNA active agent herein can also include a ligand attached to the duplex structure that aids in the delivery of the dsRNA to a target tissue or target cell.

[0047] The term "siRNA" herein is a class of double-stranded RNA molecules that can mediate the silencing of target RNA (e.g., mRNA, transcript of a gene encoding a protein) that is complementary to it. siRNAs are typically double-stranded, comprising an antisense strand that is complementary to a target RNA, and a sense strand that is complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such gene is also referred to as target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene.

[0048] The term "antisense strand" or "guide strand" refers to one oligonucleotide strand of a dsRNA that comprises a region of substantial complementarity to a target sequence (e.g., a DGAT2 mRNA).

[0049] As used herein, the term "sense strand" or "passenger strand" or "sense strand" refers to an oligonucleotide strand that comprises a region of substantial complementarity to an antisense strand as defined herein, which region of substantial complementarity is complementary to the corresponding region of the antisense strand to form a duplex region (double-stranded region) of a dsRNA.

[0050] In the present context, the term "complementary" or "complementarity" means, if not otherwise specified, the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising a second nucleotide sequence, under certain conditions, to form a duplex structure. The person skilled in the art is able to determine the conditions most suitable for the complementarity of two sequences and for the determination of this complementarity depending on the final application purpose of the hybridizing oligonucleotide or polynucleotide. Thus, in the present context, the term "complementary" or "complementarity" when used to describe the base pairing between the sense strand sequence and the antisense strand sequence of an RNAi or dsRNA, or the base pairing between the antisense strand sequence of an RNAi or dsRNA and a target sequence, is to be understood as encompassing not only the case of 100% complementarity (i.e. perfect complementarity), but also the case of less than 100% complementarity (i.e. substantial complementarity), i.e. the presence of base mismatches in the region of the duplex formed by complementarity which do not materially affect the ability of said RNAi or dsRNA to fulfill its intended function. As the person skilled in the art knows, in a duplex nucleic acid molecule, the bases on one strand are considered to be "complementarily paired" or "matched" to each other at a position, when the base on one strand forms a Watson-Crick base pair in a complementary manner with the base at the corresponding position on the other strand. For example, the purine base adenine (A) is complementarily paired to the pyrimidine base thymine (T) or uracil (U); the purine base guanine (C) is complementarily paired to the pyrimidine base cytosine (G). Accordingly, a "mismatch" means that the base on one strand at a position is not complementarily paired to the base at the corresponding position on the other strand in a duplex nucleic acid. However, it is to be understood that a nucleoside having a modification in the base moiety of the RNA nucleoside, if allowing to maintain Watson Crick base pairing, is to be considered complementarily paired as well. Thus, in the present context, nucleoside base "complementarity" encompasses Watson Crick base pairing between unmodified and modified nucleobases (see e.g. Hirao et al. (2012) Accounts of Chemical Research vol. 45 p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0051] In the context of the present application, the expression "complementary" or "complementarity" in relation to a double-stranded RNAi agent (e.g., a dsRNA such as an siRNA as described herein) preferably is no less than 70%, i.e., there are no less than 70% of the base positions in the duplex region formed by complementary hybridization that are complementary base-paired, i.e., the number of positions in the contiguous nucleotide sequence forming the duplex region in which a mismatch occurs is no more than 30%. For example, for a 21 base pair duplex region, no less than 70% complementarity means that the duplex forms no more than 6, 5, 4, 3, 2, 1, or 0 mismatched base pairs upon hybridization. Preferably, the presence of insertions and deletions is not allowed when calculating the complementarity of a contiguous nucleotide sequence in a duplex region. Accordingly, in the context of the present application, the expression "complementary (antisense strand) sequence" in relation to an RNAi agent, or "complementary (sense strand) sequence" in relation to a portion of an antisense strand, can be "fully complementary" or "substantially complementary". "Fully complementary" means that the two sequences have 100% complementarity. When a first sequence is referred to as being "substantially complementary" to a second sequence in the context of the present application, the two sequences can contain one or more, but typically no more than 30%, 20%, or 10% mismatched base pairs in a duplex formed upon hybridization, and still retain the ability to hybridize under conditions most relevant to their ultimate application (e.g., inhibition of gene expression via the RISC pathway).

[0052] "Fully complementary" as used herein means that the two strands are fully complementary to each other. It will be understood that when referring to a region of complementarity or a duplex region being fully complementary, this refers to two nucleotide strands of the same length that are fully complementary when aligned. It will thus be understood that when determining complementarity, such overhangs will not be considered mismatches when the two oligonucleotides of an RNAi or dsRNA are designed to form one or more single-stranded overhangs during hybridization. For example, for the purposes described herein, an RNAi comprising one sense oligonucleotide strand of 19 nucleotides in length and another antisense oligonucleotide strand of 21 nucleotides in length can still be considered "fully complementary" if the longer antisense oligonucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter sense oligonucleotide.

[0053] As used herein, the term "region of complementarity" refers to a region on the antisense strand that is complementary (substantially complementary or fully complementary) to a sequence as defined herein (e.g., a target sequence, e.g., a DGAT2 mRNA target sequence). Where the region of complementarity is not fully complementary (substantially complementary) to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerable mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' or 3' end of the dsRNA, e.g., the first nucleotide at the 5' end of the antisense strand can tolerate a mismatch. In some embodiments, the double stranded RNA active of the application comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA active of the application comprises no more than 4 mismatches to the target mRNA, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches to the target mRNA. In some embodiments, the antisense strand of the double stranded RNA active of the application is mismatched at the nucleotide at the 5' end to the target mRNA, e.g., the antisense strand of the double stranded RNA active of the application is U at the 5' end, without regard to whether the 3' end of the target mRNA is A, which pairs with U. In some embodiments, the antisense strand of the double stranded RNA active of the application is no more than 4 mismatches to the sense strand, e.g., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches to the sense strand. In some embodiments, the nucleotide mismatch is within, e.g., 5, 4, 3 nucleotides from the 3' end of the antisense strand or the corresponding distance from the 5' end of the sense strand. In some embodiments, the nucleotide mismatch is within, e.g., 5, 4, 3 nucleotides from the 3' end of the sense strand or the corresponding distance from the 5' end of the antisense strand. In another embodiment, the nucleotide mismatch is at, e.g., the 3' terminal nucleotide of the sense or antisense strand.

[0054] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs the duplex structure or double-stranded region of a dsRNA. A nucleotide overhang is present, for example, when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand or vice versa. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang can comprise or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the nucleotide(s) of the overhang can be present on the 5'-end, the 3'-end, or both ends of the antisense strand or the sense strand of the siRNA. In some embodiments, the overhang is present at the 3' end of the antisense strand, e.g., is 1, 2, 3, 4, or 5 nucleotides, e.g., 2 nucleotides.

[0055] "Blunt" or "blunt end" means that there are no unpaired nucleotides at the end of the dsRNA, i.e., there is no nucleotide overhang. A "blunt" dsRNA is a dsRNA that is double-stranded over its entire length, i.e., there is no nucleotide overhang at either end of the molecule. The dsRNAs of the application encompass dsRNAs that are blunt at both the 5' end and the 3' end.

[0056] As used herein, the terms "duplex region" or "duplex" or "duplex region" are used interchangeably to refer to the double-stranded structure formed by the hybridization of the sense and antisense strands in a dsRNA.

[0057] In general, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands can also comprise one or more non-ribonucleotides or modified ribonucleotides, e.g., deoxyribonucleotides or nucleotides with chemical modifications. In addition, as used herein, a "dsRNA" can comprise ribonucleotides with chemical modifications; a dsRNA can comprise substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions, or removals to the internucleosidic linkage, sugar moiety, or nucleobase, e.g., functional groups or atoms. Modifications suitable for the active agents of the application include all types of modifications disclosed herein or known in the art.

[0058] For naturally occurring oligonucleotides, the internucleosidic linkage includes a phosphate ester group that creates a phosphodiester bond between adjacent nucleosides. In this context, the term "modified internucleosidic linkage" is defined as a linkage that covalently links two nucleosides together other than a phosphodiester (PO) linkage. The nucleotide strands of the RNAi according to the application can comprise one or more internucleosidic linkages modified from the natural phosphodiester. Modifications to the internucleosidic linkage that can be contemplated according to the application include, but are not limited to: phosphorothioate linkages, phosphorodithioate linkages, methylphosphonate linkages, selenophosphonate linkages, phosphoramidate linkages, and the like. In some embodiments, the modified internucleosidic linkage in the oligonucleotides for the RNAi of the application is a phosphorothioate linkage.

[0059] As used herein, a "ligand moiety" refers to a chemical moiety conjugated to the duplex of a dsRNA that is capable of altering the distribution, targeting, or half-life of the dsRNA. References herein to "dsRNA" or "dsRNA active agent" encompass a dsRNA comprising a ligand moiety, unless the context indicates to the contrary. References herein to "siRNA" or "siRNA active agent" encompass a siRNA comprising a ligand moiety, unless the context indicates to the contrary. In some embodiments of the application, the ligand moiety is a "GalNAc ligand." As used herein, a "GalNAc ligand" refers to an asialoglycoprotein receptor (ASGPR) ligand comprising an N-acetylgalactosamine (GalNAc) or derivative moiety thereof. The term encompasses monovalent, bivalent, trivalent, tetravalent, and multivalent GalNAc ligands providing 1, 2, 3, 4, or more GalNAc or GalNAc derivative moieties. As used herein, a siRNA comprising a GalNAc or GalNAc derivative moiety is also denoted as a "GalNAc-siRNA."

[0060] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," and other similar terms, and includes inhibition at any level.

[0061] References herein to the nucleotide sequence comprised by the sense strand and / or the antisense strand of a dsRNA encompass a nucleotide sequence conjugated with a ligand moiety, unless the context indicates to the contrary.

[0062] The expression "inhibit DGAT2" refers to inhibiting the activity or expression of any DGAT2. The expression "inhibit expression of DGAT2" refers to inhibiting the expression of any DGAT2 gene as well as variants or mutants of the DGAT2 gene. Thus, the DGAT2 gene can be a wild-type DGAT2 gene, a mutant DGAT2 gene, or a transgenic DGAT2 gene in the context of a genetically manipulated cell, group of cells, or organism.

[0063] "Inhibiting DGAT2 gene expression" includes any level of inhibition of a DGAT2 gene, such as at least partial suppression of DGAT2 gene expression. DGAT2 gene expression can be assessed based on the level or change in level of any variable associated with DGAT2 gene expression, such as DGAT2 mRNA levels or DGAT2 protein levels. This level can be assessed in an individual cell or in a group of cells, including, for example, a sample derived from an individual. Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with DGAT2 expression as compared to a control level. The control level can be any type of control level utilized in the art, such as a pre-dosing baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated individual, cell, or sample.

[0064] The term "DGAT2-associated disease or disorder" as used herein refers to a disease or disorder that results from or is associated with abnormal expression and / or activity of DGAT2. The term "DGAT2-associated disease or disorder" includes a disease or disorder that results from abnormal expression of a DGAT2 gene, or a disease, disorder, or condition that can benefit from a decrease in DGAT2 gene expression, replication, or protein activity. In some embodiments, the DGAT2-associated disease or disorder is a chronic inflammatory disease. A "chronic inflammatory disease" is any disease, disorder, or condition associated with chronic inflammation. Non-limiting examples of chronic inflammatory diseases include, for example, inflammation of the liver and / or other tissues. In some embodiments, the DGAT2-associated disease or disorder is inflammation associated with metabolic dysfunction, such as steatohepatitis.

[0065] The term "effective amount" refers to the amount or dose of a dsRNA active agent or composition or combination of the application, which, when administered to a patient at one or more dosages, results in an intended effect in the patient in need of treatment or prevention. Depending on the intended effect, "therapeutically effective amount" and "prophylactically effective amount" can be included.

[0066] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the dsRNA active agent or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter by at least about 30%, even more preferably by at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0067] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than a therapeutically effective amount.

[0068] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such a cell.

[0069] An "individual" or "subject" can be used interchangeably herein and includes a mammal. Mammals include, but are not limited to, domestic and farm animals (e.g., cows, sheep, pigs, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0070] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., with which the active ingredient is administered.

[0071] The term "pharmaceutical composition" refers to a composition that is in a form suitable for administration into a subject and that does not contain additional ingredients detrimental to the active ingredients contained therein. In some embodiments, when referring to a "pharmaceutical composition," it also encompasses a pharmaceutical preparation formulated as a formulation or article.

[0072] The term "pharmaceutical combination" refers to a non-fixed combination or a fixed combination, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) a dsRNA active agent of the present application, and (ii) another therapeutic agent) are administered to the patient as separate entities either simultaneously, without specific time limitations, or sequentially with no specific time limits between the administration of the individual components. In some embodiments, the dsRNA active agent of the present application and the other therapeutic agent used in the pharmaceutical combination are administered at levels that would not be effective without co-administration of the other. The term "fixed combination" means that the two or more active ingredients are administered in a single entity. Preferably, the dosage of the two or more active ingredients and / or the time interval between the administration of the individual components are selected so that the combined use of the components results in an effect that is greater than the effect of using any of the components alone in the treatment of the disease or condition. The components can each be in the form of a separate formulation, which formulations can be the same or different. It will be appreciated that the individual components of the pharmaceutical combination need not be administered in the same pharmaceutically acceptable carrier, but can be administered in separate pharmaceutical compositions or pharmaceutical preparations.

[0073] The term "combination therapy" refers to the administration of two or more therapeutic agents, or treatment modalities, to treat a disease described herein. Such administration

[0074] As used herein, the term "other therapeutic agent" is any therapeutic agent useful in conjunction with the dsRNA active agents described herein for the prevention or treatment of a disease described herein, e.g., any therapeutic agent useful in the prevention or treatment of a DGAT2-associated disease and / or disorder, encompassing various therapeutic agents useful in the treatment of chronic inflammatory diseases.

[0075] As used herein, "treatment" means slowing, interrupting, arresting, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0076] As used herein, "prevention" includes inhibition of the onset or progression of a disease or disorder or condition or symptoms of a particular disease or disorder or condition.

[0077] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector associated with a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0078] "Subject / patient / individual sample" refers to a collection of cells or fluid obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from a fresh, frozen and / or preserved organ or tissue sample or biopsy or puncture sample; blood or any blood component; a bodily fluid, such as cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), or interstitial fluid; a cell from a subject at any time of gestation or development. The tissue sample can contain compounds not naturally admixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. In some embodiments, the subject sample described herein is tissue, e.g., liver tissue such as liver tissue homogenate, or is a liver cell.

[0079] In the present text, "optionally" or "optional" means that the event or circumstance subsequently described can or can not occur, and that the description includes the situation where the event or circumstance occurs and the situation where it does not. For example, "optionally substituted" "alkyl" includes "alkyl" as defined below and "substituted alkyl". It will be understood by those skilled in the art that for any group containing one or more substituents, such groups do not intend to introduce any substitution or substitution pattern that is spatially impractical, synthetically non-feasible, and / or inherently unstable.

[0080] In the present text, "alkyl" means straight-chained and branched, having the specified number of carbon atoms, which can be 1 to 30 carbon atoms, for example 1 to 20 carbon atoms, 1 to 16 carbon atoms, 12 to 16 carbon atoms. When referring to an alkyl residue having a specific number of carbons, all branched and straight chain forms having that number of carbons are intended to be encompassed, and optionally substituted. When an alkyl group is substituted with one or more hydroxyl groups, it can be referred to as a hydroxyalkyl group. In particular, the term "hydroxyethyl" refers to an ethyl group substituted with one hydroxyl group.

[0081] Correspondingly, the term "alkylene" denotes a divalent radical derived from an alkane, for example a straight-chain or branched alkane having the specified number of carbon atoms, which can be, for example, 0 to 30 carbon atoms. It is understood that when the number of carbon atoms is 0, i.e. C0alkylene, it denotes a valence bond. Examples thereof include C 0-20 alkylene, C 0-12 alkylene, C 0-8 alkylene, C 0-6 alkylene, etc.

[0082] In the present text, "alkenyl" means a straight-chain or branched, unsaturated hydrocarbon group containing at least one double bond, having the specified number of carbon atoms, which can be, for example, 2 to 30 carbon atoms. Specifically, alkenyl groups have 2-20, for example 2-16, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and are optionally substituted. For example, as used herein, the term "C 2-20 alkenyl" means a straight-chain or branched alkenyl group having 2 to 20 carbon atoms, for example ethenyl, propenyl, allyl, 1-butenyl, 2-butenyl, 1,3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, and the like.

[0083] In the present text, "alkynyl" refers to a straight-chain or branched-chain unsaturated hydrocarbon group comprising at least one triple bond, having a specified number of carbon atoms, e.g. 2 to 30 carbon atoms. In particular, alkynyl groups have 2 to 20, e.g. 2 to 16, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms, and are optionally substituted. For example, as used herein, the term "C2-C6 alkynyl" denotes straight-chain or branched-chain alkynyl groups having 2 to 6 carbon atoms, e.g. ethynyl, propynyl, propargyl, 1 -butynyl, 2-butynyl, 1 -pentynyl, 2-pentynyl, 3-pentynyl, 4-methyl- 1 -pentynyl, 1 -hexynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0084] The terms "alkoxy" and "alkyl-O-" are used interchangeably and denote an alkyl group as defined above attached through an oxygen atom. Preferably, the alkoxy group has 1 to 20 carbon atoms (C 1-20 alkoxy), 1 to 16 carbon atoms (C 1-16 alkoxy), 1 to 6 carbon atoms (C 1-6 alkoxy), 1 to 4 carbon atoms (C 1-4 alkoxy), or 1 to 3 carbon atoms (C 1-3 alkoxy). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propyloxy (including n-propyloxy, isopropyloxy), butyloxy (including n-butyloxy, sec-butyloxy, isobutyloxy, tert-butyloxy, and the like), pentyloxy (including n-pentyloxy, isopentyloxy, neopentyloxy, and the like), hexyloxy, heptyloxy, octyloxy, and the like.

[0085] The terms "alkylthio" and "alkyl-S-" are used interchangeably and denote an alkyl group as defined above attached through a sulfur atom. Preferably, the alkylthio group has 1 to 20 carbon atoms (C 1-20 alkylthio), 1 to 16 carbon atoms (C 1-16 alkylthio), 1 to 6 carbon atoms (C 1-6 alkylthio), 1 to 4 carbon atoms (C 1-4 alkylthio), or 1 to 3 carbon atoms (C 1-3 alkylthio). Examples of alkylthio groups include, but are not limited to, methylthio, ethylthio, propylthio, pentylthio, hexylthio, heptylthio, octylthio, and the like.

[0086] The term "cycloalkyl" denotes a fully or partially saturated, non-aromatic, monocyclic or bicyclic hydrocarbon radical consisting of carbon and hydrogen atoms, preferably fully saturated. Preferably, the cycloalkyl group has 3 to 8 ring carbon atoms (C 3-8 cycloalkyl), or 5 to 6 ring carbon atoms (C 5-6Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and the like. When a cycloalkyl group is substituted with one or more hydroxyl groups, it can be referred to as a "hydroxycycloalkyl" group, including C 3-8 hydroxycycloalkyl groups include, but are not limited to, 1-hydroxycyclopropyl, 2- hydroxycyclopropyl, 3-hydroxycyclobutyl, 4-hydroxycyclopentyl, 5-hydroxycyclopentenyl, 6- hydroxycyclohexyl, 6-hydroxycyclohexenyl, 7-hydroxycycloheptyl, 8-hydroxycyclooctyl, and the like. 5-6 hydroxycycloalkyl groups include, but are not limited to, 1-hydroxycyclopropyl, 2- hydroxycyclopropyl, 3-hydroxycyclobutyl, 4-hydroxycyclopentyl, 5-hydroxycyclopentenyl, 6- hydroxycyclohexyl, 6-hydroxycyclohexenyl, 7-hydroxycycloheptyl, 8-hydroxycyclooctyl, and the like.

[0087] In this document, the term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example 6-12, carbon atoms in the ring portion, which can be substituted or unsubstituted. Preferably, the aryl group is (C6-C12)aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which can be optionally substituted with 1-4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halo, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-O-C(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, heterocyclyl, and the like. Preferably, the aryl group is optionally substituted phenyl. 10 In this document, the term "aryl" refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6-20, for example 6-12, carbon atoms in the ring portion, which can be substituted or unsubstituted. Preferably, the aryl group is (C6-C12)aryl. Non-limiting examples include phenyl, biphenyl, naphthyl, or tetrahydronaphthyl, each of which can be optionally substituted with 1-4 substituents such as alkyl, trifluoromethyl, cycloalkyl, halo, hydroxyl, alkoxy, acyl, alkyl-C(O)-O-, aryl-O-, heteroaryl-O-, amino, mercapto, alkyl-S-, aryl-S-, nitro, cyano, carboxyl, alkyl-O-C(O)-, carbamoyl, alkyl-S(O)-, sulfonyl, sulfonamido, heterocyclyl, and the like. Preferably, the aryl group is optionally substituted phenyl.

[0088] The term "-CO-" or "-C(O)-" denotes a carbonyl group. The term "-CO-" or "-C(O)-" denotes a carbonyl group.

[0089] In this document, a bond when present in a group or moiety, denotes that the group or moiety is connected to the rest of the molecule via the wavy bond, and when present in a molecule, the chiral carbon atom to which the wavy bond is attached can have any chiral configuration, e.g., R and / or S. Additionally, when a wavy line crosses a bond from a group or moiety, e.g., in In this document, a bond

[0090] II. dsRNA active agents

[0091] The present application provides RNAi active agents, e.g., dsRNA active agents, that inhibit DGAT2. In some embodiments, the dsRNA active agent is an siRNA. In certain embodiments, the siRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of a DGAT2 gene in a cell, e.g., a liver cell, in a subject, e.g., a mammal, such as a human susceptible to or afflicted with a DGAT2-associated disease or disorder.

[0092] The endogenous RNAi (RNA interference) mechanism in organisms generally involves a series of processes, including: Dicer processing long dsRNA into short 19-21 base pair (bp) siRNAs; siRNAs binding to Ago proteins to form RNA-induced silencing complex (RISC), Ago proteins cleaving the sense strand of siRNAs and releasing it; and then the antisense strand-bound mature RISC cleaving the mRNA reverse complementary to the antisense strand through sequence complementary mechanism. According to this RNA interference mechanism, various artificial RNAi molecules with different structures have been developed, which can step into the RNAi pathway at different stages to achieve sequence-specific cleavage of target gene transcripts. See, e.g., Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211 (incorporated by reference in its entirety). Artificial RNAi molecules with such structures include, for example, siRNA molecules with duplex regions (and optionally one or two overhangs), long-chain siRNA molecules that can be Dicer enzyme substrates, short hairpin RNAs (shRNAs) that can be processed by Dicer to produce siRNA structures, and long single-stranded siRNA molecules containing only antisense strands. It can be understood that these molecular forms all fall within the scope of the RNAi active agents of the present application.

[0093] In some embodiments, the dsRNA active agents, e.g., siRNAs, of the present application (encompassing siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands) inhibit expression of a DGAT2 gene (e.g., a human DGAT2 gene) (e.g., in a cell, e.g., in a liver cell, e.g., in a human or monkey primary hepatocyte or hepatoma cell) by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%, as determined by, e.g., a method of PCR or by a protein-based method (such as by immunofluorescence analysis using, e.g., Western blot or flow cytometry techniques). In certain embodiments, the inhibition of expression is determined by the qPCR methods provided herein in a suitable biological cell, e.g., a liver cell, with a dsRNA, e.g., an siRNA, at a concentration of, e.g., about 10 nM, about 1 nM, or about 0.1 nM. In certain embodiments, the inhibition of expression is determined by the qPCR methods provided herein in a suitable biological cell line with a gradient dilution concentration of a dsRNA, e.g., an siRNA.

[0094] In some embodiments, the dsRNA active agents, e.g., siRNAs, of the present disclosure (encompassing siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands) inhibit the expression of a DGAT2 gene (e.g., a human DGAT2 gene) in vivo (e.g., in liver tissue, e.g., in mouse liver tissue) by at least about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%, particularly at low dose administration, e.g., as detected by a single subcutaneous administration to a mouse (e.g., at low dose 1 mpk, 3 mpk, 5 mpk, or 10 mpk subcutaneous administration), e.g., as described in the methods of Examples 8-12.

[0095] In some embodiments, the dsRNA active agents, e.g., siRNAs, of the present disclosure (encompassing siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands) have a long-lasting inhibitory effect on the target gene DGAT2, particularly at low dose administration, e.g., still have an inhibitory effect on the target gene DGAT2 at 4 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 11 weeks, or 12 weeks after low dose 1 mpk, 3 mpk, 5 mpk, or 10 mpk subcutaneous administration.

[0096] In some embodiments, the dsRNA active agents, e.g., siRNAs, of the present disclosure (encompassing siRNAs with modified nucleotides and siRNAs with modified nucleotides and ligands) achieve therapeutic effects in non-obese MASH models and obese mouse MASH models (e.g., leptin-deficient obese mouse MASH models), e.g., inhibit the expression of DGAT2 in liver tissue and have a long-lasting effect, effectively modulate liver triglyceride levels, and / or effectively down-regulate the expression of aspartate aminotransferase AST, down-regulate the expression of alanine aminotransferase ALT, and / or down-regulate total cholesterol TC in blood, e.g., serum.

[0097] In some embodiments, the dsRNA active agent comprises an antisense strand comprising a region of complementarity (substantially complementary or fully complementary) to at least a portion of a mRNA formed in the expression of a DGAT2 gene, e.g., a target sequence. In some embodiments, the region of complementarity is about 15 to 30 nucleotides in length, e.g., 16 to 30 nucleotides, 17 to 30 nucleotides, or 18 to 30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length). In some embodiments, the region of complementarity is between 18 and 23 nucleotides in length. In some embodiments, the region of complementarity is 19 to 23 nucleotides in length. In some embodiments, the region of complementarity is 18 to 21 nucleotides in length. In some embodiments, the region of complementarity is 18, 19, 20, or 21 nucleotides in length. In some embodiments, the region of complementarity is at least 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the antisense strand is complementary to the mRNA target sequence beginning with the second nucleotide from the 5' end. In some embodiments, the region of complementarity of the antisense strand comprises the second nucleotide from the 5' end to the 3rd, 2nd, or 1st nucleotide from the 3' end. In some embodiments, the region of complementarity of the antisense strand comprises all of the nucleotides of the antisense strand beginning with the second nucleotide from the 5' end. In some embodiments, the region of complementarity of the antisense strand comprises at least the 1st-16th, 2nd-16th, 1st-17th, 2nd-17th, 1st-18th, 2nd-18th, 1st-19th, 2nd-19th, 1st-20th, 2nd-20th, 1st-21st, or 2nd-21st nucleotides from the 5' end of the antisense strand. In some embodiments, the region of complementarity of the antisense strand comprises at least the 2nd-19th consecutive nucleotides from the 5' end of the antisense strand. In some embodiments, the region of complementarity of the antisense strand comprises or consists of the 2nd-19th, 2nd-20th, or 2nd-21st consecutive nucleotides from the 5' end of the antisense strand.

[0098] In some embodiments, the dsRNA comprises two RNA strands, an antisense strand and a sense strand, that are complementary and form a duplex structure (double-stranded region or duplex region) under conditions that would allow the dsRNA to hybridize.

[0099] In some embodiments, the antisense strand of the dsRNA comprises a region of complementarity (antisense strand complementary region) that is substantially complementary or fully complementary to a target sequence. Thus, the antisense strand complementary region of the dsRNA can be substantially complementary or fully complementary to the corresponding portion of the target sequence. The target sequence can be derived from the sequence of an mRNA formed during expression of a DGAT2 gene. In some embodiments, the antisense strand complementary region is substantially complementary to the target sequence, e.g., it is mismatched at 1, 2, 3, 4, or 5 nucleotides (preferably at 1 or 2 nucleotides at the 5' end and / or the 3' end, e.g., the first nucleotide at the 5' end of the antisense strand) to the target sequence. In some embodiments, the nucleotide sequence of the antisense strand complementary region is fully complementary to the target sequence.

[0100] In some embodiments, the antisense strand of the dsRNA is fully complementary to the corresponding portion of the target sequence beginning at the second nucleotide from the 5' end. In some embodiments, the antisense strand of the dsRNA is fully complementary to the corresponding portion of the target sequence beginning at the second nucleotide from the 5' end and ending at the 1st, 2nd, or 3rd nucleotide from the 3' end. In some embodiments, the antisense strand of the dsRNA is fully complementary to the corresponding portion of the target sequence beginning at the second nucleotide from the 5' end and ending at the last nucleotide. In some embodiments, the antisense strand of the dsRNA is fully complementary to the corresponding portion of the target sequence beginning at the 2nd-16th, 2nd-17th, 2nd-18th, 2nd-19th, 2nd-20th, or 2nd-21st nucleotide from the 5' end. In some embodiments, the antisense strand of the dsRNA is fully complementary to the corresponding portion of the target sequence beginning at the 2nd-19th, 2nd-20th, or 2nd-21st nucleotide from the 5' end. In some embodiments, the antisense strand of the dsRNA has the same number of nucleotides as the target sequence and is fully complementary to the target sequence except for the first nucleotide from the 5' end, wherein the first nucleotide of the antisense strand is U or A. In some embodiments, the nucleotide sequence of the antisense strand is fully complementary to the target sequence.

[0101] When reference is made herein to being fully complementary to a "corresponding portion of a target sequence," reference is made to the contiguous sequence of nucleotides in the target sequence that is fully complementary to the contiguous sequence of nucleotides in the antisense strand. For example, when the target sequence is 21 nucleotides and the contiguous nucleotides from 1-20 are fully complementary to the 2nd-21st nucleotides of the antisense strand, the "corresponding portion of the target sequence" refers to the contiguous nucleotides from 1-20 of the target sequence.

[0102] In some embodiments, the dsRNAs described herein target a DGAT2 gene at or near a position in the DGAT2 genome (e.g., NM_032564.5) shown in Table 1. In some embodiments, the target sequence comprises or consists of a contiguous sequence of a DGAT2 mRNA at or near a position in the DGAT2 genome (e.g., NM_032564.5) shown in Table 1.

[0103] In some embodiments, the dsRNA described herein targets any 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleotides, e.g., 19-23 contiguous nucleotides, of the nucleic acid sequence set forth in SEQ ID NO: 342 or the complement thereof, e.g., the sequence corresponding to nucleotides 663-683, 664-684, 667-687, 668-688, 670-690, 671-691, 672-692, 674-694, 675-695, 676-696, 678-698, 895-915, 897-917, 899-919, 901-921, 902-922, 903-923, 906-926, 907-927, 950-970, 1102-1122, 1458-1478, 1459-1479, 1460-1480, 1461-1481, 1462-1482, 379-399, 391-411, 405-425, 407-427, 475-495, 490-510, 494-514, 520-540, 541-561, 609-629, 725-745, 745-765, 754-774, 760-780, 824-844, 863-883, 869-889, 873-893, 883-903, 1043-1063, 1048-1068, 1055-1075, 1061-1081, 1114-1134, 1119-1139, 1124-1144, 1132-1152, 1133-1153, 1207-1227, 1277-1297, 1313-1333, 1502-1522, 1514-1534, 1580-1600, 1745-1765, 2054-2074, 2059-2079, 2063-2083, 2086-2106, 2215-2235, or 2219-2239 of the nucleic acid sequence set forth in SEQ ID NO: 342.

[0104] In some embodiments, the DGAT2 gene target sequence is selected from the group consisting of

[0105] (i) a contiguous sequence of a DGAT2 mRNA at or near (e.g., within 10 nucleotides before and after) a position of the DGAT2 genome (e.g., NM_032564.5) as shown in Table 1, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleotides at or near the position;

[0106] (ii) at least 15-35 contiguous nucleotides, e.g., 18, 19, 20, or 21 contiguous nucleotides, starting at position 663, 664, 667, 668, 670, 671, 672, 674, 675, 676, 678, 895, 897, 899, 901, 902, 903, 906, 907, 950, 1102, 1458, 1459, 1460, 1461, 1462, 379, 391, 405, 407, 475, 490, 494, 520, 541, 609, 725, 745, 754, 760, 824, 863, 869, 873, 883, 1043, 1048, 1055, 1061, 1114, 1119, 1124, 1132, 1133, 1207, 1277, 1313, 1502, 1514, 1580, 1745, 2054, 2059, 2063, 2086, 2215, or 2219 of a nucleic acid sequence set forth in the DGAT2 genome (e.g., NM_032564.5) or SEQ ID NO:342, or the complement thereof;

[0107] (iii) 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleotides of the sequence corresponding to positions 663-683, 664-684, 667-687, 668-688, 670-690, 671-691, 672-692, 674-694, 675-695, 676-696, 678-698, 895-915, 897-917, 899-919, 901-921, 902-922, 903-923, 906-926, 907-927, 950-970, 1102-1122, 1458-1478, 1459-1479, 1460-1480, 1461-1481, 1462-1482, 379-399, 391-411, 405-425, 407-427, 475-495, 490-510, 494-514, 520-540, 541-561, 609-629, 725-745, 745-765, 754-774, 760-780, 824-844, 863-883, 869-889, 873-893, 883-903, 1043-1063, 1048-1068, 1055-1075, 1061-1081, 1114-1134, 1119-1139, 1124-1144, 1132-1152, 1133-1153, 1207-1227, 1277-1297, 1313-1333, 1502-1522, 1514-1534, 1580-1600, 1745-1765, 2054-2074, 2059-2079, 2063-2083, 2086-2106, 2215-2235, or 2219-2239 in the genomic sequence of DGAT2 (e.g., NM_032564.5) or the nucleic acid sequence set forth in SEQ ID NO:342;

[0108] (iv) any 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the sequence corresponding to positions 663-683, 664-684, 667-687, 668-688, 670-690, 671-691, 672-692, 674-694, 675-695, 676-696, 678-698, 895-915, 897-917, 899-919, 901-921, 902-922, 903-923, 906-926, 907-927, 950-970, 1102-1122, 1458-1478, 1459-1479, 1460-1480, 1461-1481, 1462-1482, 379-399, 391-411, 405-425, 407-427, 475-495, 490-510, 494-514, 520-540, 541-561, 609-629, 725-745, 745-765, 754-774, 760-780, 824-844, 863-883, 869-889, 873-893, 883-903, 1043-1063, 1048-1068, 1055-1075, 1061-1081, 1114-1134, 1119-1139, 1124-1144, 1132-1152, 1133-1153, 1207-1227, 1277-1297, 1313-1333, 1502-1522, 1514-1534, 1580-1600, 1745-1765, 2054-2074, 2059-2079, 2063-2083, 2086-2106, 2215-2235, or 2219-2239 of the genomic (e.g., NM_032564.5) or nucleic acid sequence set forth in SEQ ID NO:342, preferably the nucleotide sequence corresponding to said positions; or

[0109] (v) a nucleotide sequence comprising or consisting of the nucleotide sequence set forth in any one of SEQ ID NOs: 135-201.

[0110] In some embodiments, the mRNA sequence targeted by the dsRNAs described herein (i.e., the DGAT2 gene target sequence) comprises or consists of the target sequence corresponding to a position in the DGAT2 mRNA set forth in Table 1. In some embodiments, the mRNA sequence targeted by the dsRNAs described herein (i.e., the DGAT2 gene target sequence) comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 135-201.

[0111] In some embodiments, an antisense strand of a dsRNA disclosed herein comprises a region of complementarity that is fully, substantially, or at least partially complementary to a target sequence corresponding to a location of a DGAT2 genomic sequence (e.g., NM_032564.5) set forth in Table 1 or a target sequence disclosed in Table 2 or a target sequence described herein. In some embodiments, an antisense strand of a dsRNA is identical in number of nucleotides or differs by 1, 2, or 3 nucleotides in number to a DGAT2 gene target sequence (e.g., a target sequence corresponding to a location set forth in Table 1 or a target sequence set forth in Table 2 or a target sequence described herein). In some embodiments, an antisense strand of a dsRNA is fully complementary to a DGAT2 gene target sequence (e.g., a target sequence corresponding to a location set forth in Table 1 or a target sequence set forth in Table 2 or a target sequence described herein). In some embodiments, an antisense strand comprises 1, 2, 3, 4, or 5 non-complementary sites (mismatches), e.g., mismatches at 1-3 nucleotides, to the target sequence. In some embodiments, an antisense strand of a dsRNA is fully complementary to a DGAT2 gene target sequence (e.g., a target sequence corresponding to a location set forth in Table 1 or a target sequence set forth in Table 2 or a target sequence described herein) except for 1 or 2 or 3 nucleotides. In some embodiments, an antisense strand of a dsRNA is fully complementary to a region of a DGAT2 gene target sequence (e.g., a target sequence corresponding to a location set forth in Table 1 or a target sequence set forth in Table 2 or a target sequence described herein) except for the first or second nucleotide from the 5' end to a region of the target sequence except for the first or second nucleotide from the 3' end. In some embodiments, an antisense strand of a dsRNA is fully complementary to a region of a DGAT2 gene target sequence (e.g., a target sequence corresponding to a location set forth in Table 1 or a target sequence set forth in Table 2 or a target sequence described herein) except for the first nucleotide from the 5' end to a region of the target sequence except for the first nucleotide from the 3' end. In some embodiments, an antisense strand of a dsRNA is U or A, e.g., U, at the first nucleotide from the 5' end, e.g., to more easily form a RICS complex with an Ago2 protein.

[0112] The other strand of the dsRNA (the sense strand) comprises a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure (duplex region) when combined under appropriate conditions. In some embodiments, a dsRNA comprises two RNA strands that are complementary and hybridize under conditions that would cause the dsRNA to hybridize to form a duplex structure (also referred to as a duplex region), i.e., an antisense strand and a sense strand.

[0113] In some embodiments, the sense strand and the antisense strand are each independently 15-30 nucleotides in length, for example 17-27 nucleotides, for example 19-25 nucleotides, for example 18-25 nucleotides, for example 18-24 nucleotides, for example 18-23 nucleotides, for example 19-23 nucleotides, for example 19-22 nucleotides, or for example 19-21 nucleotides. In some embodiments, the antisense strand or the sense strand is no more than 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length, independently. In some embodiments, the antisense strand or the sense strand is no less than 15, 16, 17, 18, 19, 20, or 21 nucleotides in length, independently. In some embodiments, the sense strand is 18-21 nucleotides in length (e.g., 18, 19, 20, or 21 nucleotides) and the antisense strand is 19-22 nucleotides in length (e.g., 19, 20, 21, or 22 nucleotides). In some embodiments, the sense strand is 18 or 19 nucleotides in length and the antisense strand is 19-21 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length.

[0114] In some embodiments, the antisense strand and the sense strand hybridize to form a double-stranded region. In some embodiments, the double-stranded region is 15 to 30 nucleotide pairs in length. In some embodiments, the double-stranded region is 15 to 25 nucleotide pairs or 16 to 25 nucleotide pairs in length. In some embodiments, the double-stranded region is 16 to 24 nucleotide pairs or 17 to 24 nucleotide pairs in length. In some embodiments, the double-stranded region is 17 to 23 nucleotide pairs or 18 to 23 nucleotide pairs in length. In some embodiments, the double-stranded region is 17 to 23 nucleotide pairs or 19 to 23 nucleotide pairs in length. In some embodiments, the double-stranded region is 16 to 22 nucleotide pairs, 17 to 22 nucleotide pairs, or 18 to 22 nucleotide pairs or 19 to 22 nucleotide pairs in length. In some embodiments, the double-stranded region is 16 to 21 nucleotide pairs in length, for example 16, 17, 18, 19, 20, or 21 nucleotide pairs. In some embodiments, the duplex region is 18 to 21 nucleotide pairs in length. In some embodiments, the double-stranded region is 18, 19, 20, or 21 nucleotide pairs in length, for example 19 nucleotide pairs.

[0115] In some embodiments, the double-stranded region formed by the sense and antisense strands is fully complementary. In other embodiments, the double-stranded region formed by the sense and antisense strands is substantially complementary, which can include 1, 2, 3, 4, or 5 non-complementary sites (mismatches). In some embodiments, the fully complementary double-stranded region is at least 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the fully complementary double-stranded region is between 15 and 25 nucleotide pairs, 16 and 25 nucleotide pairs, 16 and 24 nucleotide pairs, 17 and 24 nucleotide pairs, 17 and 23 nucleotide pairs, 18 and 23 nucleotide pairs, 19 and 23 nucleotide pairs, or 19 and 22 nucleotide pairs in length. In some embodiments, the fully complementary double-stranded region is 16, 17, 18, 19, 20, or 21 nucleotide pairs in length, e.g., 19 nucleotide pairs.

[0116] The dsRNAs described herein can further comprise one or more single-stranded nucleotide overhangs, e.g., 1 to 4, 2 to 4, 1 to 3, 2 to 3, 1, 2, 3, or 4 nucleotides. In some embodiments, a dsRNA having at least one nucleotide overhang has better inhibition properties relative to its blunt-ended counterpart. The nucleotide overhangs can comprise or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. The overhangs can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotides of the overhangs can be present on the 5’ end, the 3’ end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0117] In some embodiments, one or both of the sense strand and the antisense strand comprises a 3’ overhang and / or a 5’ overhang having at least 1, 2, or 3 nucleotides, e.g., one or both of the sense strand and the antisense strand comprises a 3’ overhang and / or a 5’ overhang having at least 1 nucleotide. In some embodiments, at least one strand comprises a 3’ overhang or a 5’ overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3’ overhang or a 5’ overhang of at least 2 nucleotides. In some embodiments, at least one strand comprises a 3’ overhang or a 5’ overhang of at least 3 nucleotides.

[0118] In some preferred embodiments, the antisense strand has a 3' overhang of at least 1 nucleotide and / or a 5' overhang, e.g., the antisense strand comprises a 3' overhang of 1 nucleotide and / or a 5' overhang. In some preferred embodiments, the antisense strand has a 3' overhang of at least 2 nucleotides and / or a 5' overhang, e.g., the antisense strand comprises a 3' overhang of 2 nucleotides and / or a 5' overhang. In some preferred embodiments, the antisense strand has a 3' overhang of at least 3 nucleotides and / or a 5' overhang, e.g., the antisense strand comprises a 3' overhang of 3 nucleotides and / or a 5' overhang. In preferred embodiments, the antisense strand has a 3' overhang of 1, 2, or 3 nucleotides at the 3' end, e.g., a 3' overhang of 2 nucleotides.

[0119] In some embodiments, the sense strand comprises a 5' overhang of at least 1, 2, or 3 nucleotides, and / or the antisense strand comprises a 3' overhang of at least 1, 2, or 3 nucleotides.

[0120] In some embodiments, the antisense strand of the dsRNA has an overhang at the 3' end, e.g., a 3' overhang of 2 nucleotides, and is blunt ended at the 5' end.

[0121] In some embodiments, the present application relates to a double-stranded ribonucleic acid (dsRNA) active agent for inhibiting expression of diacylglycerol O-acyltransferase 2 (DGAT2), wherein the dsRNA active agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand is fully complementary to a corresponding portion of a target sequence of a DGAT2 gene at least at positions 2-19 (e.g., 2-20 or 2-21 or full length). In some embodiments, the antisense strand is fully complementary to a target sequence of a DGAT2 gene in a region other than the first nucleoside at the 5' end, wherein the first nucleotide at the 5' end of the antisense strand is A or U, e.g., U.

[0122] In some embodiments, the dsRNA active agent of the present application comprises a sense strand and an antisense strand, wherein the sense strand comprises 19 nucleotides, the antisense strand comprises 21 nucleotides, wherein the antisense strand comprises a 3' overhang of 2 nucleotides compared to the sense strand, and wherein the sense strand is fully complementary to the antisense strand over 19 nucleotides, e.g., fully complementary over 19 consecutive nucleotides (e.g., consecutive nucleotides at positions 1-19 of the antisense strand counting from the 5' end).

[0123] In some embodiments, the dsRNA of the present application comprises a sense strand and an antisense strand forming a double-stranded region, wherein

[0124] (i) the sense strand comprises or is 19 nucleotides,

[0125] (ii) the antisense strand comprises or is 21 nucleotides and is fully complementary to a region of the target sequence of a DGAT2 gene except for the first nucleotide at the 5' end, wherein the first nucleotide of the antisense strand counting from the 5' end is A or U, e.g., U;

[0126] (iii) the antisense strand comprises a 3' overhang of 2 nucleotides compared to the sense strand, and wherein the sense strand is fully complementary to the antisense strand over 19 contiguous nucleotides, e.g., the sense strand is fully complementary to the antisense strand over nucleotides 1-19 of the antisense strand counting from the 5' end.

[0127] In one aspect of the application, the present application is directed to a double-stranded ribonucleic acid (dsRNA) active agent for inhibiting expression of diacylglycerol O-acyltransferase 2 (DGAT2), wherein the dsRNA active agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides differing by no more than 3, 2, or 1 nucleotides from any one of the nucleotide sequences of the antisense strands in Table 1, wherein the first nucleotide of the antisense strand at the 5' end is A or U, e.g., U.

[0128] In some embodiments, the dsRNA active agent comprises a sense strand comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of the sense strands in Table 1, and an antisense strand comprising at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of the antisense strands in Table 1 (e.g., corresponding to the sense strand, i.e., the antisense strand under the same siRNA name as the sense strand), wherein the first nucleotide of the antisense strand counting from the 5' end is A or U, e.g., U.

[0129] In some embodiments, the dsRNA active agent comprises a sense strand comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides differing by no more than 2 nucleotides from any one of the nucleotide sequences of the sense strands in Table 1, and an antisense strand comprising at least 15, 16, 17, 18, or 19, 20, or 21 contiguous nucleotides differing by no more than 2 nucleotides from any one of the nucleotide sequences of the antisense strands in Table 1 (e.g., corresponding to the sense strand, i.e., the antisense strand under the same siRNA name as the sense strand), wherein the first nucleotide of the antisense strand counting from the 5' end is A or U, e.g., U.

[0130] In some embodiments, the dsRNA active agent comprises a sense strand comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides differing by no more than 1 nucleotide from any one of the nucleotide sequences of the sense strands in Table 1, and an antisense strand comprising at least 15, 16, 17, 18, or 19, 20, or 21 contiguous nucleotides differing by no more than 1 nucleotide from any one of the nucleotide sequences of the antisense strands (e.g., corresponding to the sense strand, i.e., the antisense strand under the same siRNA designation as the sense strand) in Table 1, wherein the first nucleotide of the antisense strand counting from the 5' end is A or U, e.g., U.

[0131] In some specific embodiments, the antisense strand comprises a nucleotide sequence of at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-67. In some specific embodiments, the antisense strand differs by no more than 1, 2, or 3 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-67. In some specific embodiments, the antisense strand comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-67.

[0132] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15, 16, 17, 18, or 19 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 68-134. In some specific embodiments, the sense strand differs by no more than 1, 2, or 3 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 68-134. In some specific embodiments, the sense strand comprises or consists of the nucleotide sequence set forth in any one of SEQ ID NOs: 68-134.

[0133] In some embodiments, the dsRNA active agent comprises a sense strand comprising any one of the nucleotide sequences of the sense strands in Table 1, and an antisense strand comprising any one of the nucleotide sequences of the antisense strands (e.g., corresponding to the sense strand, i.e., the antisense strand under the same siRNA designation as the sense strand) in Table 1. In some embodiments, the combination of antisense and sense strands in the dsRNA active agent is as set forth for any of the combinations of antisense and sense strands shown in Table 1.

[0134] In some specific embodiments, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand and the nucleotide sequence of the sense strand comprise, respectively, SEQ ID NO: 1 / 68, SEQ ID NO: 2 / 69, SEQ ID NO: 3 / 70, SEQ ID NO: 4 / 71, SEQ ID NO: 5 / 72, SEQ ID NO: 6 / 73, SEQ ID NO: 7 / 74, SEQ ID NO: 8 / 75, SEQ ID NO: 9 / 76, SEQ ID NO: 10 / 77, SEQ ID NO: 11 / 78, SEQ ID NO: 12 / 79, SEQ ID NO: 13 / 80, SEQ ID NO: 14 / 81, SEQ ID NO: 15 / 82, SEQ ID NO: 16 / 83, SEQ ID NO: 17 / 84, SEQ ID NO: 18 / 85, SEQ ID NO: 19 / 86, SEQ ID NO: 20 / 87, SEQ ID NO: 21 / 88, SEQ ID NO: 22 / 89, SEQ ID NO: 23 / 90, SEQ ID NO: 24 / 91, SEQ ID NO: 25 / 92, SEQ ID NO: 26 / 93, SEQ ID NO: 27 / 94, SEQ ID NO: 28 / 95, SEQ ID NO: 29 / 96, SEQ ID NO: 30 / 97, SEQ ID NO: 31 / 98, SEQ ID NO: 32 / 99, SEQ ID NO: 33 / 100, SEQ ID NO: 34 / 101, SEQ ID NO: 35 / 102, SEQ ID NO: 36 / 103, SEQ ID NO: 37 / 104, SEQ ID NO: 38 / 105, SEQ ID NO: 39 / 106, SEQ ID NO: 40 / 107, SEQ ID NO: 41 / 108, SEQ ID NO: 42 / 109, SEQ ID NO: 43 / 110, SEQ ID NO: 44 / 111, SEQ ID NO: 45 / 112, SEQ ID NO: 46 / 113, SEQ ID NO: 47 / 114, SEQ ID NO: 48 / 115, SEQ ID NO: 49 / 116, SEQ ID NO: 50 / 117, SEQ ID NO: 51 / 118, SEQ ID NO: 52 / 119, SEQ ID NO: 53 / 120, SEQ ID NO: 54 / 121, SEQ ID NO: 55 / 122, SEQ ID NO: 56 / 123, SEQ ID NO: 57 / 124, SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, SEQ ID NO: 67 / 134, SEQ ID NO: 68 / 135, SEQ ID NO: 69 / 136, SEQ ID NO: 70 / 137, SEQ ID NO: 71 / 138, SEQ ID NO: 72 / 139, SEQ ID NO: 73 / 140, SEQ ID NO: 74 / 141, SEQ ID NO: 75 / 142, SEQ ID NO: 76 / 143, SEQ ID NO: 77 / 144, SEQ ID NO: 78 / 145, SEQ ID NO: 79 / 146, SEQ ID NO: 80 / 147, SEQ ID NO: 81 / 148, SEQ ID NO: 82 / 149, SEQ ID NO: 83 / 150, SEQ ID NO: 84 / 151, SEQ ID NO: 85 / 152, SEQ ID NO: 86 / 153, SEQ ID NO: 87 / 154, SEQ ID NO: 88 / 155, SEQ ID NO: 89 / 156, SEQ ID NO: 90 / 157, SEQ ID NO: 91 / 158, SEQ ID NO: 92 / 159, SEQ ID NO: 93 / 160, SEQ ID NO: 94 / 161, SEQ ID NO: 95 / 162, SEQ ID NO: 96 / 163, SEQ ID NO: 97 / 164, SEQ ID NO: 98 / 165, SEQ ID NO: 99 / 166, SEQ ID NO: 100 / 167, SEQ ID NO: 101 / 168, SEQ ID NO: 102 / 169, SEQ ID NO: 103 / 170, SEQ ID NO: 104 / 171, SEQ ID NO: 105 / 172, SEQ ID NO: 106 / 173, SEQ ID NO: 107 / 174, SEQ ID NO: 108 / 175, SEQ ID NOat least 15, 16, 17, 18, or 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, or SEQ ID NO: 67 / 134, optionally the antisense strand further has a 3' overhang of 1 or 2 nucleotides, optionally the first nucleotide at the 5' end of the antisense strand is A or U, e.g., U.

[0135] In some specific embodiments, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand and the nucleotide sequence of the sense strand comprise, respectively, SEQ ID NO: 1 / 68, SEQ ID NO: 2 / 69, SEQ ID NO: 3 / 70, SEQ ID NO: 4 / 71, SEQ ID NO: 5 / 72, SEQ ID NO: 6 / 73, SEQ ID NO: 7 / 74, SEQ ID NO: 8 / 75, SEQ ID NO: 9 / 76, SEQ ID NO: 10 / 77, SEQ ID NO: 11 / 78, SEQ ID NO: 12 / 79, SEQ ID NO: 13 / 80, SEQ ID NO: 14 / 81, SEQ ID NO: 15 / 82, SEQ ID NO: 16 / 83, SEQ ID NO: 17 / 84, SEQ ID NO: 18 / 85, SEQ ID NO: 19 / 86, SEQ ID NO: 20 / 87, SEQ ID NO: 21 / 88, SEQ ID NO: 22 / 89, SEQ ID NO: 23 / 90, SEQ ID NO: 24 / 91, SEQ ID NO: 25 / 92, SEQ ID NO: 26 / 93, SEQ ID NO: 27 / 94, SEQ ID NO: 28 / 95, SEQ ID NO: 29 / 96, SEQ ID NO: 30 / 97, SEQ ID NO: 31 / 98, SEQ ID NO: 32 / 99, SEQ ID NO: 33 / 100, SEQ ID NO: 34 / 101, SEQ ID NO: 35 / 102, SEQ ID NO: 36 / 103, SEQ ID NO: 37 / 104, SEQ ID NO: 38 / 105, SEQ ID NO: 39 / 106, SEQ ID NO: 40 / 107, SEQ ID NO: 41 / 108, SEQ ID NO: 42 / 109, SEQ ID NO: 43 / 110, SEQ ID NO: 44 / 111, SEQ ID NO: 45 / 112, SEQ ID NO: 46 / 113, SEQ ID NO: 47 / 114, SEQ ID NO: 48 / 115, SEQ ID NO: 49 / 116, SEQ ID NO: 50 / 117, SEQ ID NO: 51 / 118, SEQ ID NO: 52 / 119, SEQ ID NO: 53 / 120, SEQ ID NO: 54 / 121, SEQ ID NO: 55 / 122, SEQ ID NO: 56 / 123, SEQ ID NO: 57 / 124, SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, SEQ ID NO: 67 / 134, SEQ ID NO: 68 / 135, SEQ ID NO: 69 / 136, SEQ ID NO: 70 / 137, SEQ ID NO: 71 / 138, SEQ ID NO: 72 / 139, SEQ ID NO: 73 / 140, SEQ ID NO: 74 / 141, SEQ ID NO: 75 / 142, SEQ ID NO: 76 / 143, SEQ ID NO: 77 / 144, SEQ ID NO: 78 / 145, SEQ ID NO: 79 / 146, SEQ ID NO: 80 / 147, SEQ ID NO: 81 / 148, SEQ ID NO: 82 / 149, SEQ ID NO: 83 / 150, SEQ ID NO: 84 / 151, SEQ ID NO: 85 / 152, SEQ ID NO: 86 / 153, SEQ ID NO: 87 / 154, SEQ ID NO: 88 / 155, SEQ ID NO: 89 / 156, SEQ ID NO: 90 / 157, SEQ ID NO: 91 / 158, SEQ ID NO: 92 / 159, SEQ ID NO: 93 / 160, SEQ ID NO: 94 / 161, SEQ ID NO: 95 / 162, SEQ ID NO: 96 / 163, SEQ ID NO: 97 / 164, SEQ ID NO: 98 / 165, SEQ ID NO: 99 / 166, SEQ ID NO: 100 / 167, SEQ ID NO: 101 / 168, SEQ ID NO: 102 / 169, SEQ ID NO: 103 / 170, SEQ ID NO: 104 / 171, SEQ ID NO: 105 / 172, SEQ ID NO: 106 / 173, SEQ ID NO: 107 / 174, SEQ ID NO: 108 / 175, SEQ ID NOthe nucleotide sequences set forth in SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, or SEQ ID NO: 67 / 134, respectively.

[0136] For purposes of inhibiting expression of a target mRNA, as will be appreciated by those skilled in the art, the oligonucleotide serving as the sense strand does not participate in direct complementary binding to the target sequence, and need not have perfect base pairing with the antisense strand oligonucleotide in the duplex region. Thus, in some aspects, the sense strand (passenger strand) according to the present application can include at least one or more of the following properties: substantial complementarity to a contiguous stretch of nucleotides of the antisense strand, e.g., at least 70% complementary, at least 80% complementary, at least 90% complementary, or 100% complementary, in the hybridized duplex region with the antisense strand; 1 or more extra nucleotides forming a bulge or loop, relative to a contiguous stretch of nucleotides of the antisense strand, in the hybridized duplex region; presence of 1 or more nucleotide gaps or vacancies, relative to a contiguous stretch of nucleotides of the antisense strand, in the hybridized duplex region. Likewise, for purposes of inhibiting expression of a target mRNA, as will be appreciated by those skilled in the art, the antisense strand, which directs specific binding of RNAi to the target mRNA of interest, can also comprise a sequence that is not 100% complementary to a contiguous stretch of nucleotides of the target mRNA, e.g., the complementarity can be at least 80% complementary, at least 90% complementary, or 95% complementary; although in some cases, 100% complementarity is more preferred. According to the purposes of the present application, in some aspects, the presence of insertions and deletions is preferably not allowed when considering the sequence motif of the antisense strand to be complementary to a contiguous stretch of nucleotides of the target gene sequence. In some aspects, with respect to the sense and antisense strands of the present application, when the region of complementarity is not perfectly complementary to the contiguous stretch of nucleotides, the mismatches can be internal or terminal, e.g., 3, 2, or 1 nucleotides of mismatches at the 5' and / or 3' terminal end. In preferred embodiments, the antisense strand is complementary, e.g., perfectly complementary, to the sense strand over at least 18 or 19 or 20 or 21 contiguous nucleotides. In preferred embodiments, the antisense strand is complementary, e.g., perfectly complementary, to the sense strand over at least 18 contiguous nucleotides. In preferred embodiments, the antisense strand is complementary, e.g., perfectly complementary, to the sense strand over 19 contiguous nucleotides, e.g., the 1st-19th contiguous nucleotides counting from the 5' end of the antisense strand.

[0137] In some embodiments, the dsRNA active agent is prepared or provided as a salt, a mixed salt, or as a free acid. In some embodiments, the dsRNA active agent is prepared as a sodium salt. Such forms are within the scope of the invention disclosed herein.

[0138] Those skilled in the art will recognize that the dsRNA molecules according to the present invention can be unmodified (i.e., comprise RNA nucleosides that are natural in nature) but can also be (and preferably are) modified, so long as they retain the desired functional activity (i.e., are capable of forming the desired duplex structure and allowing or mediating specific degradation of the target RNA via the RISC pathway). Such RNA modifications can occur at the base moiety, sugar moiety, and / or phosphate linkage moiety of the nucleotides. As a non-limiting example, modified RNAi active agents can be constructed using chemical synthesis and enzymatic ligation reactions, using protocols known in the art. For example, modified RNAi active agents can be chemically synthesized using naturally occurring nucleotides or nucleotides that have been variously modified (designed to reduce off-target effects, and / or to increase the biological stability of the molecule, or to increase the physical stability of duplexes formed between the antisense and sense nucleic acids).

[0139] In some embodiments, the dsRNA active agents of the present invention comprise at least one modified nucleotide.

[0140] In this document, when referring to the amino acid sequence set forth in a SEQ ID NO of a dsRNA, the listed U, T, A, or C also encompasses modified U, T, A, or C, unless otherwise specified.

[0141] In some embodiments, the dsRNA active agent comprises one or more modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). 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 96%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. In some embodiments, substantially all of the nucleotides of the sense strand; or substantially all of the nucleotides of the antisense strand; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides in the dsRNA active agents of the present invention.

[0142] In some embodiments, all or substantially all of the nucleotides of the dsRNA active agents of the application are modified nucleotides. As described herein, a dsRNA active agent having substantially all nucleotides that are modified nucleotides refers to a dsRNA active agent having a total of 4 or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides that are ribonucleotides in the sense strand and the antisense strand. As used herein, a sense strand having substantially all nucleotides that are modified nucleotides refers to a sense strand having 2 or fewer (i.e., 0, 1, or 2) nucleotides that are ribonucleotides in the sense strand. As used herein, an antisense strand having substantially all nucleotides that are modified nucleotides refers to an antisense strand having 2 or fewer (i.e., 0, 1, or 2) nucleotides that are ribonucleotides in the antisense strand.

[0143] In some embodiments, all of the nucleotides of the sense strand and / or all of the nucleotides of the antisense strand in the dsRNA active agents are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0144] In some embodiments, the nucleotide modifications suitable for use in the dsRNA active agents of the application encompass modifications to the nucleobase, modifications to the ribose moiety, and / or modifications to the phosphate backbone. Some oligonucleotide modifications known in the art can be found in PCT Publication WO 200370918, which is incorporated by reference in its entirety.

[0145] Examples of nucleobase modifications that can be used to produce dsRNA active agents include, for example, uracil, guanine, or adenine containing nucleotides can be replaced with nucleotides containing, for example, inosine; and adenine and cytosine in the oligonucleotide can be replaced with guanine and uracil, respectively, to form G-U Wobble base pairing with the target mRNA. In addition, other examples of modified nucleobases that can be used to produce RNAi active agents include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thio-uracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Each of these modified nucleobases are contemplated by the present application.

[0146] Examples of ribose moiety modifications that can be used to produce dsRNA active agents include, for example, modification of the ribose ring structure by replacement with one of the following: a hexose ring (HNA), a threose ring (TNA), a locked nucleic acid (LNA, bicyclic ring with a base bridge between the C2 and C4 carbons of the ribose ring), or an unlocked nucleic acid (UNA, ribose ring lacking a bond between the C2 and C3 carbons). Examples of useful sugar-modified nucleosides also include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclo nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, for example, as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids. Sugar modifications also include modifications by replacing the naturally occurring 2'-OH group on the ribose ring of an RNA nucleoside with another group. In addition, a substituent can be introduced, for example, at the 2', 3', 4', or 5' position of the sugar ring.

[0147] In some embodiments, the dsRNA active agents of the present application can comprise 2' sugar modified nucleotides, such as 2' substituted nucleosides. Examples of 2' substituted modified nucleosides are 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA nucleosides (MOE), 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides. Further examples can be found, for example, in Freier and Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. In some embodiments, the RNAi active agents according to the present application comprise at least one 2'-modified nucleotide. In some embodiments, the 2'-modification is selected from the group consisting of 2'-deoxy, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-allyl, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O-N-methylacetamido (2'-O-NMA). In some embodiments, the RNAi active agents according to the present application comprise at least one 2' modified nucleoside selected from the group consisting of 2'-O-alkyl-RNA nucleosides, 2'-O-methyl-RNA nucleosides, 2'-alkoxy-RNA nucleosides, 2'-O-methoxyethyl-RNA nucleosides (MOE), 2'-amino-DNA nucleosides, 2'-fluoro-RNA nucleosides, and 2'-F-ANA nucleosides.

[0148] In some embodiments, the dsRNA active agents according to the present application can also optionally comprise chemical modifications at the 5' and / or 3' termini, i.e., non-nucleotide or nucleoside chemical moieties attached to the termini of the RNAi oligonucleotide strands (sense and / or antisense strands). Examples of chemical moieties attached at the 3' end of the oligonucleotide strand can be found, for example, disclosed in WO 2005 / 021749 and WO 2007 / 128477. Examples of chemical moieties attached at the 5' end of the oligonucleotide strand can include, but are not limited to, 5' terminal phosphate modifications, such as 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analog, and 5'-phosphorothioate (5'-PS).

[0149] In some embodiments, at least one modified nucleotide of the modified nucleotides of the dsRNA active agent suitable for use in the application is selected from the group consisting of: a deoxy nucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a 2'-5'-linked ribonucleotide (3'-RNA), an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-0-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group (e.g., a nucleoside comprising a 5' phosphorothioate group), a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate mimic, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide (GNA), a nucleotide comprising a 2' phosphate, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

[0150] In some embodiments, at least one modified nucleotide of the modified nucleotides of the dsRNA active agent suitable for use in the application is selected from the group consisting of: a non-locked nucleic acid (UNA), a locked nucleic acid (LNA), a threose nucleic acid (TNA), a HNA, a CeNA, a 2'-methoxyethyl, a 2'-0-alkyl, a 2'-0-allyl, a 2'-C-allyl, a 2'-fluoro, a 2'-deoxy, and a glycol; and combinations thereof. In some embodiments, at least one of the modified nucleotides of the modified nucleotides of the dsRNA active agent is selected from the group consisting of: a deoxy nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a nucleotide comprising a 2' phosphate, and a nucleotide comprising a phosphorothioate group; and combinations thereof.

[0151] In some embodiments, the antisense strand of a dsRNA agent of the application comprises 2'-methoxy (2'-0-methyl) modified nucleotides, e.g., all nucleotides are 2'-methoxy modified nucleotides or 1-21 nucleotides are 2' methoxy modified nucleotides, e.g., 17 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the 1st, 3rd-5th, 7th-13th, 15th, and 17th-21st nucleotides from the 5' end of the antisense strand of a dsRNA agent of the application are 2'-methoxy modified nucleotides. In some embodiments, the sense strand of a dsRNA agent of the application comprises 2'-methoxy (2'-0-methyl) modified nucleotides, e.g., all nucleotides are 2'-methoxy modified nucleotides or 1-19 nucleotides are 2' methoxy modified nucleotides, e.g., 16 nucleotides are 2'-methoxy modified nucleotides. In some embodiments, the 1st-6th and 10th-19th nucleotides from the 5' end of the sense strand of a dsRNA agent of the application are 2'-methoxy modified nucleotides. In some embodiments, in a dsRNA agent of the application, the 1st, 3rd-5th, 7th-13th, 15th, and 17th-21st nucleotides from the 5' end of the antisense strand are 2'-methoxy modified nucleotides and the 1st-6th and 10th-19th nucleotides from the 5' end of the sense strand are 2'-methoxy modified nucleotides.

[0152] In some embodiments, the antisense strand of a dsRNA agent of the application comprises 2'-fluoro modified nucleotides, e.g., 1-5 nucleotides are 2'-fluoro modified nucleotides, e.g., 4 nucleotides are 2'-fluoro modified nucleotides. In some embodiments, the 2nd, 6th, 14th, and 16th nucleotides from the 5' end of the antisense strand of a dsRNA agent of the application are 2'-fluoro modified nucleotides. In some embodiments, the sense strand of a dsRNA agent of the application comprises 2'-fluoro modified nucleotides, e.g., 1-5 nucleotides are 2'-fluoro modified nucleotides, e.g., 3 nucleotides are 2'-fluoro modified nucleotides. In some embodiments, the 7th-9th nucleotides from the 5' end of the sense strand of a dsRNA agent of the application are 2'-fluoro modified nucleotides. In some embodiments, in a dsRNA agent of the application, the 2nd, 6th, 14th, and 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro modified nucleotides and the 7th, 8th, and 9th nucleotides from the 5' end of the sense strand are 2'-fluoro modified nucleotides.

[0153] In some embodiments, one or more nucleotides of the dsRNA active agent are linked by a non-standard linkage or backbone (i.e., a modified internucleoside linkage or a modified backbone). In some embodiments, the modified internucleoside linkage is a covalent internucleoside linkage that comprises a non-phosphate. In some embodiments, the modified internucleoside linkage or backbone includes, but is not limited to: a 5'-phosphorothioate group (denoted herein as lower case "s"), a chiral phosphorothioate, a phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate or 3'-alkylene phosphonate), a chiral phosphonate, a phosphinate, a phosphoramidate (e.g., 3'-amino phosphoramidate, aminoalkylphosphoramidate, or thio-phosphoramidate), a thioalkyl-phosphonate, a thioalkylphosphotriester, a morpholino linkage, a boranophosphates having normal 3'-5' linkages, a boranophosphate analog of a 2'-5' linkage, or a boranophosphate having an inverted polarity wherein the adjacent nucleotide pair is 3'-5' to 5'-3' or 2'-5' to 5'-2' linkage. In some embodiments, the modified internucleoside linkage or backbone is free of a phosphorus atom. In some embodiments, the modified internucleoside linkage free of a phosphorus atom includes, but is not limited to: a short chain alkyl or cycloalkyl inter-sugar linkage, a mixed heteroatom and alkyl or cycloalkyl inter-sugar linkage, or one or more short chain heteroatom or heterocyclic inter-sugar linkages. In some embodiments, the modified internucleoside backbone includes, but is not limited to: a siloxane backbone, a sulfide backbone, a sulfoxide backbone, a sulfone backbone, a formacetyl and thioformacetyl backbone, a methylene formacetyl and thioformacetyl backbone, an alkene containing backbone, a sulfamate backbone, a methylene imino and methylene hydrazino backbone, a sulfonate and sulfonamide backbone, a amide backbone, and other backbones having mixed N, O, S and CH2components.

[0154] In some embodiments, the sense strand of the dsRNA active agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the antisense strand of the dsRNA active agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, both the sense and antisense strands can each independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of the dsRNA active agent can contain 1, 2, 3, or 4 phosphorothioate linkages. In some embodiments, the antisense strand of the dsRNA active agent can contain 1, 2, 3, or 4 phosphorothioate linkages. In some embodiments, both the sense and antisense strands can each independently contain 1, 2, 3, or 4 phosphorothioate linkages.

[0155] In some embodiments, the dsRNA active agent sense strand contains 2 phosphorothioate internucleoside linkages. In some embodiments, the 2 phosphorothioate internucleoside linkages are between nucleotides 1-3 counting from the 5’ end of the sense strand. In some embodiments, the dsRNA active agent antisense strand contains 4 phosphorothioate internucleoside linkages. In some embodiments, the 4 phosphorothioate internucleoside linkages are between nucleotides 1-3 counting from the 5’ end and between nucleotides 1-3 counting from the 3’ end of the antisense strand. In some embodiments, the dsRNA active agent contains 2 phosphorothioate internucleoside linkages in the sense strand and 4 phosphorothioate internucleoside linkages in the antisense strand. In some embodiments, the dsRNA active agent contains 2 phosphorothioate internucleoside linkages in the sense strand, the 2 phosphorothioate internucleoside linkages being between nucleotides 1-3 counting from the 5’ end of the sense strand; and 4 phosphorothioate internucleoside linkages in the antisense strand, the 4 phosphorothioate internucleoside linkages being between nucleotides 1-3 counting from the 5’ end and between nucleotides 1-3 counting from the 3’ end of the antisense strand.

[0156] Other ways of modifying the dsRNA active agent can also be found in the modifications listed in, for example, WO2023044094A1, WO2023245060A2, WO2018 / 027106, WO2024 / 197017A2, or WO2023 / 076451A1, which are incorporated herein in their entirety.

[0157] In some embodiments, the modified nucleotides in the sense and antisense strands of the dsRNA active agent have the following modification pattern:

[0158] Antisense strand:

[0159] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0160] and / or

[0161] Sense strand:

[0162] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm,

[0163] wherein,

[0164] Nf = any 2’-fluoro modified nucleotide;

[0165] Nm = any 2’-methoxy nucleotide;

[0166] s represents a phosphorothioate linkage between two nucleotides.

[0167] In some embodiments, the antisense strand in the dsRNA active agent comprises or consists of a modified nucleotide sequence as any one of the antisense strands in Table 2 of the specification, and / or the sense strand comprises or consists of a modified nucleotide sequence as any one of the sense strands in Table 2 of the specification.

[0168] In some embodiments, the combination of modified antisense and sense strands in the dsRNA active agent comprises any combination of antisense and sense strands as shown in Table 2 of the specification.

[0169] In some embodiments, the dsRNA active agent is any of the dsRNA active agents shown in Table 2.

[0170] In some specific embodiments, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the modified nucleotide sequence of the antisense strand and the modified nucleotide sequence of the sense strand comprise SEQ ID NO: 202 / SEQ ID NO: 269, SEQ ID NO: 203 / SEQ ID NO: 270, SEQ ID NO: 204 / SEQ ID NO: 271, SEQ ID NO: 205 / SEQ ID NO: 272, SEQ ID NO: 206 / SEQ ID NO: 273, SEQ ID NO: 207 / SEQ ID NO: 274, SEQ ID NO: 208 / SEQ ID NO: 275, SEQ ID NO: 209 / SEQ ID NO: 276, SEQ ID NO: 210 / SEQ ID NO: 277, SEQ ID NO: 211 / SEQ ID NO: 278, SEQ ID NO: 212 / SEQ ID NO: 279, SEQ ID NO: 213 / SEQ ID NO: 280, SEQ ID NO: 214 / SEQ ID NO: 281, SEQ ID NO: 215 / SEQ ID NO: 282, SEQ ID NO: 216 / SEQ ID NO: 283, SEQ ID NO: 217 / SEQ ID NO: 284, SEQ ID NO: 218 / SEQ ID NO: 285, SEQ ID NO: 219 / SEQ ID NO: 286, SEQ ID NO: 220 / SEQ ID NO: 287, SEQ ID NO: 221 / SEQ ID NO: 288, SEQ ID NO: 222 / SEQ ID NO: 289, SEQ ID NO: 223 / SEQ ID NO: 290, SEQ ID NO: 224 / SEQ ID NO: 291, SEQ ID NO: 225 / SEQ ID NO: 292, SEQ ID NO: 226 / SEQ ID NO: 293, SEQ ID NO: 227 / SEQ ID NO: 294, SEQ ID NO: 228 / SEQ ID NO: 295, SEQ ID NO: 229 / SEQ ID NO: 296, SEQ ID NO: 230 / SEQ ID NO: 297, SEQ ID NO: 231 / SEQ ID NO: 298, SEQ ID NO: 232 / SEQ ID NO: 299, SEQ ID NO: 233 / SEQ ID NO: 300, SEQ ID NO: 234 / SEQ ID NO: 301, SEQ ID NO: 235 / SEQ ID NO: 302, SEQ ID NO: 236 / SEQ ID NO: 303, SEQ ID NO: 237 / SEQ ID NO: 304, SEQ ID NO: 238 / SEQ ID NO: 305, SEQ ID NO: 239 / SEQ ID NO: 306, SEQ ID NO: 240 / SEQ ID NO: 307, SEQ ID NO: 241 / SEQ ID NO: 308, SEQ ID NO: 242 / SEQ ID NO: 309, SEQ ID NO: 243 / SEQ ID NO: 310, SEQ ID NO: 244 / SEQ ID NO: 311, SEQ ID NO: 245 / SEQ ID NO: 312, SEQ ID NO: 246 / SEQ ID NO: 313, SEQ ID NO: 247 / SEQ ID NO: 314, SEQ ID NO: 248 / SEQ ID NO: 315, SEQ ID NO: 249 / SEQ ID NO: 316, or SEQ ID NO: 250 / SEQ ID NO: 317, respectively.ID NO: 305, SEQ ID NO: 239 / SEQ ID NO: 306, SEQ ID NO: 240 / SEQ ID NO: 307, SEQ ID NO: 241 / SEQ ID NO: 308, SEQ ID NO: 242 / SEQ ID NO: 309, SEQ ID NO: 243 / SEQ ID NO: 310, SEQ ID NO: 244 / SEQ ID NO: 311, SEQ ID NO: 245 / SEQ ID NO: 312, SEQ ID NO: 246 / SEQ ID NO: 313, SEQ ID NO: 247 / SEQ ID NO: 314, SEQ ID NO: 248 / SEQ ID NO: 315, SEQ ID NO: 249 / SEQ ID NO: 316, SEQ ID NO: 250 / SEQ ID NO: 317, SEQ ID NO: 251 / SEQ ID NO: 318, SEQ ID NO: 252 / SEQ ID NO: 319, SEQ ID NO: 253 / SEQ ID NO: 320, SEQ ID NO: 254 / SEQ ID NO: 321, SEQ ID NO: 255 / SEQ ID NO: 322, SEQ ID NO: 256 / SEQ ID NO: 323, SEQ ID NO: 257 / SEQ ID NO: 324, SEQ ID NO: 258 / SEQ ID NO: 325, SEQ ID NO: 259 / SEQ ID NO: 326, SEQ ID NO: 260 / SEQ ID NO: 327, SEQ ID NO: 261 / SEQ ID NO: 328, SEQ ID NO: 262 / SEQ ID NO: 329, SEQ ID NO: 263 / SEQ ID NO: 330, SEQ ID NO: 264 / SEQ ID NO: 331, SEQ ID NO: 265 / SEQ ID NO: 332, SEQ ID NO: 266 / SEQ ID NO: 333, SEQ ID NO: 267 / SEQ ID NO: 334, or SEQ ID NO: 268 / SEQ ID NO: 335.at least 15, 16, 17, 18, or 19 contiguous modified nucleotides in the nucleotide sequence set forth in SEQ ID NO: 335, optionally the antisense strand further has a 3' overhang of 1 or 2 nucleotides, optionally the first nucleotide at the 5' end of the antisense strand is a modified A or U, for example a modified U, for example Um or Am, for example Um.

[0171] In some specific embodiments, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the modified nucleotide sequence of the antisense strand and the modified nucleotide sequence of the sense strand comprise SEQ ID NO: 202 / SEQ ID NO: 269, SEQ ID NO: 203 / SEQ ID NO: 270, SEQ ID NO: 204 / SEQ ID NO: 271, SEQ ID NO: 205 / SEQ ID NO: 272, SEQ ID NO: 206 / SEQ ID NO: 273, SEQ ID NO: 207 / SEQ ID NO: 274, SEQ ID NO: 208 / SEQ ID NO: 275, SEQ ID NO: 209 / SEQ ID NO: 276, SEQ ID NO: 210 / SEQ ID NO: 277, SEQ ID NO: 211 / SEQ ID NO: 278, SEQ ID NO: 212 / SEQ ID NO: 279, SEQ ID NO: 213 / SEQ ID NO: 280, SEQ ID NO: 214 / SEQ ID NO: 281, SEQ ID NO: 215 / SEQ ID NO: 282, SEQ ID NO: 216 / SEQ ID NO: 283, SEQ ID NO: 217 / SEQ ID NO: 284, SEQ ID NO: 218 / SEQ ID NO: 285, SEQ ID NO: 219 / SEQ ID NO: 286, SEQ ID NO: 220 / SEQ ID NO: 287, SEQ ID NO: 221 / SEQ ID NO: 288, SEQ ID NO: 222 / SEQ ID NO: 289, SEQ ID NO: 223 / SEQ ID NO: 290, SEQ ID NO: 224 / SEQ ID NO: 291, SEQ ID NO: 225 / SEQ ID NO: 292, SEQ ID NO: 226 / SEQ ID NO: 293, SEQ ID NO: 227 / SEQ ID NO: 294, SEQ ID NO: 228 / SEQ ID NO: 295, SEQ ID NO: 229 / SEQ ID NO: 296, SEQ ID NO: 230 / SEQ ID NO: 297, SEQ ID NO: 231 / SEQ ID NO: 298, SEQ ID NO: 232 / SEQ ID NO: 299, SEQ ID NO: 233 / SEQ ID NO: 300, SEQ ID NO: 234 / SEQ ID NO: 301, SEQ ID NO: 235 / SEQ ID NO: 302, SEQ ID NO: 236 / SEQ ID NO: 303, SEQ ID NO: 237 / SEQ ID NO: 304, SEQ ID NO: 238 / SEQ ID NO: 305, SEQ ID NO: 239 / SEQ ID NO: 306, SEQ ID NO: 240 / SEQ ID NO: 307, SEQ ID NO: 241 / SEQ ID NO: 308, SEQ ID NO: 242 / SEQ ID NO: 309, SEQ ID NO: 243 / SEQ ID NO: 310, SEQ ID NO: 244 / SEQ ID NO: 311, SEQ ID NO: 245 / SEQ ID NO: 312, SEQ ID NO: 246 / SEQ ID NO: 313, SEQ ID NO: 247 / SEQ ID NO: 314, SEQ ID NO: 248 / SEQ ID NO: 315, SEQ ID NO: 249 / SEQ ID NO: 316, or SEQ ID NO: 250 / SEQ ID NO: 317, respectively.NO: 235 / SEQ ID NO: 302, SEQ ID NO: 236 / SEQ ID NO: 303, SEQ ID NO: 237 / SEQ ID NO: 304, SEQ ID NO: 238 / SEQ ID NO: 305, SEQ ID NO: 239 / SEQ ID NO: 306, SEQ ID NO: 240 / SEQ ID NO: 307, SEQ ID NO: 241 / SEQ ID NO: 308, SEQ ID NO: 242 / SEQ ID NO: 309, SEQ ID NO: 243 / SEQ ID NO: 310, SEQ ID NO: 244 / SEQ ID NO: 311, SEQ ID NO: 245 / SEQ ID NO: 312, SEQ ID NO: 246 / SEQ ID NO: 313, SEQ ID NO: 247 / SEQ ID NO: 314, SEQ ID NO: 248 / SEQ ID NO: 315, SEQ ID NO: 249 / SEQ ID NO: 316, SEQ ID NO: 250 / SEQ ID NO: 317, SEQ ID NO: 251 / SEQ ID NO: 318, SEQ ID NO: 252 / SEQ ID NO: 319, SEQ ID NO: 253 / SEQ ID NO: 320, SEQ ID NO: 254 / SEQ ID NO: 321, SEQ ID NO: 255 / SEQ ID NO: 322, SEQ ID NO: 256 / SEQ ID NO: 323, SEQ ID NO: 257 / SEQ ID NO: 324, SEQ ID NO: 258 / SEQ ID NO: 325, SEQ ID NO: 259 / SEQ ID NO: 326, SEQ ID NO: 260 / SEQ ID NO: 327, SEQ ID NO: 261 / SEQ ID NO: 328, SEQ ID NO: 262 / SEQ ID NO: 329, SEQ ID NO: 263 / SEQ ID NO: 330, SEQ ID NO: 264 / SEQ ID NO: 331, SEQ ID NO: 265 / SEQ ID NO: 332, SEQ ID NO: 266 / SEQ ID NO: 333, SEQ ID NO: 267 / SEQ ID NO: 334, or SEQ ID NO: 268 / SEQ ID NO: 335.

[0172] As used herein (e.g., in Tables 1, 2, and 3), the following symbols are used to represent modified nucleotides, targeting groups, and linking groups. Unless otherwise indicated by the sequence, these monomers are linked to one another by 5'-3'-phosphodiester linkages (when the first of the two nucleotides is a phosphorothioate (s), s represents a phosphorothioate linkage between the two nucleotides) when present in an oligonucleotide:

[0173] T = 5'-methyluridine-3'-phosphorothioate

[0174] Nf = any 2'-fluoro modified nucleotide

[0175] Nfs = any 2'-fluoro modified nucleoside-3' phosphorothioate

[0176] Af = 2'-fluoro adenosine-3'-phosphorothioate

[0177] Afs = 2'-fluoro adenosine-3'-phosphorothioate

[0178] Cf = 2'-fluoro cytidine-3'-phosphorothioate

[0179] Cfs = 2'-fluoro cytidine-3'-phosphorothioate

[0180] Gf = 2'-fluoro guanosine-3'-phosphorothioate

[0181] Gfs = 2'-fluoro guanosine-3'-phosphorothioate

[0182] Tf = 2'-fluoro-5'-methyluridine-3'-phosphorothioate

[0183] Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate

[0184] Uf = 2'-fluoro uridine-3'-phosphorothioate

[0185] Ufs = 2'-fluoro uridine-3'-phosphorothioate

[0186] Nm = any 2'-methoxy nucleotide

[0187] Nms = any 2'-methoxy nucleoside-3'-phosphorothioate

[0188] Am = 2'-methoxy adenosine-3'-phosphorothioate

[0189] Ams = 2'-methoxy adenosine-3'-phosphorothioate

[0190] Tm = 2'-methoxy thymidine-3'-phosphorothioate

[0191] Tms = 2'-methoxy thymidine-3'-phosphorothioate

[0192] Um = 2'-methoxyuridine-3'-phosphate

[0193] Ums = 2'-methoxyuridine-3'-phosphorothioate

[0194] Gm = 2'-methoxyguanosine-3'-phosphate

[0195] Gms = 2'-methoxyguanosine-3'-phosphorothioate

[0196] Cm = 2'-methoxy cytidine-3'-phosphate

[0197] Cms = 2'-methoxy cytidine-3'-phosphorothioate

[0198] In some embodiments, the dsRNA active agents of the present application further comprise a ligand. When the present application refers to "dsRNA" or "dsRNA active agents", it also encompasses dsRNA conjugated to a ligand, also referred to as dsRNA-ligand conjugates.

[0199] As described herein, "ligand" refers to a chemical moiety conjugated to the oligonucleotide of the dsRNA, which is capable of altering the distribution, targeting or half-life of the dsRNA. In preferred embodiments, such ligand provides enhanced affinity for the selected target (e.g. molecule, cell or cell type, compartment (e.g. cellular or organ compartment, tissue, organ or region of the body)) compared to, for example, the dsRNA in the absence of said ligand.

[0200] In some embodiments, the ligand modulates or enhances the pharmacokinetic properties of the dsRNA by improving the cellular distribution, bioavailability, metabolism, excretion, permeability and / or cellular uptake of the nucleotides. In particular, the ligand can direct the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the dsRNA in such organ, tissue or cell type. At the same time, the ligand can serve to reduce the activity (e.g. off-target activity or activity) of the dsRNA in non-target cell types, tissues or organs.

[0201] Vajinder Kumar, Targeted delivery of oligonucleotides using multivalent protein-carbohydrate interactions, Cite this: Chem. Soc. Rev., 2023, 52, 1273; Rosemary Kanasty, Delivery materials for siRNA therapeutics, NATURE MATERIALS, VOL 12, NOVEMBER 2013; Wanyi Tai, Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery, Molecules 2019, 24, 2211; doi:10.3390 / molecules24122211; and WO 93 / 07883, WO 2013 / 033230, WO 2023044094A1, WO 2023245060A2, WO 2018 / 027106, WO 2012083185A2, WO 2015021092, WO 2018044350A1, WO 2024 / 197017A2, or WO 2023 / 076451A1, which are incorporated herein by reference, provide descriptions of ligands suitable for use with dsRNAs and conjugation modification approaches.

[0202] In some embodiments, ligands for use with RNAi active agents of the present application, such as dsRNA active agents, can be selected from sugars, cell surface receptor ligands, antibodies, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0203] In some embodiments, ligands for use with RNAi active agents of the present application, such as dsRNA active agents, are sugars, including but not limited to galactose, lactose, N-acetylgalactosamine, mannose, and mannose-6-phosphate. Sugar ligands can be used to enhance delivery or activity in a range of tissues such as the liver and / or muscle. In some embodiments, the ligand is a monosaccharide.

[0204] In some embodiments, the ligand for the RNAi active agent, e.g., dsRNA active agent, of the application is an asialoglycoprotein receptor (ASGPR) ligand. In particular, the ASPGR ligand comprises a moiety selected from galactose or a galactose derivative (e.g., galactosamine, N-formyl galactosamine, N-acetyl galactosamine (GalNAc), N-propionyl galactosamine, N-n-butyryl galactosamine, N-isobutyryl galactosamine, etc.). The ASPGR ligand can or can not have a linking group (also referred to as a "linker").

[0205] In this context, when the galactose derivative in the ligand is N-acetyl galactosamine (GalNAc) or a GalNAc derivative, the ligand is also referred to as a GalNAc ligand, encompassing monovalent, bivalent, trivalent or tetravalent GalNAc ligands capable of providing 1, 2, 3 or 4 GalNAc or GalNAc derivative moieties.

[0206] In some embodiments, in the dsRNA active agent of the application formed between a dsRNA oligonucleotide and a ligand containing a galactose derivative (e.g., GalNAc) as a targeting group, the molar ratio of the dsRNA oligonucleotide to the galactose derivative (e.g., GalNAc) can be any suitable ratio, e.g., 1:1.

[0207] In some embodiments, the ligand, e.g., monosaccharide, is N-acetylgalactosamine (GalNAc) or a derivative thereof. GalNAc ligands comprising one or more N-acetylgalactosamine (GalNAc) or a derivative thereof are described, e.g., in US 8,106,022, the complete contents of which are hereby incorporated by reference. In some embodiments, the GalNAc ligand is used as a ligand to target the dsRNA active agent to a particular cell. In some embodiments, the GalNAc ligand targets the dsRNA to hepatocytes, e.g., by a ligand that specifically targets asialoglycoprotein receptors of hepatocytes, e.g., hepatocytes (hepatocyte). Exemplary GalNac ligands suitable for delivery of dsRNA include, e.g., Alnylam’s Tri-Touch GalNAc delivery system (see, e.g., PCT / US2008 / 085574, US8828956B2) or “(1+1+1) trivalent GalNac” (non-nucleosidic trivalent GalNAc) (see, e.g., US986788B2, Rajeev, Kallanthottathil G et al. “Hepatocyte-specific delivery of siRNAs conjugated to novel non-nucleosidic trivalent N-acetylgalactosamine elicits robust gene silencing in vivo.” Chembiochem : a European journal of chemical biology vol. 16, 6 (2015): 903-8. doi:10.1002 / cbic.201500023); Dicerna’s GalXC TM Platform or GalXC-Plus TM (involving tetraloop structure coupled to monovalent GalNAc) (see, e.g., WO2016100401A1), or Arrowhead’s Dynamic PolyConjugates (DPC TM containing polymer PBAVE) or TriM TM (Targeted RNNI molecule). The cited documents are incorporated herein in their entirety.

[0208] Commonly used ligands for conjugation to dsRNA can also be found, for example, in those disclosed in WO2023044094A1, WO2023245060A2, WO2018 / 027106, WO2012083185A2, WO2015021092, WO2018044350A1, WO2024 / 197017A2, or WO2023 / 076451A1, the entire contents of which are hereby incorporated by reference.

[0209] In some embodiments, the dsRNA active agents of the present application include one GalNAc or GalNAc derivative linked to the oligonucleotide of the dsRNA active agent. In certain embodiments, the dsRNA active agents of the present application include one or more (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked to the nucleotides of the dsRNA active agent via a monovalent linker.

[0210] In some embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative. In some embodiments, the ligand is one or more GalNAc derivatives linked via a monovalent, bivalent, or trivalent branched linker. In some embodiments, the nucleotides of the dsRNA are conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate or phosphorothioate group. In some embodiments, the ligand is conjugated to the 5’ or 3’ end of the sense and / or antisense strand of the dsRNA active agent. Preferably, the ligand is conjugated to the 3’ end of the sense strand of the dsRNA active agent.

[0211] In some embodiments, the RNAi active agents of the application, such as dsRNA active agents, include one or more GalNAc or GalNAc derivatives as ligands. The GalNAc or GalNAc derivatives can be attached to the RNAi oligonucleotide through a linker, such as a bivalent, trivalent, or tetravalent branch point linker (branching linker). In some embodiments, the ligand is conjugated to the 5' or 3' end of the sense strand and / or antisense strand of the dsRNA active agent. In some embodiments, a GalNAc ligand is conjugated to the 3' end of the sense strand of the RNAi. In some embodiments, a GalNAc ligand is attached to the oligonucleotide at the 3' end of the sense strand of the RNAi through a linker. In some embodiments, a GalNAc ligand is conjugated to the 5' end of the sense strand. In some embodiments, a GalNAc ligand is attached to the oligonucleotide at the 5' end of the sense strand of the RNAi through a linker. In some embodiments, a GalNAc ligand is conjugated to the 3' end of the antisense strand. In some embodiments, a GalNAc ligand is attached to the oligonucleotide at the 3' end of the antisense strand of the RNAi through a linker.

[0212] In some embodiments, the GalNAc or GalNAc derivative is attached to the RNAi of the application through a bivalent linker. In other embodiments, the GalNAc or GalNAc derivative is attached to the RNAi of the application through a trivalent linker. In other embodiments, the GalNAc or GalNAc derivative is attached to the RNAi of the application through a tetravalent linker.

[0213] In some embodiments, one or more of the GalNAc or GalNAc derivatives can be individually attached to the RNAi oligonucleotide via a linker independently of any other GalNAc or GalNAc derivative. In some embodiments, any two or more of the GalNAc or GalNAc derivatives can be attached to the RNAi oligonucleotide via one common linker moiety in a tandem cluster. Further, dsRNA active agents including multiple GalNAc or GalNAc derivatives attached thereto in independent and / or in a tandem cluster are also included in the application.

[0214] In some embodiments, the oligonucleotide of the dsRNA is conjugated to the ligand moiety comprising N-acetylgalactosamine through a phosphoester group or a phosphorothioate group.

[0215] In some embodiments, the ligand of the dsRNA active agent useful in the present application is attached to the 5' and / or 3' terminal nucleotide of the sense strand and, optionally, the 5' and / or 3' terminal nucleotide of the antisense strand of the dsRNA of the present application, optionally, through a phosphorothioate linkage or a phosphate linkage to the nucleotide. In some embodiments, the dsRNA active agent is conjugated to the ligand through a phosphate group or a phosphorothioate group, preferably a phosphate group, for example, the phosphate internucleoside linkage or the phosphorothioate internucleotide linkage is at the 3' terminal end of the sense strand or the antisense strand; or the phosphorothioate internucleotide linkage is at the 5' terminal end of the sense strand or the antisense strand; or the phosphorothioate internucleotide linkage is at both the 5' and 3' terminal ends of the sense strand, and / or the phosphorothioate internucleotide linkage is at both the 5' and 3' terminal ends of the antisense strand.

[0216] In some cases, the ligand of the present application can also be conjugated to the internal sequence of the oligonucleotide of the dsRNA. In some embodiments, the ligand can be attached to the phosphate group, 2'-position hydroxyl group, or the base of the nucleotide. In other embodiments, the ligand can be attached to the 3'-position hydroxyl group of the nucleotide, in which case the nucleotides are linked by 2'-5' phosphodiester linkage. In some embodiments, when the ligand of the present application is attached to the end of the oligonucleotide chain of the dsRNA (such as siRNA), the ligand is usually attached to the phosphate group of the nucleotide; when the ligand is attached to the internal sequence of the oligonucleotide of the dsRNA (such as siRNA), the ligand is usually attached to the sugar ring of the ribose or the base.

[0217] The ligand of the present application can be linked directly to the oligonucleotide strand of the dsRNA of the present application or via a linking moiety (e.g., a linker or a linking group, such as a linking group contained in the ligand). In some embodiments of the present application, the dsRNA active agent of the present application can optionally comprise a linker between the oligonucleotide strand of the dsRNA and the ligand. In some embodiments, the linker is a bio-cleavable linker. In some embodiments, the linker need not be bio-cleavable. In some embodiments, the linker can comprise a branching region. In the present context, the term “branching region” means a moiety of a compound that is capable of covalently coupling two or more entities together. In some embodiments, a linker with a branching region can be used to conjugate multiple entities such as N-acetylgalactosamine moieties to the oligonucleotide of the dsRNA of the present application. Linkers with branching regions useful for this purpose are known in the art, including but not limited to, amino acids (including natural amino acids and non-natural amino acids), peptides and derivatives thereof, sugar units and derivatives thereof, aryl-substituted compounds and derivatives thereof, substituted hydrocarbyl groups and derivatives thereof, triazole-containing derivatives, and the like. See, e.g., CN104651408A, CN113286888A, WO2015 / 173208, and WO2023 / 076451. In some embodiments of the present application, when referring to “ligand” or “ligand moiety”, it can also be a ligand or ligand moiety comprising a linker.

[0218] In some embodiments, the present application also relates to an RNA-induced silencing complex (RISC) comprising the antisense strand of any one of the dsRNA active agents described herein.

[0219] In some embodiments, there is provided an RNAi active agent, e.g., a dsRNA active agent, comprising one or more (e.g., 1) ligand linked to a dsRNA oligonucleotide of the present application, wherein each of the ligands independently has the structure of Formula (I):

[0220] wherein,

[0221] each Gal independently represents a terminal galactose derivative;

[0222] L represents a linker;

[0223] n is an integer selected from 1, 2, 3, and 4; and

[0224] the wavy line indicates linkage to the oligonucleotide of the dsRNA of the present application via this bond. It is understood that the ligand is linked to the 5’ end and / or 3’ end of the sense strand and / or the antisense strand of the oligonucleotide of the dsRNA of the present application, preferably via a phosphonate bond or a phosphorothioate bond.

[0225] In some embodiments, the linker L can have or not have branching moieties. In some embodiments, the branching moieties can present as di-branching, tri-branching or other multi-branching shapes.

[0226] In some embodiments, each Gal is linked to the oligonucleotide of the dsRNA of the application via a linker L independently of the other Gal(s).

[0227] Thus, in some embodiments, each ligand of formula (I) of the application independently has the structure of formula (I-a):

[0228] wherein,

[0229] L A1 represents a linker for connecting the Gal moiety to L A2 moiety;

[0230] L A2 represents a 2-valent, 3-valent, 4-valent or 5-valent linker for connecting n Gal-L A1 moieties to the RNAi of the application;

[0231] each Gal independently represents a terminal galactose derivative; and

[0232] n is an integer selected from 1, 2, 3 and 4; and

[0233] the wavy line represents the attachment to the oligonucleotide of the dsRNA of the application via this valence bond. It is understood that the ligand is attached to the 5’- and / or 3’-end of the sense strand and / or the antisense strand and optionally the 3’-end of the antisense strand of the oligonucleotide of the dsRNA of the application, preferably by a phosphonate or thiophosphonate linkage.

[0234] In the dsRNA active agents of the application comprising a ligand of formula (I) or a subformula thereof such as (I-a), each Gal independently represents a GalNAc (N-acetylgalactosamine) or a GalNAc derivative. In some embodiments, each Gal independently represents a galactose derivative moiety having the structure:

[0235] wherein,

[0236] each R1independently is H or a hydroxyl protecting group,

[0237] R2is selected from hydrogen, hydroxyl, C 1-20 alkyl, C 2-20 alkenyl, C 1-20 alkoxy, C 1-20 alkylthio, -NR a R b , C 6-20 aryl-C0-8 alkylene-O-, C 6-20 aryl-C 0-8 alkylene-S-, and CH3O-(CH2CH2O) q -CH2CH2O-, wherein R a and R b each independently is H or C 1-20 alkyl, q represents an integer from 1 to 16, and wherein the aryl is optionally substituted with one or more C 1-8 alkyl; and

[0238] wavy bond indicates attachment to the remainder of the molecule via that bond.

[0239] In the present context, suitable hydroxyl protecting groups are known to those skilled in the art and include, but are not limited to, acetyl (Ac), benzoyl (Bz), phenoxyacetyl, pivaloyl, methoxymethyl (MOM), methylthiomethyl (MTM), phenoxy methyl (BOM), 2-methoxyethoxy methyl (MEM), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxybenzyloxy methyl (PMBM), triphenylmethyl (Tr), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), isobutyryl, trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), and isopropyldimethylsilyl. In some embodiments, the hydroxyl protecting group can be selected from acetyl (Ac), benzoyl (Bz), phenoxyacetyl, pivaloyl, monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), isobutyryl, t-butyldimethylsilyl, t-butyldiphenylsilyl (TBDPS), triisopropylsilyl, and isopropyldimethylsilyl.

[0240] Accordingly, in some embodiments, each R1is independently H, acetyl (Ac), benzoyl (Bz), monomethoxytrityl (MMTr), dimethoxytrityl (DMTr), or t-butyldiphenylsilyl (TBDPS). Preferably, each R1is independently H, acetyl (Ac), benzoyl (Bz), dimethoxytrityl (DMTr), or t-butyldiphenylsilyl (TBDPS). In some embodiments, each R1is independently H or benzoyl (Bz). In some embodiments, each R1is independently H.

[0241] In some embodiments, each R2is independently selected from the group consisting of: hydrogen, hydroxyl, C 1-6 alkyl, C 2-6 alkenyl, C1-16 alkoxy (e.g., C 1-6 alkoxy), C 1-6 alkylthio, -NR a R b , phenyl-C 0-4 alkylene-O-, phenyl-C 0-4 alkylene-S-, and CH3O-(CH2CH2O) q -CH2CH2O-, wherein R a and R b each independently is H or C 1-6 alkyl such as C 1-4 alkyl, q represents an integer from 1 to 12, and wherein the phenyl is optionally substituted with one or more C 1-4 alkyl groups. In some embodiments, each R2independently is C 1-16 alkoxy, e.g., C 1-6 alkoxy.

[0242] In some embodiments, each Gal independently represents preferably wherein each variable is as defined herein, and the wavy bond indicates attachment to the remainder of the molecule via that bond.

[0243] In some embodiments, each Gal independently represents preferably wherein each variable is as defined herein, and the wavy bond indicates attachment to the remainder of the molecule via that bond.

[0244] In some embodiments, in formula (I-a), L A1 each independently represents a linker having the structure:

[0245] -(CH2) m1 -C(O)-NH-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0246] -(CH2) m1 -C(O)-NH-(CH2) m2 -C(O)-NH-(CH2) m3 -;

[0247] -(CH2) m1 -NH-C(O)-(CH2) m2 -NH-C(O)-(CH2) m3 -;

[0248] -(CH2) m1-NH-C(O)-(CH2) m2 -C(O)-NH-(CH2) m3

[0249] wherein each of ml, m2 and m3 is independently 1, 2, 3, 4, 5, 6, 7 or 8; and wherein the left side of the group is connected to Gal and the right side is connected to the rest of the molecule.

[0250] In some embodiments, ml is preferably 3, 4, 5, 6, 7 or 8, more preferably 3, 4, 5 or 6, most preferably 4.

[0251] In some embodiments, m2 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3.

[0252] In some embodiments, m3 is preferably 1, 2, 3, 4, 5 or 6, more preferably 1, 2 or 3, most preferably 2.

[0253] In some embodiments, L A1 each independently represents a linker selected from the group consisting of:

[0254] wherein each of ml and m2 is independently 1, 2, 3, 4, 5, 6, 7 or 8; and wherein position 1 of the group is connected to Gal and position 2 is connected to the rest of the molecule.

[0255] In some embodiments, L A1 each independently represents a linker selected from the group consisting of:

[0256] wherein position 1 of the group is connected to Gal and position 2 is connected to the rest of the molecule.

[0257] In some embodiments, L A2 represents a 2-, 3- or 4-valent linker comprising a monomethylol methane, dimethylol methane or trimethylol methane moiety, wherein the L A2 is connected to the L A1 is connected to the rest of the molecule of the dsRNA of the application via the methane carbon atom (directly or indirectly).

[0258] In some embodiments, L A2 represents a linker having a structure selected from the group consisting of:

[0259] wherein L A3 represents nothing or a spacer; the oxygen atom on the left side of the group is connected to the L A1 ​partially linked by an ether linkage, the right side is linked to the oligonucleotide of the dsRNA of the invention. The carbon atom marked with an asterisk can be considered to be L A2 the branching point of the moiety.

[0260] In some embodiments, L A3 represents a spacer having the structure:

[0261] preferably

[0262] wherein q is an integer selected from 1 to 16, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16; preferably q is an integer selected from 1 to 12, for example an integer selected from 8 to 12; the 1 -position of said group is connected to the branching point in the moiety A2 the 2-position is connected to the oligonucleotide of the dsRNA of the invention, for example by a phosphonate linkage or a phosphorothioate linkage.

[0263] In some embodiments, L A3 represents a spacer having the structure:

[0264] preferably

[0265] wherein the 1 -position of said group is connected to the branching point in the moiety A2 the 2-position is connected to the oligonucleotide of the dsRNA of the invention, for example by a phosphonate linkage or a phosphorothioate linkage.

[0266] In some embodiments, the ligand of formula (I) or (I-a) each independently has the structure of formula (la-i):

[0267] wherein the variables are as defined herein, for example as defined in formula (I) or (I-a); the wavy line indicates the connection to the oligonucleotide of the dsRNA via this valence bond. It will be appreciated that the ligand is connected to the 5'-end and / or 3'-end of the sense strand and / or the antisense strand of the oligonucleotide of the dsRNA of the invention, preferably by a phosphonate linkage or a phosphorothioate linkage. In a preferred embodiment, each R1independently is H; each of m1and m2independently is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 3 or 4; q is an integer selected from 1 to 16, preferably an integer selected from 1 to 12, more preferably an integer from 8 to 12, for example 8, 9, 10, 11 or 12.

[0268] In particular, the ligand of formula (I) or (I-a) each independently is a L96 moiety having the structure:

[0269] wherein the wavy line indicates attachment to the oligonucleotide of the dsRNA via the valence bond. It is understood that the ligand is attached to the 5' end and / or 3' end of the sense strand and / or the antisense strand of the oligonucleotide of the dsRNA of the application, preferably via a phosphonate bond or a phosphorothioate bond. In some embodiments, the ligand is attached to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the application.

[0270] In some embodiments, the dsRNA active agent of the application having a ligand of formula (Ia-i) is shown below:

[0271] wherein, indicates the dsRNA oligonucleotide duplex as described herein, 3' indicates the 3' end of the sense strand of the dsRNA, X indicates oxygen or sulfur, and the other variables are as defined herein.

[0272] In particular, the dsRNA active agent of the application having a ligand of structure L96 is shown below:

[0273] wherein, indicates the dsRNA oligonucleotide duplex as described herein, 3' indicates the 3' end of the sense strand of the dsRNA, X indicates oxygen or sulfur.

[0274] In some embodiments, in the dsRNA active agent of the application, the dsRNA oligonucleotide duplex is attached to one ASGPR ligand, wherein the ASGPR ligand has any structure selected from formula (I) or any subformula thereof, for example a structure selected from any one of (I-a), (Ia-i), or L96, and the ASGPR ligand is attached to the 5' end or 3' end of the sense strand and / or the antisense strand of the oligonucleotide of the dsRNA of the application. Preferably, the ligand is attached via a phosphonate bond or a phosphorothioate bond.

[0275] In some embodiments, in the dsRNA active agent of the application, the dsRNA oligonucleotide duplex is attached to one ASGPR ligand, wherein the ASGPR ligand has the structure of formula (I-a), preferably formula (Ia-i), more preferably L96, and the ASGPR ligand is attached to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the application. Preferably, the ligand is attached via a phosphonate bond or a phosphorothioate bond.

[0276] In some embodiments, the ligand is L96, see for example WO2009073809 and WO2009082607, which are incorporated herein by reference in their entirety.

[0277] In some embodiments, in the dsRNA active agents of the application, the double-stranded linkage of the dsRNA oligonucleotide is linked with a GalNac ligand, such as a ligand having the structure of L96, and the ligand is linked to the 3' end of the sense strand of the oligonucleotide of the dsRNA of the application, wherein the ligand is linked via a phosphonate linkage or a phosphorothioate linkage, preferably a phosphonate linkage.

[0278] In some embodiments, the ligand is

[0279] In some embodiments, the dsRNA active agents comprise the ligand conjugated nucleotides, as shown in the following schematic

[0280] and wherein X is O or S, and the double-stranded structure is a nucleotide strand of the dsRNA, such as a double-stranded region formed by a sense strand and an antisense strand.

[0281] In some embodiments, the antisense strand in the dsRNA active agents comprises any one of the nucleotide sequences in the antisense strands of Table 1 (encompassing nucleotide sequences with modified nucleotides), and / or the sense strand comprises any one of the nucleotide sequences in the sense strands of Table 1 (encompassing nucleotide sequences with modified nucleotides), wherein the sense strand is conjugated at the 3' end with one of the ligands of the application, such as an ASGPR ligand or a GalNac ligand, such as having the structure of formula (I-a), preferably formula (la-i), more preferably L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0282] In some embodiments, the combination of antisense strand and sense strand in the dsRNA active agents is shown by any combination of the nucleotide sequences (encompassing nucleotide sequences with modified nucleotides) of the antisense strands and the nucleotide sequences (encompassing nucleotide sequences with modified nucleotides) of the sense strands shown in Table 1, wherein the sense strand is conjugated at the 3' end with one of the GalNac ligands of the application, such as an ASGPR ligand or a GalNac ligand, such as having the structure of formula (I-a), preferably formula (la-i), more preferably L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0283] In some embodiments, the dsRNA active agents are any of the dsRNA active agents comprising the nucleotide sequences (encompassing nucleotide sequences with modified nucleotides) shown in Table 1, and wherein the sense strand is conjugated at the 3' end with one of the GalNac ligands of the application, such as an ASGPR ligand or a GalNac ligand, such as having the structure of formula (I-a), preferably formula (la-i), more preferably L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0284] In some embodiments, the antisense strand in the dsRNA active agent comprises any of the modified nucleotide sequences in the antisense strands in Table 2 of the specification, and / or the sense strand comprises any of the modified nucleotide sequences in the sense strands in Table 2 of the specification, wherein the sense strand is conjugated at the 3’ end to a ligand of the disclosure, such as an ASGPR ligand or a GalNac ligand, for example having the structure of Formula (I-a), preferably Formula (la-i), more preferably L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0285] In some embodiments, the combination of antisense and sense strands in the dsRNA active agent is as shown for any of the combinations of antisense and sense strands shown in Table 2, wherein the sense strand is conjugated at the 3’ end to a GalNac ligand of the disclosure, such as an ASGPR ligand or a GalNac ligand, for example having the structure of Formula (I-a), preferably Formula (la-i), more preferably having the structure of L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0286] In some embodiments, the dsRNA active agent is any of the dsRNA active agents shown in Table 2, and wherein the sense strand is conjugated at the 3’ end to a GalNac ligand of the disclosure, such as an ASGPR ligand or a GalNac ligand, for example having the structure of Formula (I-a), preferably Formula (la-i), more preferably having the structure of L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0287] In some embodiments, the antisense strand in the dsRNA active agent comprises any of the modified nucleotide sequences in the antisense strands in Table 3 of the specification, and / or the sense strand comprises any of the modified nucleotide sequences in the sense strands in Table 3 of the specification, wherein the sense strand is conjugated at the 3’ end to a ligand of the disclosure, such as an ASGPR ligand or a GalNac ligand, for example having the structure of Formula (I-a), preferably Formula (la-i), more preferably L96, preferably the ligand is linked via a phosphonate linkage or a phosphorothioate linkage.

[0288] In some embodiments, the combination of modified antisense and sense strands in the dsRNA active agent of the disclosure is as shown for the combinations of antisense and sense strands shown in Table 3. In some embodiments, the sense strand and the antisense strand in the dsRNA active agent comprise, or alternatively consist of, the modified nucleotide sequence of the sense strand and the modified nucleotide sequence of the antisense strand, respectively, in any of the combinations of sense and antisense strands selected from the combinations of sense and antisense strands in Table 3, conjugated at the 3’ end to the L96 ligand structure via a phosphonate linkage.

[0289] In some embodiments, the dsRNA active agents conjugated to a ligand of the present disclosure are the dsRNA active agents set forth in Table 3. In some embodiments, the combination of antisense and sense strands in the dsRNA active agents are set forth as any of the combinations of antisense and sense strands set forth in Table 3. In some embodiments, the dsRNA active agents are any of the dsRNA active agents set forth in Table 3.

[0290] In some specific embodiments, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the antisense and sense strands respectively comprise at least 15, 16, 17, 18, or 19 consecutive modified nucleotides in the nucleotide sequence set forth in any one of SEQ ID NO: 203 / 336, SEQ ID NO: 204 / 337, SEQ ID NO: 206 / 338, SEQ ID NO: 207 / 339, SEQ ID NO: 215 / 340, or SEQ ID NO: 225 / 341, wherein the sense strand is linked to L96 via a phosphate linkage (e.g., the 3’ terminal nucleotide of the sense strand is linked to L96 via a phosphate linkage), optionally the antisense strand further has a 3’ overhang of 1 or 2 nucleotides, optionally the first nucleotide at the 5’ end of the antisense strand is a modified A or U, e.g., a modified U, e.g., Um or Am.

[0291] In some specific embodiments, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the antisense and sense strands respectively comprise or consist of the nucleotide sequence set forth in any one of SEQ ID NO: 203 / 336, SEQ ID NO: 204 / 337, SEQ ID NO: 206 / 338, SEQ ID NO: 207 / 339, SEQ ID NO: 215 / 340, or SEQ ID NO: 225 / 341.

[0292] In some specific embodiments, the dsRNA active agent comprises a sense strand and an antisense strand, wherein

[0293] a. the sense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 336 conjugated at the 3’ end with the L96 structural ligand, and the antisense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 203;

[0294] b. the sense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 337 conjugated at the 3’ end with the L96 structural ligand, and the antisense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 204;

[0295] c. the sense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 338 conjugated at the 3' end with a L96 structure ligand, and the antisense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 206;

[0296] d. the sense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 339 conjugated at the 3' end with a L96 structure ligand, and the antisense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 207;

[0297] e. the sense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 340 conjugated at the 3' end with a L96 structure ligand, and the antisense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 215; or

[0298] f. the sense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 341 conjugated at the 3' end with a L96 structure ligand, and the antisense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 225.

[0299] In some embodiments, the dsRNA active agents of the present application are GalNAc-siRNA conjugates selected from the group consisting of:

[0300] In some embodiments, the ligand is a lipid or lipid-based molecule. In one embodiment, such lipid or lipid-based molecule binds to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into target cells or cell membranes, or (c) can be used to modulate binding to serum proteins, such as HSA. In some embodiments, the ligand is a lipid nanoparticle, such as an LNP. Exemplary LNP delivery systems are, for example, DLin-DMA, DLin-MC3-DMA, L319, PNP (peptide nanoparticles) delivery platform, EDV (EnGeneIC Dream Vector) endogenous delivery vehicle nanocell platform.

[0301] In some embodiments, antibodies can also be applied as ligands for delivery of dsRNA, such as antibody oligonucleotide conjugate drugs (AOCs).

[0302] In some embodiments, a polymeric matrix can also be used to co-deliver the dsRNA, such as the LODER (LOcal Drug EluteR) delivery platform.

[0303] In some embodiments, the ligand is attached to the 5' and / or 3' terminal nucleotide of the sense strand and, optionally, the 5' and / or 3' terminal nucleotide of the antisense strand of the dsRNA of the application, optionally, through a phosphorothioate linkage or a phosphodiester linkage to the nucleotide, such as through an internucleotide linkage. In some embodiments, the dsRNA active agent is conjugated to the ligand through a phosphodiester group or a phosphorothioate group, such as the phosphorothioate internucleotide linkage is at the 3' terminal of the sense strand or the antisense strand; or the phosphorothioate internucleotide linkage is at the 5' terminal of the sense strand or the antisense strand; or the phosphorothioate internucleotide linkage is at both the 5' and 3' terminal of the sense strand, and / or the phosphorothioate internucleotide linkage is at both the 5' and 3' terminal of the antisense strand.

[0304] In some cases, the ligand can also be conjugated to an internal sequence of the oligonucleotide of the dsRNA. In some embodiments, the ligand can be attached to the phosphate group, 2'-position hydroxyl group, or the base of the nucleotide. In other embodiments, the ligand can be attached to the 3'-position hydroxyl group of the nucleotide, when the nucleotides are linked by 2'-5' phosphodiester linkage. In some embodiments, when the ligand is attached to the terminal of the nucleotide chain of the dsRNA (such as siRNA), the ligand is usually attached to the phosphate group of the nucleotide; when the ligand is attached to an internal sequence of the nucleotide of the dsRNA (such as siRNA), the ligand is usually attached to the sugar ring of the ribose or the base.

[0305] The ligand can be directly linked to the nucleotide duplex of the dsRNA of the application or linked via a linking moiety (e.g., a linker). In some embodiments of the application, the dsRNA of the application can optionally comprise a linker between the nucleotide strand of the dsRNA and the ligand. In some embodiments, the linker is a bio-cleavable linker. In some embodiments, the linker need not be bio-cleavable. In some embodiments, the linker can comprise a branching region. In this context, the term “branching region” means a moiety of a compound that is capable of covalently coupling two or more entities together. In some embodiments, a linker with a branching region can be used to conjugate multiple entities such as N-acetylgalactosamine moieties to the oligonucleotide of the dsRNA of the application. Linkers with branching regions useful for this purpose are known in the art, including but not limited to, amino acids (including natural amino acids and non-natural amino acids), peptides and derivatives thereof, saccharide units and derivatives thereof, aryl-substituted compounds and derivatives thereof, substituted hydrocarbyl groups and derivatives thereof, triazole-containing derivatives, and the like. See, e.g., CN104651408A, CN113286888A, WO2015 / 173208, and WO2023 / 076451.

[0306] The RNAi active agents of the application, e.g., dsRNA active agents, can be delivered or introduced by any means known in the art (e.g., to cells in vitro, to test animals, or to humans). In some embodiments, the RNAi active agents of the application, e.g., dsRNA active agents, are delivered to a subject in a manner such that the RNAi active agents, e.g., dsRNA active agents, can hybridize to the mRNA of DGAT2 within a cell comprising DGAT2 and inhibit expression by inhibiting transcription. Examples of routes of delivery of the RNAi active agents, e.g., dsRNA active agents, include direct injection at a tissue site. Alternatively, the RNAi active agents, e.g., dsRNA active agents, can be modified to target selected cells and then administered systemically. The nucleic acid molecules of the application can be delivered to cells using carriers that are generally known in the art and described, e.g., in US20070111230 (the entire contents of which are incorporated herein).

[0307] The present application also relates to an RNA-induced silencing complex (RISC) comprising the antisense strand of any one of the dsRNA active agents according to the present application.

[0308] The various embodiments and examples described herein, and any combination thereof, are equally applicable to the dsRNA active agents of the present application. For example, the variable definitions described for Formula (I) (e.g., Gal) can be equally applied to other RNAi active agents, including dsRNA active agents. Moreover, the various aspects and / or embodiments and / or examples and / or features described herein can be combined in any manner, whereby the resulting technical solutions are also included within the scope of the present application.

[0309] III. Preparation of dsRNA active agents

[0310] The dsRNA can be synthesized by standard methods known in the art. The double stranded RNA active agents of the present application can be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. In some embodiments, the individual strands of the dsRNA, such as siRNA, can be prepared using solution phase or solid phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide strands comprising non-natural or modified nucleotides can be readily prepared.

[0311] In some embodiments, the dsRNA of the present application is prepared by RNA solid phase synthesis. RNA solid phase synthesis is a commonly used technique for synthesizing RNA molecules that allows for the stepwise construction of RNA strands on a solid support. This method has the advantages of high throughput, high efficiency, and automation, and is widely used in the biotechnology and research fields. In some embodiments, RNA solid phase synthesis comprises the following basic methods and steps:

[0312] 1. Template recognition:

[0313] In solid phase synthesis, a template is first required, usually a single-stranded DNA that contains the sequence complementary to the desired RNA sequence. This template DNA is immobilized on a solid support, such as controlled pore glass (CPG) or polystyrene beads.

[0314] 2. Transcription initiation:

[0315] The RNA polymerase recognizes and binds to the promoter sequence on the template DNA. In solid phase synthesis, this process usually does not require a primer, as the RNA polymerase can start synthesizing the RNA strand directly at the promoter region.

[0316] 3. Transcription elongation:

[0317] Once the RNA polymerase binds to the promoter, it begins to synthesize the RNA strand on the template DNA. In this process, the RNA polymerase moves along the DNA template, adding nucleotide triphosphates (NTPs) complementary to the template DNA bases one by one. Each time an NTP is added, the RNA strand is extended by one nucleotide at the 3' end.

[0318] 4. Cycling synthesis:

[0319] A key feature of solid-phase synthesis is that multiple cycles of synthesis can be performed. Each cycle involves the addition of a new NTP, removal of unreacted NTPs, and elution and re-binding of the RNA polymerase. This process can be automated, greatly improving the efficiency of synthesis.

[0320] 5. Transcription termination:

[0321] When the RNA strand reaches the desired length, the transcription process needs to be terminated. This is typically achieved by the addition of a specific termination signal or chemical.

[0322] 6. Post-treatment:

[0323] After synthesis is complete, the RNA strand needs to be released from the solid support and any unreacted NTPs, protecting groups, and other impurities need to be removed. This is typically achieved by chemical or enzymatic methods, such as the use of specific enzymes to cleave the linkage on the solid support.

[0324] 7. Purification and analysis:

[0325] Finally, the synthesized RNA needs to be purified by appropriate methods (such as gel electrophoresis, column chromatography, etc.). The purified RNA can be verified for its length, purity, and sequence accuracy by various analytical methods (such as capillary electrophoresis, mass spectrometry, etc.).

[0326] In some embodiments, the solid support is a blank solid support, e.g., a blank CPG solid support. In some embodiments, the solid support is a solid support comprising a ligand.

[0327] IV. Pharmaceutical compositions

[0328] In some embodiments, the present application provides a composition comprising a dsRNA active agent of the present application or a pharmaceutically acceptable salt thereof, preferably the composition is a pharmaceutical composition or a pharmaceutical formulation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, a composition, e.g., a pharmaceutical composition, comprises a dsRNA active agent of the present application in combination with one or more other therapeutic agents.

[0329] The present application also includes compositions (including pharmaceutical compositions) comprising a dsRNA active agent of the present application or a pharmaceutically acceptable salt thereof. These compositions can further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as are known in the art.

[0330] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents, and the like, which are physiologically compatible. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol or combinations thereof.

[0331] For the use of pharmaceutical excipients and their purposes, see also "Handbook of Pharmaceutical Excipients", 8thEdition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago.

[0332] The compositions of the application can be in a variety of forms. These forms include, for example, liquid, semi-solid and solid dosage

[0333] Pharmaceutical or pharmaceutical compositions comprising the dsRNA active agents of the application can be prepared by mixing the dsRNA active agents of the application having the desired degree of purity with one or more optional pharmaceutically acceptable excipients.

[0334] In some embodiments, the dsRNA active agents according to the application can be present in a non-buffered solution, preferably the non-buffered solution is saline or water. In other embodiments, the dsRNA active agents are in a buffered solution, for example wherein the buffered solution comprises acetate, citrate, prolamine, carbonate or phosphate or any combination thereof, such as phosphate buffered saline (PBS).

[0335] The pharmaceutical compositions or formulations of the application can also contain more than one active ingredient desired for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In some embodiments, the other active ingredients are, for example, various therapeutic agents for the treatment of chronic inflammatory diseases. The active ingredients are suitably present in amounts effective for the purpose intended.

[0336] In some embodiments, the pharmaceutical compositions or formulations of the application can be contained in a vial, or contained in a syringe.

[0337] V. Pharmaceutical Combinations and Kits

[0338] In some embodiments, the present application also provides a pharmaceutical combination or pharmaceutical combination product comprising a dsRNA active agent of the application, and one or more other therapeutic agents.

[0339] It is another object of the application to provide a kit-of-parts comprising a pharmaceutical combination of the application, preferably said kit is in the form of a pharmaceutical dosage unit. Thereby dosage units can be provided according to a dosing regimen or pharmaceutical administration interval.

[0340] In one embodiment, the kit-of-parts of the application comprises within the same package:

[0341] - a first container containing a pharmaceutical composition comprising a dsRNA active agent of the application;

[0342] - a second container containing a pharmaceutical composition comprising a further therapeutic agent.

[0343] In some embodiments, the further therapeutic agent, e.g. any therapeutic agent effective in the prevention or treatment of a DGAT2-associated disease and / or disorder, encompasses various therapeutic agents used in the treatment of chronic inflammatory diseases.

[0344] VI. Uses and methods

[0345] One aspect of the application provides a method of inhibiting the expression and / or activity of a DGAT2 gene in a cell, comprising contacting said cell with a dsRNA active agent, a pharmaceutical composition, a pharmaceutical combination product of the application, whereby the expression of said DGAT2 gene in said cell is inhibited. In some embodiments, said cell is in a subject. In some embodiments, said cell is a hepatocyte. In some embodiments, said subject is suffering from a DGAT2-associated disease and / or disorder.

[0346] In some embodiments, contacting the cell with the dsRNA active agent inhibits expression (e.g., mRNA levels) of DGAT2 by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%. In some embodiments, inhibiting expression of DGAT2 reduces the level of DGAT2 protein in the liver of the subject by at least 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%. In some embodiments, administration of the dsRNA active agent to a subject results in a reduction in DGAT2 concentration or content (e.g., in the liver, e.g., in liver tissue or liver cells), or a reduction in accumulation of DGAT2 protein or a reduction in DGAT2 protein content in the subject (e.g., in the liver, e.g., in liver tissue or liver cells). In some embodiments, administration of the dsRNA active agent to a subject results in a reduction in DGAT2 expression or protein content in the liver, e.g., in liver tissue or liver cells, of the subject.

[0347] In one aspect, the present application provides methods of preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a dsRNA active agent, pharmaceutical composition, pharmaceutical combination, or kit of the present application. In some embodiments, the disease is a DGAT2-associated disease and / or disorder.

[0348] In some embodiments, the present application relates to a dsRNA active agent, pharmaceutical composition, pharmaceutical combination, or kit of the present application for use in therapy, e.g., for use in treating a DGAT2-associated disease and / or disorder.

[0349] In some embodiments, the present application relates to a method of treating a disease, e.g., a DGAT2-associated disease or disorder mentioned herein, or use for such treatment, or use for the manufacture of a medicament for such treatment, utilizing a dsRNA active agent, pharmaceutical composition, pharmaceutical combination, or kit of the present application.

[0350] In some embodiments, the disease or disorder is associated with increased expression or activity of DGAT2. In some embodiments, the disease or disorder is caused by gene expression (e.g., abnormal expression) of DGAT2. In some embodiments, the disease or disorder is an indication that would benefit from a reduction in diacylglycerol O-acyltransferase 2 (DGAT2) expression and / or activity.

[0351] In some embodiments, "increased expression or activity of DGAT2" refers to increased expression (e.g., nucleic acid or protein level) or activity of DGAT2 in a subject's tissue or cell, e.g., liver or liver cell. In some embodiments, the DGAT2 associated disease and / or disorder is a patient having DGAT2 with medium or high expression in the liver or liver cells. In some embodiments, the DGAT2 associated disease and / or disorder is a patient having an amount of DGAT2 in the liver that is higher than the amount of DGAT2 in the liver or body fluid of a healthy control. In some embodiments, the DGAT2 associated disease and / or disorder is a patient having lipid droplet accumulation in the liver due to increased expression or activity of DGAT2.

[0352] In some embodiments, the DGAT2 associated disease and / or disorder is a chronic inflammatory disease, e.g., in the liver and other tissues. In some embodiments, the DGAT2 associated disease and / or disorder is a metabolic dysfunction related chronic inflammatory disease. In one instance, the chronic inflammatory disease is a chronic inflammatory liver disease. In one instance, the chronic inflammatory liver disease is selected from the group consisting of liver fat accumulation, liver inflammation, liver fibrosis, fatty liver disease (steatosis), fatty hepatitis such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and cirrhosis. In one embodiment, the chronic inflammatory disease is a chronic inflammatory liver disease, e.g., inflammation of the liver, liver fibrosis, fatty hepatitis such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic steatohepatitis (ASH), alcoholic liver disease (ALD), HCV-related cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin insensitivity, and / or diabetes.

[0353] The dsRNA active agents of the application or compositions or medicaments or formulations comprising the same can also be administered in combination with one or more other therapies, e.g., other treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or disorders mentioned herein. Accordingly, the present application also relates to combination therapies of the dsRNA active agents of the application or compositions or medicaments or formulations comprising the same with one or more other therapies.

[0354] In other aspects, the present application provides the use of the dsRNA active agents of the application or compositions or medicaments or formulations comprising the same or combination products in the manufacture or preparation of a medicament for the uses described herein, e.g., for preventing or treating the relevant diseases or disorders mentioned herein.

[0355] In some embodiments, the method or use further comprises determining the level of DGAT2 (e.g. protein or nucleic acid level, e.g. mRNA level) in the subject or in the liver or liver cells prior to administering the dsRNA active agent. In some embodiments, the level of DGAT2 is compared to the level of DGAT2 (e.g. protein or nucleic acid level, e.g. mRNA level) in a healthy subject or in a healthy liver or liver cells, and if the level of DGAT2 in the subject or in the liver or liver cells is higher than the level of DGAT2 in the healthy control, the subject is administered the dsRNA active agent or a composition or medicament or formulation or combination product comprising the same.

[0356] The subject can be a mammal, for example, a primate, preferably, a higher primate, e.g., a human (e.g., an individual having or at risk of having a disease described herein).

[0357] In one embodiment, the subject has or is at risk of having a disease described herein.

[0358] Combination therapies of the application encompass combined administration (e.g., two or more therapeutic agents are included in the same formulation or separate formulations), and separate administration, in which case, administration of a dsRNA active agent of the application or a composition or medicament or formulation comprising the same can precede, coincide with, and / or follow the administration of the other therapeutic agent and / or active agent.

[0359] In some embodiments, other therapeutic agents that can be combined or co-administered with a dsRNA active agent or medicament or formulation or composition of the application can be any therapeutic agent effective in preventing or treating a DGAT2-related disease and / or disorder, encompassing various therapeutic agents used in the treatment of chronic inflammatory diseases. Examples

[0360] Example 1 siRNA synthesis

[0361] RNA solid-phase synthesis is a commonly used technique for synthesizing RNA molecules that allows for the stepwise construction of RNA strands on a solid support. This method has the characteristics of high throughput, high efficiency and automation, and is widely used in biotechnology and research fields. The following is the synthesis of the DGAT2 RNAi agent duplex shown in Table 2 according to the following method: All RNAs and 2'-modified phosphoramidites were purchased from Shanghai Zhiwei Technology Development Co., Ltd. Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5'-O-dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidites have the same protecting groups as the 2'-O-methyl phosphoramidites. The abasic (3'-O-dimethoxytrityl-2'-deoxyribo-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Shanghai Zhiwei Technology Development Co., Ltd. The target ligand containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3A) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM, dissolved in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM, dissolved in acetonitrile) was used as the activator solution. The coupling time was 12 minutes (RNA), 15 minutes (target ligand), 90 seconds (2'OMe), and 60 seconds (2'F). To introduce the phosphorothioate linkage, 100 mM 3-phenyl 1,2,4-dithiazoline-5-one (POS, commercially available from PolyOrg, Inc., Leominster, MA, USA) dissolved in anhydrous acetonitrile was used.

[0362] 1. Preparation of siRNA without ligand

[0363] 1.1 Synthesis of the sense strand (SS strand)

[0364] By solid phase phosphoramidite synthesis method, using blank CPG solid phase carrier as the starting cycle, according to the order of nucleotide arrangement of the sense strand, nucleoside monomers or nucleotide analog monomers are connected one by one from 3'-5' direction. Each connection of nucleoside monomers or nucleotide analog monomers contains four-step reactions of deprotection, coupling, capping, oxidation or thio. The synthesis scale of 5 μmol of oligonucleic acid is as follows:

[0365] Nucleoside monomers or nucleotide analog monomers provide 0.05 mol / L acetonitrile solution. The conditions of each reaction are the same, i.e. the temperature is 25°C, 3% trichloroacetic acid-dichloromethane solution is used for deprotection, and deprotection is performed for 3 times; the activator used for coupling reaction is 0.25 mol / L ETT-acetonitrile solution, and coupling is performed for 2 times; capping uses 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v), and capping is performed for 2 times; oxidation uses 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v), and oxidation is performed for 2 times; thio uses 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1 / 1, v / v), and thio is performed for 2 times.

[0366] 1.2 Synthesis of antisense strand (AS strand)

[0367] By solid phase phosphoramidite synthesis method, using blank CPG solid phase carrier as the starting cycle, according to the order of nucleotide arrangement of the antisense strand, nucleoside monomers or nucleotide analog monomers are connected one by one from 3'-5' direction. Each connection of nucleoside monomers or nucleotide analog monomers contains four-step reactions of deprotection, coupling, capping, oxidation or thio. The synthesis conditions of 5 μmol of oligonucleic acid of the antisense strand are the same as those of the sense strand.

[0368] 1.3 Purification and annealing of oligonucleic acid

[0369] 1.3.1 aminolysis

[0370] The synthesized solid phase carrier (sense strand or antisense strand) is added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and the reaction is performed at 35°C (or 55°C) constant temperature water bath for 16 hours (or 8 hours). The solid phase carrier is washed with ethanol / water three times, 1 mL each time, and the filtrate is concentrated by centrifugation. The crude product is purified.

[0371] 1.3.2 purification

[0372] Crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5 PW 13 μιη column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, containing 20% acetonitrile and buffer B was identical to buffer A with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled and then run on size exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 gel, using either filtered DI water or a running buffer of 100 mM ammonium bicarbonate, pH 6.7, containing 20% acetonitrile.

[0373] 1.3.3 Annealing

[0374] Sense strands (SS strands) were mixed with antisense strands (AS strands) in a molar ratio (SS strands / AS strands = 1 / 1.05) according to Table 2, heated to 70-95 degrees in a water bath for 3-5 min, allowed to cool to room temperature naturally, and the system was lyophilized to obtain the product. Some RNAi agents were lyophilized and stored at -15 to -25 °C. The duplex concentration was determined by measuring the absorbance of a solution in 1x phosphate buffered saline using a UV-Vis spectrometer. The solution absorbance at 260 nm was then multiplied by a conversion factor and a dilution factor to determine the duplex concentration. Unless otherwise noted, all conversion factors were 0.037 mg / (mL-cm). For some experiments, the conversion factor was calculated from the extinction coefficient determined experimentally.

[0375] 2. Preparation of siRNA active agents linked to GalNac ligands

[0376] The synthesis of the antisense strand is carried out by the solid phase phosphoramidite method, using a solid support as the starting cycle, and the nucleoside monomers are connected one by one in the order of 3'-5' according to the nucleotide sequence of the antisense strand. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiation. The synthesis conditions of the 1 μmol oligonucleotide of the antisense strand are the same as those of the sense strand.

[0377] 2.2 Synthesis of the antisense strand (AS strand)

[0378] The synthesis of the antisense strand is carried out by the solid phase phosphoramidite method, using a solid support as the starting cycle, and the nucleoside monomers are connected one by one in the order of 3'-5' according to the nucleotide sequence of the antisense strand. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thiation. The synthesis conditions of the 1 μmol oligonucleotide of the antisense strand are the same as those of the sense strand.

[0379] 2.3 Purification and annealing of the oligonucleotide

[0380] 2.3.1 Aminolysis

[0381] The synthesized solid support (sense strand or antisense strand) is added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and the reaction is carried out at 35°C (or 55°C) constant temperature water bath for 16 hours (or 8 hours). The solid support is washed with ethanol / water three times, 1 mL each time, and the filtrate is concentrated by centrifugation. The crude product is purified.

[0382] 2.3.2 Purification

[0383] The methods of purification and desalting are well known to those skilled in the art. For example, a strong anion filler column can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification. The product is collected and subjected to gel filler purification column for desalting, and the elution system is pure water.

[0384] 2.3.3 Annealing

[0385] The sense strand (SS strand) chain and antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated to 70-95°C in a water bath for 3-5 min, and naturally cooled to room temperature. The system was freeze-dried to obtain the product.

[0386] Table 1: Sense and antisense strands of DGAT2 unmodified double-stranded RNA

[0387] Table 2: Sense and antisense strands of DGAT2 modified double-stranded RNA

[0388] The naked sequence of the sequence corresponding to each SEQ ID NO. is listed in the sequence listing in ST26 format.

[0389] Table 3: Sense and antisense strands of DGAT2 modified GalNac-siRNA

[0390] The naked sequence of the sequence corresponding to each SEQ ID NO. is listed in the sequence listing in ST26 format.

[0391] Example 2 Inhibition of human DGAT2 in Hep3B cells by siRNA

[0392] Hep3B2.1-7 cells (Sbiotech (Shanghai) Biotechnology Co., Ltd., Cat# iCell-h091) were cultured in EMEM medium (ATCC, Cat# 30-2003) containing 10% fetal bovine serum at 37°C, 5% CO2.

[0393] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat# 13778150) according to the following procedure: siRNA was prepared to 1 mM working solution with DEPC-treated water. Solution A was prepared, each part of solution A contained 1 μΐ of siRNA working solution and 9 μΐ of Opti-MEM medium (GIBCO, Cat#31985070). Solution B was prepared, each part of solution B contained 0.3 μΐ of Lipofectamine RNAiMAX and 9.7 μΐ of Opti-MEM medium. After mixing solution A and B, the mixture was placed in a 96-well plate and incubated at room temperature for 20 min, then 80 μΐ of Hep3B cells were added, each well contained 15000 cells, the final concentration of siRNA was 10 nM. Each siRNA and negative control were detected in two wells, respectively. After transfection for 24 hours, the cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat# CL132), and reverse transcription and qPCR were performed according to the kit instructions, and the DGAT2 mRNA level was determined. The DGAT2 mRNA level was corrected according to the GAPDH internal standard mRNA level. The DGAT2 mRNA expression level was calculated by the ΔΔCt relative quantification method, expressed as the percentage of the remaining expression of DGAT2 mRNA in the cells treated with negative siRNA control group, and the calculation formula was as follows:

[0394] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0395] ΔΔCt = ΔCt (dosing group) - ΔCt (negative siRNA control group)

[0396] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0397] Table 4: Single dose screening of dsRNA drugs against DGAT2 in Hep3B cells

[0398] The results of Table 4 show the inhibition of DGAT2 mRNA after transfection of siRNA molecules in Table 2 in Hep3B.

[0399] Example 3 Inhibition of human DGAT2 in Hep3B cells by siRNA

[0400] Hep3B2.1-7 cells (iCell Bioscience Co., Ltd., Cat#iCell-h091) were cultured in EMEM medium (ATCC, Cat#30-2003) containing 10% fetal bovine serum at 37°C, 5% CO2.

[0401] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150) according to the following procedure: siRNA was prepared into 1 mM working solution with DEPC-treated water. Solution A was prepared, each containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (GIBCO, Cat#31985070). Solution B was prepared, each containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. After mixing solution A and solution B, the mixture was placed in a 96-well plate and incubated at room temperature for 20 min, followed by the addition of 80 μl of Hep3B cells, 15000 cells per well, and the final concentration of siRNA was 10 nM. Each siRNA and negative control was tested in two wells, respectively. After transfection for 24 hours, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine the DGAT2 mRNA level. The DGAT2 mRNA level was corrected according to the GAPDH internal mRNA level. The DGAT2 mRNA expression level was calculated by the ΔΔCt relative quantification method, expressed as the percentage of the residual expression of DGAT2 mRNA in the cells treated with the negative siRNA (negative control, PBS) control group, and the calculation formula was as follows:

[0402] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0403] ΔΔCt = ΔCt (dosing group) - ΔCt (negative siRNA control group)

[0404] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0405] The results of Figure 1 (two wells and average values are listed for each sample, respectively) show the single-point inhibition of DGAT2 mRNA after transfection of siRNA molecules in Hep3B, indicating that the detected siRNA molecules can effectively inhibit the expression level of DGAT2 mRNA in Hep3B.

[0406] Example 4 Inhibition of human DGAT2 in Hep3B cells by siRNA

[0407] Hep3B2.1-7 cells (iCell Bioscience Co., Ltd., Cat# iCell-h091) were cultured in EMEM medium (ATCC, Cat# 30-2003) containing 10% fetal bovine serum at 37°C, 5% CO2.

[0408] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat# 13778150) according to the following procedure: siRNA was prepared into 1 mM working solution with DEPC-treated water. Solution A was prepared, each containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (GIBCO, Cat# 31985070). Solution B was prepared, each containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. After mixing solution A and solution B, the mixture was incubated at room temperature for 20 min, and then 80 μl of Hep3B cells (15000 cells per well) were added, with a final siRNA stock concentration of 10 nM, followed by 7 wells of 10-fold gradient dilution. After 24 hours of transfection, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat# CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine the DGAT2 mRNA level. The DGAT2 mRNA level was corrected according to the GAPDH internal standard mRNA level. The DGAT2 mRNA expression level was calculated by the ΔΔCt relative quantification method, expressed as the percentage of the residual expression of DGAT2 mRNA relative to the cells treated with negative siRNA control (PBS), and the calculation formula was as follows:

[0409] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0410] ΔΔCt = ΔCt (dose group) - ΔCt (negative siRNA control group)

[0411] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0412] Normalized mRNA remaining = (mRNA relative expression level per well / negative control well relative expression level) * 100

[0413] The results of Figure 2 demonstrate the normalized suppression of DGAT2 mRNA after transfection of siRNA molecules in Hep3B.

[0414] Example 5 Inhibition of human DGAT2 in human primary hepatocytes by siRNA

[0415] Activity screening of siRNA in human primary hepatocytes was performed using a concentration gradient.

[0416] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat# 13778150) according to the product manual, with a final siRNA concentration of 10 nM. Equal volume of diluted Lipofectamine RNAiMAX and siRNA in Opti-MEM medium (GIBCO, Cat#31985070) was mixed and incubated at room temperature for 20 min, then added to a 96-well plate at a volume of 20 μl per well, followed by the addition of 80 μl of thawed human primary hepatocytes, 40,000 cells per well, with a final siRNA stock concentration of 10 nM, followed by a 6-fold gradient dilution of 7 wells. After 24 hours of transfection, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat# CL132), and reverse transcription and qPCR were performed according to the kit manual. DGAT2 mRNA levels were determined. DGAT2 mRNA levels were corrected according to GAPDH internal standard mRNA levels. DGAT2 mRNA expression levels were calculated by the ΔΔCt relative quantification method, expressed as the percentage of residual expression of DGAT2 mRNA in cells treated with negative siRNA control, and the calculation formula is as follows:

[0417] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0418] ΔΔCt = ΔCt (dose group) - ΔCt (negative siRNA control group)

[0419] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0420] The results of Figure 3 demonstrate the inhibition of DGAT2 mRNA after transfection of siRNA molecules in human primary hepatocytes. XD000515 and XD000533 have better inhibition effect than DGAT2 ASO-Ionis ION224 (ISIS769355 in WO2017011276A1) at different concentrations.

[0421] Example 6 Inhibition of monkey DGAT2 in monkey primary hepatocytes by siRNA

[0422] siRNAs were screened for activity in monkey primary hepatocytes using a concentration gradient.

[0423] siRNAs were transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat# 13778150) according to the product manual, with a final siRNA concentration of 10 nM. Lipofectamine RNAiMAX and siRNA diluted in Opti-MEM medium (GIBCO, Cat#31985070) were mixed at equal volume, incubated at room temperature for 20 min, and added to a 96-well plate at a volume of 20 μΐ per well, followed by the addition of 80 μΐ of thawed monkey primary hepatocytes with a cell density of 40,000 cells per well, and a final siRNA stock concentration of 6.25 nM, followed by a 5-fold dilution gradient in 14 wells. After 24 hours of transfection, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat# CL132), and reverse transcription and qPCR were performed according to the kit manual. DGAT2 mRNA levels were determined. DGAT2 mRNA levels were corrected according to the GAPDH internal standard mRNA level. DGAT2 mRNA expression levels were calculated by the ΔΔCt relative quantification method, and expressed as the percentage of residual expression of DGAT2 mRNA in cells treated with negative siRNA control, according to the following formula:

[0424] ΔCt = Ct (target gene) - Ct (internal standard gene)

[0425] ΔΔCt = ΔCt (dose group) - ΔCt (negative siRNA control group)

[0426] Relative expression level of DGAT2 mRNA = 2 -ΔΔCt × 100%

[0427] The results of Figure 4 show the inhibition of DGAT2 mRNA after transfection of siRNA molecules in monkey primary hepatocytes. XD000515 and XD000533 have better inhibitory effects than DGAT2 ASO-Ionis ION224 (ISIS769355 in WO2017011276A1) at different concentrations.

[0428] Example 7 Inhibition of rat DGAT2 in Hepa-1c1c7 cells by siRNA

[0429] The siRNA was screened for activity in mouse hepatoma cells using a concentration gradient.

[0430] Hepa-1c1c7 cells (Nanjing Keyeite Science and Technology Co., Ltd., Cat#CBP60231) were cultured in MEM Alpha medium (Gibco, Cat#12571063) containing 10% fetal bovine serum at 37°C, 5% CO2.

[0431] siRNA was transfected using Lipofectamine RNAiMAX (ThermoFisher, Cat#13778150) according to the following method: siRNA was prepared into a 1 μM working solution with DEPC water. Solution A was prepared, each containing 1 μl of siRNA working solution and 9 μl of Opti-MEM medium (GIBCO, Cat#31985070). Solution B was prepared, each containing 0.3 μl of Lipofectamine RNAiMAX and 9.7 μl of Opti-MEM medium. After mixing solution A and solution B, the mixture was placed in a 96-well plate and incubated at room temperature for 20 min, then 80 μl of Hepa-1c1c7 cells were added, with 15000 cells per well, and the final concentration of siRNA stock solution was 10 nM, followed by 10-fold gradient dilution in 7 wells. After transfection for 24 hours, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions to determine the DGAT2 mRNA level. The DGAT2 mRNA level was corrected according to the GAPDH internal mRNA level. The DGAT2 mRNA expression level was calculated by the ΔΔCt relative quantification method, expressed as the percentage of residual expression of DGAT2 mRNA in cells treated with negative siRNA control group, and the calculation formula was as follows:

[0432] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0433] ΔΔCt = ΔCt (dose group) - ΔCt (negative siRNA control group)

[0434] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0435] Normalized mRNA remaining (normalized level) = (mRNA relative expression level per well / negative control well relative expression level) * 100

[0436] The results of Figure 5 show the normalized inhibition of DGAT2 mRNA after transfection of siRNA molecules in Hepa-1c1c7. XD000512, XD000515, XD000516, XD000533 and XD000551 all show a significant inhibitory effect.

[0437] Example 8 Inhibition of murine DGAT2 in vivo in mice by siRNA

[0438] The drug was initially screened by knocking down the DGAT2 gene in the liver of wild-type C57 mice.

[0439] C57BL / 6N mice, female, 35-41 days old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., grade SPF, quality inspection unit Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number 110011241105958463. The mice were acclimated for 3 days after arrival, and then the study began.

[0440] Dosing: This test set up PBS group, XD000509.1, XD000512.1, XD000515.1, XD000516.1, XD000533.1, XD000551.1, a total of 7 groups, 9 mice in each group, subcutaneous single dose and frequency as shown in Table 5, PBS group as control group, on the 0th day of the experiment, D7, D21, D35 mice were euthanized with carbon dioxide, and the mouse liver tissue was taken, 3 mice from each group each time, a total of 21 mice each time, and stored in PBS wet ice.

[0441] When detecting the mRNA expression level of DGAT2 in mice, liver cells were obtained by digesting the liver, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions. The DGAT2 mRNA level was determined. The DGAT2 mRNA level was corrected according to the GAPDH internal standard mRNA level. The DGAT2 mRNA expression level was calculated by the ΔΔCt relative quantification method, expressed as the percentage of residual expression of DGAT2 mRNA in the liver of mice treated with PBS control group, and the calculation formula was as follows:

[0442] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0443] ΔΔCt = ΔCt (dose group) - ΔCt (negative siRNA control group)

[0444] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0445] Table 5: Dosing conditions for each group

[0446] The results of Figure 6 show the inhibition of DGAT2 mRNA in C57BL / 6N mice. The results show that after 7 days of 5mpk administration, XD000533.1 and XD000551.1 can achieve mRNA remaining of less than 10% in mice, showing very good knockdown effect, XD000512.1, XD000515.1, and XD000516.1 can achieve DGAT2 mRNA remaining of about 25%, and XD000509.1 can achieve DGAT2 mRNA remaining of about 50%; after 21 days of 5mpk administration, XD000515.1 and XD000533.1 can achieve mRNA remaining of less than 10% in mice, showing very good knockdown effect, XD000551.1 can achieve DGAT2 mRNA remaining of about 15%, which is higher than before 7 days, XD000512.1 and XD000516.1 can achieve DGAT2 mRNA remaining of about 20%, and XD000509.1 can achieve DGAT2 mRNA remaining of about 85%; after 35 days of 5mpk administration, XD000515.1 and XD000533.1 can achieve mRNA remaining of about 12% in mice, showing very good knockdown effect, XD000551.1 can achieve DGAT2 mRNA remaining of about 15%, XD000512.1 and XD000516.1 can achieve DGAT2 mRNA remaining of about 50%, and XD000509.1 can achieve DGAT2 mRNA remaining of about 75%. In summary, XD000515.1 and XD000533.1 can both show very stable knockdown effect in mice for a long period of time.

[0447] Example 9 Inhibition of mouse DGAT2 in vivo by siRNA in mice

[0448] This experiment preliminarily screened drugs by knocking down DGAT2 gene in the liver of C57BL / 6N mice

[0449] C57BL / 6N mice:

[0450] C57BL / 6N mice, female, 42-62 days old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., grade SPF, quality inspection unit Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number 110011241107462085. The mice were acclimated for 3 days after arrival, and then the study began.

[0451] Dosing:

[0452] The experiment was set up with PBS group, XD000515.1-0.3 mg / kg, XD000515.1-1 mg / kg, XD000515.1-3 mg / kg, XD000533.1-0.3 mg / kg, XD000533.1-1 mg / kg, XD000533.1-3 mg / kg, a total of 7 groups, each group has 12 mice, the right ear of the mouse is marked with ear tag, subcutaneous single dose, the specific dose and frequency are shown in Table 6, the PBS group is used as a control group, the mice are dosed on day 0 of the experiment, and the mice are euthanized with carbon dioxide on days 7, 21, 35, and 49, and the mouse liver tissue is placed in PBS wet ice.

[0453] When detecting the mRNA expression level of DGAT2 in mice, liver cells were obtained by digesting the liver, cell RNA was extracted using FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Vazyme, Cat#CL132), and reverse transcription and qPCR were performed according to the kit instructions. The DGAT2 mRNA level was determined. The DGAT2 mRNA level was corrected according to the GAPDH internal mRNA level. The DGAT2 mRNA expression level was calculated by the ΔΔCt relative quantification method, expressed as the percentage of residual expression of DGAT2 mRNA in the liver of mice treated with PBS control group, and the calculation formula is as follows:

[0454] ΔCt = Ct (target gene) - Ct (internal reference gene)

[0455] ΔΔCt = ΔCt (dosing group) - ΔCt (negative siRNA control group)

[0456] DGAT2 mRNA relative expression level = 2 -ΔΔCt × 100%

[0457] Table 6. Experimental design

[0458] The results of Figure 7 show the inhibition of DGAT2 mRNA in C57BL / 6N mice, and the results show that XD000515.1 and XD000533.1 exhibit significant inhibition in vivo and have long-term dosing potential.

[0459] Example 10 Inhibition of mouse DGAT2 in vivo by siRNA in mice

[0460] Pharmacodynamics of DGAT2-siRNA in C57 non-obese MASH model

[0461] C57BL / 6N mice, 8-9 weeks old, male, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., grade SPF, quality inspection unit Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number 110011241107461000. The mice were quarantined for 3 days after arrival, and then the study began.

[0462] Feed: MCD methionine and choline-deficient mouse chow (US item number: 59580, specification: 5 kg)

[0463] Dosing: This experiment set up Normal group (PBS dosing), Model group (PBS dosing), XD000515.1 group, XD000533.1 group, a total of 4 groups, 6 mice in each group, of which the Normal group was fed with normal feed, and the rest of the groups were fed with MCD feed. The administration method was subcutaneous injection, and only one dose of drug was given on the 0th day of the experiment. The specific dosing information is shown in Table 71. The experiment lasted for 4 weeks, and the mice were weighed every Tuesday and Friday. On the 27th day of the experiment, the experiment was ended. The mice were euthanized with carbon dioxide, and the liver was weighed, a part of which was used for pathological section: liver oil red stain (Oil red stain). A part was used for in vitro detection, such as qPCR detection as shown in Example 9.

[0464] Table 7: Dosing information for each group

[0465] The results of liver oil red stain (Oil red stain) are shown in the lower panel of Figure 8, which shows that the MASH model was successfully established. The results of the upper panel of Figure 8 show the inhibition of DGAT2 mRNA in C57 mice. The results show that XD000515.1 and XD000533.1 significantly inhibit DGAT2 mRNA in vivo in mice, and the knockdown can still be maintained at 4 weeks, indicating a long-acting potential for drug administration.

[0466] Example 11 Inhibition of mouse DGAT2 in vivo by siRNA in mice

[0467] Drug screening was performed by knocking down the DGAT2 gene in the liver of ob / ob mice (leptin-deficient obese mice).

[0468] C57BL / 6J mice, male 7-8 weeks old, purchased from Jiangsu Jizhu Yaoke Biotechnology Co., Ltd., grade SPF, quality inspection unit Suzhou Xishan Biotechnology Co., Ltd., certificate number A202410240291; ob / ob mice, male 7-8 weeks old, purchased from Jiangsu Jizhu Yaoke Biotechnology Co., Ltd., grade SPF, quality inspection unit Suzhou Xishan Biotechnology Co., Ltd., certificate number A202410240229.

[0469] Dosing: This experiment sets Normal group, Model group, XD000515.1 group, XD000533.1 group, a total of 4 groups. The Normal group has 12 C57BL / 6J mice, and the rest of the groups each have 14 mice, which are all ob / ob mice. The specific dosing dose and frequency are shown in the table below. C57BL / 6J mice are fed with normal feed, and ob / ob mice are fed with Amylin diet (GAN diet) for 12 weeks, and both are provided with free drinking water. The body weight of the mice is monitored twice a week; body fat is monitored every three weeks; 100uL of blood is collected from the eye socket every two weeks, and the serum is collected for serum alanine transferase / aspartate aminotransferase / total cholesterol / triglyceride level detection. The mice are euthanized with carbon dioxide at 0 days, 6 weeks and 12 weeks after the start of the experiment, and the liver tissue of the mice is taken. The following tests are performed respectively:

[0470] (1) The mRNA remaining in the liver at 6 weeks and 12 weeks, the experimental procedure is referred to qPCR detection of Example 9, and the results are shown in Figure 9;

[0471] (2) Hepatic TG detection (Suzhou Guochen Biological Technology Co., Ltd.), the results are shown in Figure 10;

[0472] (3) Blood biochemical data AST / ALT / TC (Suzhou Guochen Biological Technology Co., Ltd.), the results are shown in Figure 11.

[0473] The statistical method is as follows: ##P<0.01, ###P<0.001, ****P<0.0001 by one-way ANOVA, *vs. WT (C57, normal feed), #vs. MASH model (ob / ob, GAN feed)

[0474] Table 8: Dosing information of each group

[0475] The results of Figure 9 show that the inhibition of DGAT2 mRNA in ob / ob mice, XD000515.1 and XD000533.1 can significantly inhibit the expression of DGAT2 mRNA in vivo, and the knockdown can still be maintained at 12 weeks, with long-term administration potential.

[0476] The results of Figure 10 show that the liver triglyceride level is significantly down-regulated at 6 weeks, and the liver TG is also down-regulated at 12 weeks.

[0477] The results of Figure 11 show that the blood biochemical data AST / ALT / TC expression is down-regulated to different degrees at 12 weeks, and MASH is partially relieved.

[0478] Example 12 Inhibition of mouse DGAT2 in vivo by siRNA in mice

[0479] Detection of the effect of DGAT2-siRNA in vivo in hDGAT2-KI mice

[0480] hDGAT2-KI (C57) mice, female, about 5 weeks old, purchased from Bio-Swift Biotech Co., Ltd., grade SPF, quality inspection unit Suzhou Xishan Biotechnology Co., Ltd., certificate number B202507010603. The mice were quarantined for 3 days after arrival, and then the study began.

[0481] Dosing: This test sets up PBS group, DGAT2 ASO-1 mpk group, DGAT2 ASO-3 mpk group, DGAT2 ASO-10 mpk group, XD000515.1-1 mpk group, XD000515.1-3 mpk group, XD000515.1-10 mpk group, a total of 7 groups, 9 mice in each group. The administration method is subcutaneous injection, and only one drug is given at the beginning of the experiment. The specific dosing and frequency are shown in Table 9. The experiment lasted for 6 weeks, and at Day 14, Day 28, and Day 42, three mice were selected from each group and euthanized with carbon dioxide, and the liver was taken for qPCR. The steps are described in Example 9.

[0482] Table 9: Dosing information for each group

[0483] DGAT2 ASO: ISIS769355 in WO2017011276A1

[0484] The results of FIG. 12 show that the inhibition of DGAT2 mRNA in DGAT2 KI mice, wherein XD000515.1 can significantly inhibit the expression of DGAT2 mRNA in vivo, and the inhibition effect of XD000515.1 high dose 10 mg reaches about 90% on Day 14 and Day 28. At the same time, the three single doses of the positive control ASO are equivalent to the PBS group, and no significant inhibition effect is detected. In summary, the knockdown effect of XD000515.1 is better than that of ASO.

Claims

1. A dsRNA active agent, wherein the dsRNA active agent comprises a sense strand and an antisense strand that can form a duplex region, wherein the antisense strand comprises a region of complementarity that is complementary to a target sequence and the region of complementarity of the antisense strand comprises no more than 3, 2, or 1 nucleotide mismatches with the target sequence, wherein the target sequence is selected from (i) a target sequence corresponding to positions 670-690 or 899-919 of a DGAT2 mRNA; (ii) a mRNA target sequence set forth in SEQ ID NO: 139 or SEQ ID NO: 148; (iii) a mRNA target sequence set forth in any one of SEQ ID NOs: 135-201; or (iv) a target sequence corresponding to a position in a DGAT2 mRNA set forth in Table 1; optionally, the dsRNA active agent is for use in inhibiting expression of a gene encoding DGAT2.

2. A dsRNA active agent, wherein the dsRNA active agent comprises a sense strand and an antisense strand that can form a duplex region, wherein the antisense strand comprises a sequence that is complementary to a target sequence of a mRNA encoding DGAT2 and comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3, 2, or 1 nucleotides from any one of the nucleotide sequences of the antisense strands in Table 1 or the nucleotide sequence of any one of the antisense strands set forth in SEQ ID NOs: 1-67, optionally, the dsRNA active agent is for use in inhibiting expression of a gene encoding DGAT2, optionally, the target sequence of the mRNA is as defined in claim 1.

3. The dsRNA active agent of claim 2, wherein the nucleotide sequence of the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3, 2, or 1 nucleotides from the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO:

14.

4. The dsRNA active agent of any one of claims 1-3, wherein the antisense strand comprises a region of complementarity to a target sequence that is between 15 and 30 nucleotides, between 18 and 30 nucleotides, or between 18 and 23 nucleotides in length, for example, the region of complementarity is 18, 19, 20, or 21 nucleotides in length; optionally, the region of complementarity comprises at least nucleotides 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21, for example, 2-21, from the 5’ end of the antisense strand.

5. The dsRNA active agent of any one of claims 1 to 4, wherein the antisense strand has the same number of nucleotides as the target sequence and is mismatched to the target sequence at least at the first nucleotide at the 5' end, e.g., is fully complementary to the corresponding portion of the mRNA target sequence except for the first nucleotide at the 5' end; or the nucleotide sequence of the antisense strand is fully complementary to the target sequence.

6. The dsRNA active agent of any one of claims 1 to 5, wherein the first nucleotide at the 5' end of the antisense strand is A or U, e.g., U.

7. The dsRNA active agent of any one of claims 1 to 6, wherein the dsRNA active agent comprises a sense strand comprising at least 15, 16, 17, 18, or 19 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of the sense strands in Table 1 or any one of the sense nucleotide sequences set forth in SEQ ID NOs: 68-134.

8. The dsRNA active agent of claim 7, wherein the sense strand comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence set forth in SEQ ID NO: 72 or SEQ ID NO:

81.

9. The dsRNA active agent of any one of claims 1 to 8, wherein the double-stranded region formed by the sense strand and the antisense strand is fully complementary or can comprise 1, 2, 3, 4, or 5 non-complementary sites; optionally, the fully complementary double-stranded region is between 15 to 25 nucleotide pairs, 16 to 25 nucleotide pairs, 16 to 24 nucleotide pairs, 16 to 22 nucleotide pairs, 17 to 24 nucleotide pairs, 17 to 23 nucleotide pairs, 18 to 23 nucleotide pairs, or 19 to 22 nucleotide pairs in length, e.g., the fully complementary double-stranded region is 16, 17, 18, or 19 nucleotide pairs in length, e.g., 19 nucleotide pairs in length.

10. The dsRNA active agent of any one of claims 1 to 9, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, e.g., 17-27 nucleotides, e.g., 19-25 nucleotides, e.g., 19-23 nucleotides, or e.g., 19-21 nucleotides in length, e.g., the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length.

11. The dsRNA active agent of any one of claims 1 to 10, wherein the sense strand and / or the antisense strand comprises a 3' overhang or a 5' overhang of at least 1, 2, or 3 nucleotides, e.g., only the antisense strand comprises a 3' overhang of 2 nucleotides.

12. The dsRNA active agent of any one of claims 1 to 11, comprising a sense strand and an antisense strand forming a double-stranded region, wherein (i) the sense strand comprises or is 19 nucleotides, (ii) the antisense strand comprises or is 21 nucleotides and is fully complementary to a region of a target sequence of an mRNA encoding DGAT2 except for the first nucleotide counting from the 5' end, wherein the first nucleotide of the 5' end of the antisense strand is A or U, e.g., U; (iii) the antisense strand comprises a 3' overhang of 2 nucleotides compared to the sense strand, and wherein the sense strand is fully complementary to the antisense strand over 19 nucleotides, e.g., fully complementary over 19 consecutive nucleotides; e.g., the sense strand is fully complementary to the antisense strand over nucleotides 1-19 counting from the 5' end of the antisense strand.

13. The dsRNA active agent of any one of claims 1 to 12, wherein the dsRNA active agent comprises a sense strand and an antisense strand, the nucleotide sequence of the sense strand comprises any one of the nucleotide sequences of the sense strands in Table 1, and / or, the nucleotide sequence of the antisense strand comprises any one of the nucleotide sequences of the antisense strands in Table 1.

14. The dsRNA active agent of any of claims 1 to 13, wherein the dsRNA active agent comprises an antisense strand and a sense strand, wherein the antisense and sense strands comprise, respectively, SEQ ID NO: 1 / 68, SEQ ID NO: 2 / 69, SEQ ID NO: 3 / 70, SEQ ID NO: 4 / 71, SEQ ID NO: 5 / 72, SEQ ID NO: 6 / 73, SEQ ID NO: 7 / 74, SEQ ID NO: 8 / 75, SEQ ID NO: 9 / 76, SEQ ID NO: 10 / 77, SEQ ID NO: 11 / 78, SEQ ID NO: 12 / 79, SEQ ID NO: 13 / 80, SEQ ID NO: 14 / 81, SEQ ID NO: 15 / 82, SEQ ID NO: 16 / 83, SEQ ID NO: 17 / 84, SEQ ID NO: 18 / 85, SEQ ID NO: 19 / 86, SEQ ID NO: 20 / 87, SEQ ID NO: 21 / 88, SEQ ID NO: 22 / 89, SEQ ID NO: 23 / 90, SEQ ID NO: 24 / 91, SEQ ID NO: 25 / 92, SEQ ID NO: 26 / 93, SEQ ID NO: 27 / 94, SEQ ID NO: 28 / 95, SEQ ID NO: 29 / 96, SEQ ID NO: 30 / 97, SEQ ID NO: 31 / 98, SEQ ID NO: 32 / 99, SEQ ID NO: 33 / 100, SEQ ID NO: 34 / 101, SEQ ID NO: 35 / 102, SEQ ID NO: 36 / 103, SEQ ID NO: 37 / 104, SEQ ID NO: 38 / 105, SEQ ID NO: 39 / 106, SEQ ID NO: 40 / 107, SEQ ID NO: 41 / 108, SEQ ID NO: 42 / 109, SEQ ID NO: 43 / 110, SEQ ID NO: 44 / 111, SEQ ID NO: 45 / 112, SEQ ID NO: 46 / 113, SEQ ID NO: 47 / 114, SEQ ID NO: 48 / 115, SEQ ID NO: 49 / 116, SEQ ID NO: 50 / 117, SEQ ID NO: 51 / 118, SEQ ID NO: 52 / 119, SEQ ID NO: 53 / 120, SEQ ID NO: 54 / 121, SEQ ID NO: 55 / 122, SEQ ID NO: 56 / 123, SEQ ID NO: 57 / 124, SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, SEQ ID NO: 67 / 134, SEQ ID NO: 68 / 135, SEQ ID NO: 69 / 136, SEQ ID NO: 70 / 137, SEQ ID NO: 71 / 138, SEQ ID NO: 72 / 139, SEQ ID NO: 73 / 140, SEQ ID NO: 74 / 141, SEQ ID NO: 75 / 142, SEQ ID NO: 76 / 143, SEQ ID NO: 77 / 144, SEQ ID NO: 78 / 145, SEQ ID NO: 79 / 146, SEQ ID NO: 80 / 147, SEQ ID NO: 81 / 148, SEQ ID NO: 82 / 149, SEQ ID NO: 83 / 150, SEQ ID NO: 84 / 151, SEQ ID NO: 85 / 152, SEQ ID NO: 86 / 153, SEQ ID NO: 87 / 154, SEQ ID NO: 88 / 155, SEQ ID NO: 89 / 156, SEQ ID NO: 90 / 157, SEQ ID NO: 91 / 158, SEQ ID NO: 92 / 159, SEQ ID NO: 93 / 160, SEQ ID NO: 94 / 161, SEQ ID NO: 95 / 162, SEQ ID NO: 96 / 163, SEQ ID NO: 97 / 164, SEQ ID NO: 98 / 165, SEQ ID NO: 99 / 166, SEQ ID NO: 100 / 167, SEQ ID NO: 101 / 168, SEQ ID NO: 102 / 169, SEQ ID NO: 103 / 170, SEQ ID NO: 104 / 171, SEQ ID NO: 105 / 172, SEQ ID NO: 106 / 173, SEQ ID NO: 107 / 174, SEQ ID NO: 108 / 175, SEQ IDthe nucleotide sequences set forth in SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, or SEQ ID NO: 67 / 134, or consisting of the nucleotide sequences set forth in SEQ ID NO: 58 / 125, SEQ ID NO: 59 / 126, SEQ ID NO: 60 / 127, SEQ ID NO: 61 / 128, SEQ ID NO: 62 / 129, SEQ ID NO: 63 / 130, SEQ ID NO: 64 / 131, SEQ ID NO: 65 / 132, SEQ ID NO: 66 / 133, or SEQ ID NO: 67 / 134, respectively; Optionally, the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 5, and the sense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 72; or the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 14, and the sense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO:

81.

15. The dsRNA active agent of any one of claims 1 to 14, wherein at least one nucleotide in the dsRNA active agent is a modified nucleotide, Optionally, wherein all nucleotides of the sense strand except 1 or 2 nucleotides are modified nucleotides; or all nucleotides of the antisense strand except 1 or 2 nucleotides are modified nucleotides; or all nucleotides of the sense strand except 1 or 2 nucleotides and all nucleotides of the antisense strand except 1 or 2 nucleotides are modified nucleotides; Optionally, all nucleotides of the sense strand are modified nucleotides; or all nucleotides of the antisense strand are modified nucleotides; or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

16. The dsRNA active agent of claim 15, wherein at least one of the modified nucleotides is selected from the group consisting of LNA, HNA, TNA, CeNA, deoxynucleotides, 3' terminal deoxythymidine (dT) nucleotides, 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, 2'-5'-linked ribonucleotides (3'-RNA), unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-0-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-0-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, tetrahydropyran modified nucleotides, 1,5- anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides comprising phosphorothioate groups, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphate, nucleotides comprising 5'-phosphate mimics, vinyl-phosphonate nucleotides, heat unstable nucleotides, glycol modified nucleotides (GNA), nucleotides containing 2'-phosphate, and 2-O-(N-methylacetamide) modified nucleotides; and combinations of one or more thereof; optionally, the antisense strand has 2'-methoxy modified nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 from the 5' end, and the sense strand has 2'-methoxy modified nucleotides at positions 1-6 and 10-19 from the 5' end; optionally, the antisense strand has 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5' end, and the sense strand has 2'-fluoro modified nucleotides at positions 7, 8, and 9 from the 5' end.

17. The dsRNA active agent of any one of claims 1-16, wherein the antisense strand comprises phosphorothioate linkages, and / or the sense strand comprises phosphorothioate linkages, optionally wherein the phosphorothioate internucleotide linkages are between positions 1-3 from the 5' end of the sense strand, and between positions 1-3 from the 5' end and between positions 1-3 from the 3' end of the antisense strand.

18. The dsRNA active agent of any one of claims 15-17, wherein the modified nucleotides of the antisense strand and sense strand have the following modification pattern: Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm; and / or Sense strand: NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm, wherein, Nf = any 2'-fluoro modified nucleotide; Nm = any 2'-methoxy nucleotide; ​ s represents a phosphorothioate linkage between two nucleotides.

19. The dsRNA active agent of any one of claims 15 to 18, wherein the modified nucleotide sequence of the antisense strand comprises the modified nucleotide sequence of any one of the antisense strands in Table 2 of the specification, and / or the modified nucleotide sequence of the sense strand comprises the modified nucleotide sequence of any one of the sense strands in Table 2 of the specification; Optionally, the dsRNA active agent comprises a combination of the modified nucleotide sequence of the antisense strand and the modified nucleotide sequence of the sense strand as shown in Table 2; Optionally, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the modified nucleotide sequence of the antisense strand and the modified nucleotide sequence of the sense strand comprise SEQ ID NO: 202 / SEQ ID NO: 269, SEQ ID NO: 203 / SEQ ID NO: 270, SEQ ID NO: 204 / SEQ ID NO: 271, SEQ ID NO: 205 / SEQ ID NO: 272, SEQ ID NO: 206 / SEQ ID NO: 273, SEQ ID NO: 207 / SEQ ID NO: 274, SEQ ID NO: 208 / SEQ ID NO: 275, SEQ ID NO: 209 / SEQ ID NO: 276, SEQ ID NO: 210 / SEQ ID NO: 277, SEQ ID NO: 211 / SEQ ID NO: 278, SEQ ID NO: 212 / SEQ ID NO: 279, SEQ ID NO: 213 / SEQ ID NO: 280, SEQ ID NO: 214 / SEQ ID NO: 281, SEQ ID NO: 215 / SEQ ID NO: 282, SEQ ID NO: 216 / SEQ ID NO: 283, SEQ ID NO: 217 / SEQ ID NO: 284, SEQ ID NO: 218 / SEQ ID NO: 285, SEQ ID NO: 219 / SEQ ID NO: 286, SEQ ID NO: 220 / SEQ ID NO: 287, SEQ ID NO: 221 / SEQ ID NO: 288, SEQ ID NO: 222 / SEQ ID NO: 289, SEQ ID NO: 223 / SEQ ID NO: 290, SEQ ID NO: 224 / SEQ ID NO: 291, SEQ ID NO: 225 / SEQ ID NO: 292, SEQ ID NO: 226 / SEQ ID NO: 293, SEQ ID NO: 227 / SEQ ID NO: 294, SEQ ID NO: 228 / SEQ ID NO: 295, SEQ ID NO: 229 / SEQ ID NO: 296, SEQ ID NO: 230 / SEQ ID NO: 297, SEQ ID NO: 231 / SEQ ID NO: 298, SEQ ID NO: 232 / SEQ ID NO: 299, SEQ ID NO: 233 / SEQ ID NO: 300, SEQ ID NO: 234 / SEQ ID NO: 301, SEQ ID NO: 235 / SEQ ID NO: 302, SEQ ID NO: 236 / SEQ ID NO: 303, SEQ ID NO: 237 / SEQ ID NO: 304, SEQ ID NO: 238 / SEQ ID NO: 305, SEQ ID NO: 239 / SEQ ID NO: 306, SEQ ID NO: 240 / SEQ ID NO: 307, SEQ ID NO: 241 / SEQ ID NO: 308, SEQ ID NO: 242 / SEQ ID NO: 309, SEQ ID NO: 243 / SEQ ID NO: 310, SEQ ID NO: 244 / SEQ ID NO: 311, SEQ ID NO: 245 / SEQ ID NO: 312, SEQ ID NO: 246 / SEQ ID NO: 313, SEQ ID NO: 247 / SEQ ID NO: 314, SEQ ID NO: 248 / SEQ ID NO: 315, SEQ ID NO: 249 / SEQ ID NO: 316, or SEQ ID NO: 250 / SEQ ID NO: 317, respectively.a modified nucleotide sequence set forth in, or respectively consisting of, SEQ ID NO: 302 / SEQ ID NO: 303, SEQ ID NO: 304 / SEQ ID NO: 305, SEQ ID NO: 306 / SEQ ID NO: 307, SEQ ID NO: 308 / SEQ ID NO: 309, SEQ ID NO: 310 / SEQ ID NO: 311, SEQ ID NO: 312 / SEQ ID NO: 313, SEQ ID NO: 314 / SEQ ID NO: 315, SEQ ID NO: 316 / SEQ ID NO: 317, SEQ ID NO: 318 / SEQ ID NO: 319, SEQ ID NO: 320 / SEQ ID NO: 321, SEQ ID NO: 322 / SEQ ID NO: 323, SEQ ID NO: 324 / SEQ ID NO: 325, SEQ ID NO: 326 / SEQ ID NO: 327, SEQ ID NO: 328 / SEQ ID NO: 329, SEQ ID NO: 330 / SEQ ID NO: 331, SEQ ID NO: 332 / SEQ ID NO: 333, SEQ ID NO: 334 / SEQ ID NO: 335, or SEQ ID NO: 336 / SEQ ID NO: 337; and / or a modified nucleotide sequence set forth in, or respectively consisting of, SEQ ID NO: 302 / SEQ ID NO: 303, SEQ ID NO: 304 / SEQ ID NO: 305, SEQ ID NO: 306 / SEQ ID NO: 307, SEQ ID NO: 308 / SEQ ID NO: 309, SEQ ID NO: 310 / SEQ ID NO: 311, SEQ ID NO: 312 / SEQ ID NO: 313, SEQ ID NO: 314 / SEQ ID NO: 315, SEQ ID NO: 316 / SEQ ID NO: 317, SEQ ID NO: 318 / SEQ ID NO: 319, SEQ ID NO: 320 / SEQ ID NO: 321, SEQ ID NO: 322 / SEQ ID NO: 323, SEQ ID NO: 324 / SEQ ID NO: 325, SEQ ID NO: 326 / SEQ ID NO: 327, SEQ ID NO: 328 / SEQ ID NO: 329, SEQ ID NO: 330 / SEQ ID NO: 331, SEQ ID NO: 332 / SEQ ID NO: 333, SEQ ID NO: 334 / SEQ ID NO: 335, or SEQ ID NO: 336 / SEQ ID NO:

337. Preferably, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the modified nucleotide sequence of the antisense strand and the modified nucleotide sequence of the sense strand respectively comprise or consist of the modified nucleotide sequence as shown in SEQ ID NO: 206 / SEQ ID NO: 273 or SEQ ID NO: 215 / SEQ ID NO:

282.

20. The dsRNA active agent of any one of claims 1 to 19, further comprising a ligand, for example the ligand is linked to a nucleotide at the 5’ and / or 3’ end of the sense strand of the dsRNA and optionally a nucleotide at the 5’ and / or 3’ end of the antisense strand, optionally the ligand is linked to the nucleotide via a phosphorothioate group or a phosphate group; Optionally, the ligand is a ligand capable of targeting delivery of the dsRNA active agent to liver tissue or hepatocytes, for example a ligand capable of delivering the dsRNA molecule to the asialoglycoprotein receptor (ASGPR) of liver tissue or hepatocytes, for example it comprises a moiety selected from galactose or a galactose derivative (e.g. galactosamine, N-formyl galactosamine, N-acetyl galactosamine (GalNAc), N-propionyl galactosamine, N-n-butyryl galactosamine, N-isobutyryl galactosamine, etc.); Preferably, the ligand comprises one or more GalNAc or GalNAc derivatives.

21. The dsRNA active agent of any of claims 1 to 20, wherein the oligonucleotide of the dsRNA active agent is conjugated to one or more (e.g., 1) ligand, wherein the ligand each independently has the structure of Formula (I): wherein, each Gal independently represents a terminal galactose derivative; L represents a linker; n is an integer selected from 1, 2, 3 and 4; and the wavy line represents attachment to the oligonucleotide of the dsRNA via this valence bond, preferably the ligand is attached to the 5’ and / or 3’ end of the sense strand and / or the antisense strand of the oligonucleotide of the dsRNA, preferably via a phosphoester bond or a phosphorothioate bond; Optionally, each of the ligands independently has the structure of Formula (Ia-i): wherein, the wavy line represents attachment to the oligonucleotide of the dsRNA via this valence bond, preferably the ligand is attached to the 5’ and / or 3’ end of the sense strand and / or the antisense strand of the oligonucleotide of the dsRNA, preferably via a phosphoester bond or a phosphorothioate bond, each R1 independently is H; each of m1 and m2 independently is 1, 2, 3, 4, 5, 6, 7 or 8, preferably 3 or 4; q is an integer selected from 1 to 16, preferably an integer selected from 1 to 12, more preferably an integer from 8 to 12, for example 8, 9, 10, 11 or 12. Optionally, each of said ligands is independently an L96 moiety having the structure: wherein the wavy line indicates attachment to the oligonucleotide of the dsRNA via the valence bond; Preferably, the ligand is attached to the 3' end of the sense strand of the oligonucleotide of the dsRNA; Preferably, the attachment is via a phosphonate or thiophosphonate linkage.

22. The dsRNA active agent of claim 20 or 21, wherein the antisense strand in the dsRNA active agent comprises the modified nucleotide sequence of any one of the antisense strands in Table 3, and / or the sense strand comprises the modified nucleotide sequence of any one of the sense strands in Table 3 conjugated at the 3' end to the L96 ligand structure via a phosphonate linkage; Optionally, the antisense strand and sense strand respectively comprise or consist of the modified nucleotide sequence of the sense strand and the modified nucleotide sequence of the antisense strand in any one of the combinations of sense and antisense strands in Table 3 conjugated at the 3' end to the L96 ligand structure via a phosphonate linkage; Optionally, the combination of sense and antisense strands in the dsRNA active agent is selected from any of the combinations of sense and antisense strands as shown in Table 6; Optionally, the dsRNA active agent comprises an antisense strand and a sense strand, wherein the antisense strand and sense strand respectively comprise or consist of the nucleotide sequence set forth in any one of SEQ ID NO: 203 / 336, SEQ ID NO: 204 / 337, SEQ ID NO: 206 / 338, SEQ ID NO: 207 / 339, SEQ ID NO: 215 / 340, or SEQ ID NO: 225 / 341; Optionally, the dsRNA active agent comprises a sense strand and an antisense strand, wherein a. the sense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 336 conjugated at the 3' end to the L96 structure ligand, and the antisense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 203; b. the sense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 337 conjugated at the 3' end to the L96 structure ligand, and the antisense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 204; c. the sense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 338 conjugated at the 3' end to the L96 structure ligand, and d. the sense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 339 conjugated at the 3' end to the L96 structure ligand, and the antisense strand comprises or consists of the modified nucleotide sequence set forth in SEQ ID NO: 207; e. the sense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO: 340 conjugated with a L96 structure ligand at the 3’ end, and the antisense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO: 215; or f. the sense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO: 341 conjugated with a L96 structure ligand at the 3’ end, and the antisense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO:

225.

23. The dsRNA active agent of claim 22, comprising a sense strand and an antisense strand, wherein the sense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO: 338 conjugated with a L96 structure ligand at the 3’ end, and the antisense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO: 206; or the sense strand comprises or consists of a modified nucleotide sequence as set forth in SEQ ID NO: 340 conjugated at the 3' terminus with a L96 structure ligand, and the antisense strand comprises or consists of the modified nucleotide sequence of SEQ ID NO:

215.

24. A cell containing the dsRNA active agent of any one of claims 1 to 23.

25. A pharmaceutical composition comprising the dsRNA active agent of any one of claims 1 to 23 and a pharmaceutically acceptable carrier, optionally the pharmaceutical composition is for inhibiting expression of a DGAT2 gene; optionally, the dsRNA active agent is in a non-buffered solution, for example the non-buffered solution is saline or water; or the dsRNA active agent is in a buffered solution, for example wherein the buffered solution comprises acetate, citrate, prolamine, carbonate or phosphate or any combination thereof, such as phosphate buffered solution (PBS).

26. A pharmaceutical combination comprising the dsRNA active agent of any one of claims 1 to 23 and one or more other therapeutic agents, for example any therapeutic agent effective in preventing or treating a DGAT2-related disease and / or disorder, encompassing various therapeutic agents for treating a chronic inflammatory disease (e.g. a chronic inflammatory liver disease, for example selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatosis), steatohepatitis, for example non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) or cirrhosis of the liver).

27. A method of inhibiting expression of a DGAT2 gene in a cell, the method comprising contacting the cell with the dsRNA active agent of any one of claims 1 to 23 or the pharmaceutical composition of claim 25 or the pharmaceutical combination of claim 26, thereby inhibiting expression of the DGAT2 gene in the cell, optionally the cell is in a subject, optionally the subject is a mammal, for example a human; preferably the cell is a liver cell. Optionally, the subject has a DGAT2-associated disease and / or disorder, for example the DGAT2-associated disease is a chronic inflammatory disease, for example a chronic inflammatory liver disease, for example selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatosis), steatohepatitis, for example nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), or cirrhosis; Optionally, the contacting of the cell with the dsRNA active agent inhibits expression of DGAT2 by at least 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%, or the inhibition of DGAT2 expression reduces the level of DGAT2 protein in the liver (e.g., liver tissue or liver cells) of the subject by at least 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, or about 94%.

28. A method of preventing or treating a condition for which a reduction in DGAT2 expression and / or activity would be beneficial or a DGAT2-associated disease and / or disorder in a subject, comprising administering to the subject a dsRNA active agent according to any one of claims 1-23 or a pharmaceutical composition according to claim 25 or a pharmaceutical combination according to claim 26; Optionally, the DGAT2-associated disorder is selected from a chronic inflammatory disease, for example a chronic inflammatory liver disease, for example selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatosis), steatohepatitis, for example nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), or cirrhosis; Optionally, the administration of the dsRNA active agent to the subject results in a reduction in DGAT2 concentration or content (e.g., in liver tissue or liver cells) or a reduction in DGAT2 protein accumulation or content (e.g., in liver tissue or liver cells) in the subject; Optionally, the method further comprises administering to the subject one or more additional therapeutic agents, for example any therapeutic agent effective for preventing or treating a DGAT2-associated disease and / or disorder, including various therapeutic agents for treating a chronic inflammatory disease, for example a chronic inflammatory liver disease, for example selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatosis), steatohepatitis, for example nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), or cirrhosis; Optionally, the method further comprises determining the level of DGAT2 in a sample from the subject.

29. Use of a dsRNA active agent according to any one of claims 1-23 or a pharmaceutical composition according to claim 25 or a pharmaceutical combination according to claim 26 for the manufacture of a medicament for the prevention or treatment of a DGAT2 related disease and / or disorder having an indication that would benefit from a reduction of DGAT2 expression and / or activity; Optionally, the DGAT2 related disorder is selected from a chronic inflammatory disease, such as a chronic inflammatory liver disease, for example selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatosis), steatohepatitis, such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) or cirrhosis; Optionally, the administration of the dsRNA active agent or the pharmaceutical to the subject results in a reduction of DGAT2 concentration or content (for example in liver tissue or hepatocytes) or a reduction of DGAT2 protein accumulation or content (for example in liver tissue or hepatocytes) in the subject; Optionally, the medicament is for administration in combination with one or more other therapeutic agents, such as any therapeutic agent effective for the prevention or treatment of a DGAT2 related disease and / or disorder, including various therapeutic agents for the treatment of a chronic inflammatory disease, such as a chronic inflammatory liver disease, for example selected from liver fat accumulation, hepatitis, liver fibrosis, fatty liver disease (steatosis), steatohepatitis, such as non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) or cirrhosis; Optionally, the level of DGAT2 in a sample from the subject is determined prior to administration of the medicament.

30. A kit comprising a dsRNA active agent according to any one of claims 1-23 or a pharmaceutical composition according to claim 25 or a pharmaceutical combination according to claim 26.

31. A vial or syringe comprising a dsRNA active agent according to any one of claims 1-23 or a pharmaceutical composition according to claim 25 or a pharmaceutical combination according to claim 26.

32. An RNA-induced silencing complex (RISC) comprising the antisense strand of any one of the dsRNA active agents according to any one of claims 1 to 23.

Images