Oligonucleotide compositions and methods of use thereof

Abstract

To provide designed PNPLA3 oligonucleotides, compositions and the methods thereof.SOLUTION: Provided oligonucleotide compositions provide improved single-stranded RNA interference and / or RNase H-mediated knockdown. Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications or patterns thereof, conjugation with additional chemical moieties, and / or stereochemistry and / or patterns thereof, can have significant impact on oligonucleotide properties and activities. In some embodiments, the present disclosure provides methods for treating diseases using provided oligonucleotide compositions, for example, in RNA interference and / or RNase H-mediated knockdown.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 514,769, filed June 2, 2017, and U.S. Provisional Patent Application No. 62 / 670,698, filed May 11, 2018, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Oligonucleotides targeting PNPLA3 (PNPLA3 oligonucleotides) are useful in a variety of applications (e.g., therapeutic applications). The applications of naturally occurring nucleic acids (e.g., unmodified DNA or unmodified RNA) can be limited, for example, by the susceptibility of such naturally occurring nucleic acids to the action of endonucleases and exonucleases. Summary of the Invention

[0003] In particular, the present disclosure includes the recognition that controlling structural elements of a PNPLA3 oligonucleotide can significantly affect the properties and / or activity of the PNPLA3 oligonucleotide, such as chemical modifications (e.g., sugar modifications, base modifications, and / or internucleotide linkage modifications) or patterns thereof, alterations in stereochemistry (e.g., stereochemistry of the backbone chiral internucleotide linkages) or patterns thereof, and / or conjugation with additional chemical moieties (e.g., lipid moieties, targeting moieties, carbohydrate moieties, moieties that bind to the asialoglycoprotein receptor or ASGPR (e.g., GalNAc moieties), etc.). In some embodiments, the properties and / or activities include, but are not limited to, involvement in or induction of a decrease in the expression, activity, or levels of the PNPLA3 gene or its gene product, such decrease being mediated, for example, by RNA interference (RNAi interference), single-stranded RNA interference (ssRNAi), RNase H-mediated knockdown, steric hindrance of translation, etc.

[0004] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. [Brief description of the drawings]

[0005] [Figure 1A] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1B] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1C] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1D] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1E] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1F] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1G] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1H] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1I] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1J] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1K] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Figure 1L] Schematic diagrams of various ssRNAi and hybrid formats are shown. [Diagram 2] Schematic diagrams of various antisense oligonucleotide formats are shown. [Figure 3A]Examples of multimeric formats are shown. The oligonucleotides can be linked directly and / or via a linker. As shown, the multimers can include oligonucleotide monomers of the same or different structures / types. In some embodiments, the monomers of the multimer are ssRNAi agents. In some embodiments, the monomers of the multimer are RNase H-dependent antisense oligonucleotides (ASOs). The monomers can be linked through various positions, for example, the monomers can be linked through the 5' end, the 3' end, or positions intervening therebetween. [Figure 3B] Examples of chemical methods are provided for linking monomers to form multimers, which can exert their function via different pathways. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The present invention may be understood more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein.

[0007] It is to be understood that this invention is not limited to particular synthetic methods, which may, of course, vary, and 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.

[0008] In some embodiments, the present disclosure includes the recognition that stereochemistry (particularly the stereochemistry of backbone chiral center) can unexpectedly improve the properties of PNPLA3 oligonucleotide.Contrary to the previous observation that some structural elements that increase stability can also reduce activity (e.g., RNA interference), the present disclosure shows that controlling stereochemistry can surprisingly increase stability without significantly reducing activity.

[0009] In some embodiments, the disclosure provides oligonucleotides having certain 5' end structures.

[0010] In some embodiments, the present disclosure provides 5' end structures that, when used in accordance with the present disclosure, may generate oligonucleotides with enhanced biological activity (eg, RNAi activity).

[0011] In some embodiments, the present disclosure includes the recognition that various additional chemical moieties (such as lipid moieties and / or carbohydrate moieties) may be incorporated into an oligonucleotide to improve one or more PNPLA3 oligonucleotide properties (such as knockdown of a PNPLA3 target gene or its gene product). In some embodiments, the additional chemical moieties are optional. In some embodiments, a PNPLA3 oligonucleotide may include more than one additional chemical moiety. In some embodiments, a PNPLA3 oligonucleotide may include two or more additional chemical moieties, the additional chemical moieties being the same or different, or of the same category (e.g., targeting moiety, carbohydrate moiety, moiety that binds to ASPGR, lipid moiety, etc.), or of different categories. In some embodiments, certain additional chemical moieties facilitate delivery of the oligonucleotide to a desired cell, tissue, and / or organ. In some embodiments, certain additional chemical moieties facilitate internalization of the oligonucleotide and / or increase the stability of the oligonucleotide.

[0012] In some embodiments, the disclosure provides PNPLA3 oligonucleotide compositions that achieve allele-specific suppression, whereby a transcript derived from one allele of a particular target gene is selectively knocked down relative to at least one other allele of the same gene.

[0013] In some embodiments, the present disclosure shows that certain provided structural elements, techniques, and / or features are particularly useful for PNPLA3 oligonucleotides (e.g., RNAi agents) that are involved in and / or induce RNAi mechanisms.However, in any case, the teachings of the present disclosure are not limited to oligonucleotides that are involved in or function via any particular mechanism.In some embodiments, the present disclosure relates to any oligonucleotide, which functions via any mechanism and includes any sequence, structure, or format (or part thereof) described herein. In some embodiments, the present disclosure provides PNPLA3 oligonucleotides, which function via any mechanism and include any sequence, structure, or format (or a portion thereof) described herein, including but not limited to any 5'-terminal structure, 5'-terminal region, first region (including but not limited to a seed region), second region (including but not limited to a post-seed region), and 3'-terminal region (which may be a 3'-terminal dinucleotide and / or a 3'-terminal cap), optional additional chemical moieties (including but not limited to a targeting moiety, a carbohydrate moiety, a moiety that binds APGR, and a lipid moiety), stereochemistry or stereochemistry pattern, modification or modification pattern, internucleotide linkage or internucleotide linkage pattern, sugar(s) modification or sugar modification pattern, base(s) modification or base modification pattern. In some embodiments, the provided oligonucleotides may participate in (e.g., induce) an RNAi mechanism. In some embodiments, provided oligonucleotides may participate in RNase H (ribonuclease H) mechanisms. In some embodiments, provided oligonucleotides may act as translation inhibitors (e.g., sterically block translation). In some embodiments, provided oligonucleotides may be therapeutic. In some embodiments, provided oligonucleotides are useful in therapeutic, diagnostic, research, and / or nanomaterial applications.In some embodiments, a target is a particular allele with respect to which modification of expression and / or activity of one or more products (e.g., RNA and / or protein products) is intended. In many embodiments, a target allele is one whose presence and / or expression is associated with (e.g., correlates with) the presence, onset, and / or severity of one or more diseases and / or conditions. Alternatively, or in addition, in some embodiments, a target allele is one whose modification of the level and / or activity of one or more gene products is correlated with amelioration of one or more aspects of a disease and / or condition (e.g., delayed onset, reduced severity, responsiveness to other treatments, etc.).

[0014] In some embodiments, different alleles of the same gene exist where the presence and / or activity of a particular allele (disease-associated allele) is associated (e.g., correlated) with the presence, onset, and / or severity of one or more disorders, diseases, and / or conditions, and such different alleles are not so associated, or are associated to a lesser extent (e.g., exhibit a less significant or statistically insignificant correlation). In some such embodiments, the oligonucleotides and methods thereof described herein may preferentially or specifically target the associated allele compared to one or more less / unassociated allele(s), thereby mediating allele-specific suppression.

[0015] In some embodiments, the PNPLA3 target sequence is a sequence to which the PNPLA3 oligonucleotide described herein binds. In many embodiments, the target sequence is identical to or an exact complement of the sequence of the provided oligonucleotide or the sequence of consecutive residues present therein (e.g., the provided oligonucleotide comprises a target binding sequence that is identical to or an exact complement of the target sequence). In some embodiments, the target binding sequence is an exact complement of the target sequence of a transcription product (e.g., pre-mRNA, mRNA, etc.). The target binding sequence / target sequence can be of various lengths for the provided oligonucleotide having the desired activity and / or properties. In some embodiments, the target binding sequence / target sequence comprises 5-50 bases. In some embodiments, a small number of differences / mismatches between (relevant portions of) the PNPLA3 oligonucleotide and its target sequence are tolerated, including but not limited to the 5' and / or 3' terminal regions of the target sequence and / or oligonucleotide sequence. In many embodiments, the target sequence is present in the transcription products (eg, mRNA and / or pre-mRNA) produced from the target gene.

[0016] In some embodiments, the target sequence comprises one or more allelic sites (i.e., positions where allelic variation occurs in the target gene). In some embodiments, the allelic site is a mutation. In some embodiments, the allelic site is a SNP. In some such embodiments, the provided oligonucleotides bind preferentially or specifically to one allele compared to one or more other alleles. In some embodiments, the provided oligonucleotides bind preferentially to disease-associated alleles. For example, in some embodiments, the PNPLA3 oligonucleotides provided herein (or target binding sequence portions thereof) have a sequence that is at least partially identical to the target sequence of a particular allelic version, or is the exact complement of the target sequence of a particular allelic version.

[0017] Unless otherwise specified, all sequences (including but not limited to base sequences and chemical patterns, modification patterns, and / or stereochemical patterns) are presented in 5' to 3' order.

[0018] In some embodiments, the disclosure provides compositions and methods related to target-specific oligonucleotides, including or comprising a PNPLA3 oligonucleotide having a base sequence of any oligonucleotide disclosed herein, or a region of at least 15 consecutive nucleotides of a base sequence of any oligonucleotide disclosed herein, wherein the first nucleotide of the base sequence or the first nucleotide of the at least 15 consecutive nucleotides can be optionally replaced by a T or a DNA T. In some embodiments, the oligonucleotide has the ability to induce ssRNAi.

[0019] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkage, sugar, and / or base modifications.

[0020] In some embodiments, the nucleotide is a natural nucleotide. In some embodiments, the nucleotide is a modified nucleotide. In some embodiments, the nucleotide is a nucleotide analog. In some embodiments, the base is a modified base. In some embodiments, the base is a protected nucleobase (such as a protected nucleobase used in oligonucleotide synthesis). In some embodiments, the base is a base analog. In some embodiments, the sugar is a modified sugar. In some embodiments, the sugar is a sugar analog. In some embodiments, the internucleotide linkage is a modified internucleotide linkage. In some embodiments, the nucleotide comprises a base, a sugar, and an internucleotide linkage, each of which is independently and optionally of natural or non-natural origin. In some embodiments, the nucleoside comprises a base and a sugar, each of which is independently and optionally of natural or non-natural origin. Non-limiting examples of nucleotides include DNA (2'-deoxy) nucleotides and RNA (2'-OH) nucleotides, as well as those that comprise one or more modifications in the base, sugar, and / or internucleotide linkage. Non-limiting examples of sugars include ribose and deoxyribose, and ribose and deoxyribose with 2'-modifications, including but not limited to, 2'-F, LNA, 2'-OMe, and 2'-MOE modifications. In some embodiments, the internucleotide linkage can have the structure of Formula I, as described in the present disclosure. In some embodiments, the internucleotide linkage does not contain phosphorus, but is a moiety that serves to link two natural or unnatural sugars.

[0021] In some embodiments, the present disclosure provides chiral-controlled PNPLA3 oligonucleotide compositions that more strongly induce reduction in expression, activity, and / or levels of the PNPLA3 gene or its gene product, single-stranded RNA interference, and / or RNase H-mediated knockdown when compared to a reference condition, such as the absence of the composition or the presence of a reference composition (e.g., a stereoirregular composition of oligonucleotides having the same base sequence and chemical modifications).

[0022] In some embodiments, the PNPLA3 oligonucleotide composition comprising a plurality of oligonucleotides is stereoirregular in that the plurality of oligonucleotides do not share a common stereochemistry at any chiral internucleotide bond.In some embodiments, the PNPLA3 oligonucleotide composition comprising a plurality of oligonucleotides is chiral controlled in that the plurality of oligonucleotides share a common stereochemistry at one or more chiral internucleotide bonds.In some embodiments, the PNPLA3 oligonucleotide composition comprising a chiral controlled first plurality of oligonucleotides has reduced susceptibility to endonuclease and exonuclease compared to the PNPLA3 oligonucleotide composition comprising a stereoirregular first plurality of oligonucleotides.

[0023] In some embodiments, the composition comprises multimers of two or more of any of a first plurality of PNPLA3 oligonucleotides and / or a second plurality of oligonucleotides, wherein the first plurality of oligonucleotides and the second plurality of oligonucleotides are capable of independently inducing knockdown of the same or different targets via RNA interference and / or RNase H-mediated knockdown independently.

[0024] In some embodiments, the PNPLA3 oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a first plurality of oligonucleotides) is chiral controlled in that the plurality of oligonucleotides independently share a common stereochemistry at one or more chiral internucleotide bonds.In some embodiments, the plurality of oligonucleotides share a common stereochemistry at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral internucleotide bonds, each of which is independently Rp or Sp.In some embodiments, the plurality of oligonucleotides share a common stereochemistry at each chiral internucleotide bond. In some embodiments, a chiral internucleotide linkage is referred to as a chiral controlled internucleotide linkage when a given level of oligonucleotides in a composition share a common stereochemical configuration (independently Rp or Sp).

[0025] In some embodiments, at least 5 internucleotide linkages are chiral controlled. In some embodiments, at least 10 internucleotide linkages are chiral controlled. In some embodiments, at least 15 internucleotide linkages are chiral controlled. In some embodiments, each chiral internucleotide linkage is chiral controlled.

[0026] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of PNPLA3 oligonucleotides comprising: 1) consensus sequence, 2) common patterns of skeletal bonds, and 3) Common patterns of skeletal chiral centers Share The compositions are substantially pure preparations of single oligonucleotides in that a given level of oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0027] In some embodiments, the common pattern of backbone chiral centers comprises at least one internucleotide linkage that includes a chiral center that is chiral controlled.

[0028] In some embodiments, the level and / or diastereopurity of an oligonucleotide can be determined by analytical methods (eg, chromatographic methods, spectroscopic methods, spectroscopic methods, or any combination thereof).

[0029] In particular, the present disclosure includes the recognition that stereoirregular PNPLA3 oligonucleotide preparations contain multiple separate chemical entities that differ from each other (e.g., in the stereochemical structure (or stereochemistry) of individual backbone chiral centers within the oligonucleotide chain). If the stereochemistry of the backbone chiral centers is not controlled, stereoirregular oligonucleotide preparations are uncontrolled compositions with an indeterminate level of oligonucleotide stereoisomers. Although such stereoisomers may have the same base sequence and / or chemical modification, such stereoisomers are different chemical entities at least because of their different backbone stereochemistry, and such stereoisomers may have different properties (e.g., nuclease susceptibility, activity, distribution, etc.) as shown herein. In some embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its length, its backbone bonding pattern, and its backbone chiral center pattern. In some embodiments, the present disclosure shows that the improved properties and activities achieved by controlling the stereochemistry within PNPLA3 oligonucleotides may be comparable or even better than those achieved by using chemical modifications.

[0030] In some embodiments, the PNPLA3 oligonucleotide comprises, in 5' to 3' order, a 5' terminal region, a seed region, a post-seed region, and a 3' terminal region, and optionally further comprises an additional chemical moiety.

[0031] In some embodiments, the 5' terminal region is the entire portion of the PNPLA3 oligonucleotide located 5' to the seed region. In some embodiments, the 3' terminal region is the entire portion of the PNPLA3 oligonucleotide located 3' to the post-seed region.

[0032] In some embodiments, the 5' terminal structure is a 5' terminal group.

[0033] In some embodiments, the 5' terminal structure comprises a 5' end group.

[0034] In some embodiments, provided oligonucleotides may include a 5' terminal region, 5' terminal structure, 5' terminal group, 5' terminal nucleoside, or 5' terminal nucleotide, as described herein or known in the art.

[0035] In some embodiments, the 5' terminal structure, 5' terminal region, 5' nucleotide portion, seed region, post-seed region, 3' terminal dinucleotide, and / or 3' terminal cap independently have any structure described herein or known in the art. In some embodiments, any structure of the 5' terminal described herein or known in the art, and / or any structure of the 5' nucleotide portion described herein or known in the art, and / or any structure of the seed region described herein or known in the art, and / or any structure of the post-seed region described herein or known in the art, and / or any structure of the 3' terminal dinucleotide described herein or known in the art, and / or any structure of the 3' terminal cap described herein or known in the art can be combined.

[0036] In some embodiments, the oligonucleotide provided comprises one or more blocks. In some embodiments, the oligonucleotide provided comprises one or more blocks, where a block comprises one or more consecutive nucleosides, and / or nucleotides, and / or sugars, or bases, and / or internucleotide linkages. In some embodiments, a block comprises the entire seed region or a portion thereof. In some embodiments, a block comprises the entire post-seed region or a portion thereof.

[0037] In some embodiments, the oligonucleotides provided are blockmers.

[0038] In some embodiments, the oligonucleotides provided are altmers that include alternating blocks. In some embodiments, blockmers or altmers can be defined by chemical modifications (including presence or absence), such as, for example, base modifications, sugar modifications, internucleotide linkage modifications, stereochemistry, and the like, or patterns thereof.

[0039] In some embodiments, provided oligonucleotides include one or more sugar modifications. In some embodiments, the sugar modification is at the 2' position. In some embodiments, the sugar modification is selected from 2'-F, 2'-OMe, and 2'-MOE. 2'-F is also known as 2'fluoro. 2'-OMe is also known as 2'-O-methyl. 2'-MOE is also known as 2'-methoxyethyl or MOE.

[0040] In some embodiments, the PNPLA3 oligonucleotide contains only two 2'-Fs. In some embodiments, the PNPLA3 oligonucleotide contains only two 2'-Fs, and these two nucleotides are located at the 2nd and 14th positions.

[0041] In some embodiments, the PNPLA3 oligonucleotide contains only two 2'-Fs, these two nucleotides are at the second and fourteenth positions, and the first nucleotide is 2'-deoxy.

[0042] In some embodiments, the PNPLA3 oligonucleotide contains only two 2'-Fs, which are at the second and fourteenth positions, and the first nucleotide is a 2'-deoxyT.

[0043] In some embodiments, the PNPLA3 oligonucleotide contains only two 2'-Fs, these two nucleotides are at the second and fourteenth positions, the first nucleotide is 2'-deoxy, and the 5' terminal structure is -OH.

[0044] In some embodiments, the PNPLA3 oligonucleotide contains only two 2'-Fs, these two nucleotides are present at the second and fourteenth positions, the first nucleotide is a 2'-deoxy T, and the 5' terminal structure is -OH.

[0045] In some embodiments herein, when referring to a PNPLA3 oligonucleotide, "first" (e.g., the first nucleotide) refers to the 5' end of the oligonucleotide, and "last" or "terminal" (e.g., the last or terminal nucleotide) refers to the 3' end.

[0046] In some embodiments, provided oligonucleotides contain alternating sugars with a particular modification and sugars with no modification or different modifications, hi some embodiments, sugars with a particular modification are found in one or more blocks.

[0047] In some embodiments, the oligonucleotides provided include one or more blocks that alternate between sugars with a particular 2'-modification and sugars that, independently, have no modification or a different modification. In some embodiments, the oligonucleotides provided include one or more blocks that alternate between sugars with a 2'-F modification and sugars that, independently, have no modification or a different modification. In some embodiments, the oligonucleotides provided include one or more blocks that alternate between sugars with a 2'-OMe modification and sugars that, independently, have no modification or a different modification. In some embodiments, the oligonucleotides provided include one or more blocks that alternate between sugars with a 2'-OMe modification and sugars that, independently, have no modification or a different modification.

[0048] In some embodiments the sugar blocks are ff, fffm, fffmm, fffmmm, fffmmmm, fffmmmmm, fffmmmmmm, fffmmmmmmf, fffmmmmmmff, fffmmmmmmffm, fffmmmmmmffmm, fffmmmmmmffmmf, fffmmmmmmffmmf, fffmmmmmmffmmfm, fffmmmmmmffmmfmf, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfm, fffmmmmmm mffmmfmfmfmm, ffmmffmm, ffmmmmmmffmmfmfmfmmm, fmfmfmfmfmfmfm, fmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmf mfm, fmfmfmfmfmfmfmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf mfmm, fmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmm, fmfm fmfmfmfmmm, fmmffmm, fmmmmmmffmmfmfmfmmm, mff, mffm, mffmf, mffmff, mffmffm, mffmmffmm, mfmfm, mfmfmfmfmfffmfmfmfmmm, mfmfmfmfmfmfmfm, mf mfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmfmfmmm, mfmfmfmfmfmfmfmfmm, mf mfmfmfmfmfmfmm, mfmfmfmfmfmfmm, mfmfmfmfmfmfmmm, mfmfmfmfmfmmm, mfm fmfmfmfmmmfm, mfmfmfmfmmmm, mfmfmfmfmmm, mfmfmfmfmmmfmfm, mfmfmfmfmmmfmmm, mfmfmfmmmmmmfm, mfmfmfmmm, mfmfmfmmmfmfmfm, mfmfmfmmmf mfmmm, mfmfmfmmmfmmmfm, mfmfmfmmmmmfmfm, mfmfmmm, mfmfmmmfmfmfmfm, mfmfmmmfmfmfmmm, mfmfmmmfmfmmmfm, mfmfmmmfmmmfmfm, mfmfmmmmmfmfmfm,mfmmm, mfmmmfmfmfmfmfm, mfmmmfmfmfmfmmm, mfmmmfmfmfmmmfm, mfmmmfmfmmmfmfm, mfm mmfmmmfmfmfm, mfmmmfmmmfmfmfm, mfmmmmmfmfmfmfm, mmffm, mmffmm, mmffmm, mmffmmf, m mffmmff, mmffmmffm, mmffmmffmm, mmffmmfmfmfmmm, mmm, mmmffmmfmfmfmmm, mmmfmfmfmfmfmfm, mmmfmfmfmfmfmmm, mmmfmfmfmfmmmfm, mmmfmfmfmmmfmfm, mmmfmfmmmfmfmfm, mmm fmmmfmfmfm, mmmmffmmfmfmmmm, mmm, mmmm, mmmmm, mmmmmffmmfmfmmmm, mmmmmmfmfmfmfm, mmmmmm, mmmmmmffmmfmfmfmfm, mmmmmm, mmmmmmffmmfmfmfmm, mfmf, mfmf, mfmfmf, fmfmfm, fmfmfmf, dfdf, dfdfdf, dfdfdfdf, fdfdfdf, fdfdfdfd, dfdfmfmf, dfmfmf, mfdfmf, or dfmfdf, where m indicates 2'-OMe, f indicates 2'-F and d indicates no substitution at the 2' position. In some embodiments, the seed region and / or the post-seed region may comprise a block of sugar modifications.

[0049] In some embodiments, the block is a stereochemical block. In some embodiments, the block is an Rp block, in that each internucleotide bond of the block is Rp. In some embodiments, the seed region block is an Rp block. In some embodiments, the post-seed region block is an Rp block. In some embodiments, the block is an Sp block, in that each internucleotide bond of the block is Sp. In some embodiments, the seed region block is an Sp block. In some embodiments, the post-seed region block is an Sp block. In some embodiments, the provided oligonucleotide comprises both an Rp block and an Sp block. In some embodiments, the provided oligonucleotide comprises one or more Rp blocks, but does not comprise an Sp block. In some embodiments, the provided oligonucleotide comprises one or more Sp blocks, but does not comprise an Rp block. In some embodiments, the provided oligonucleotide comprises one or more PO blocks, and each internucleotide bond of the block is a natural phosphate bond.

[0050] In some embodiments, the seed region block is an Sp block, and the sugar moieties each comprise a 2'-F modification. In some embodiments, the seed region block is an Sp block, and the internucleotide linkages are each modified internucleotide linkages, and the sugar moieties each comprise a 2'-F modification. In some embodiments, the seed region block is an Sp block, and the internucleotide linkages are each phosphorothioate linkages, and the sugar moieties each comprise a 2'-F modification. In some embodiments, the seed region block comprises four or more nucleoside units. In some embodiments, a nucleoside unit is a nucleoside. In some embodiments, the seed region block comprises five or more nucleoside units. In some embodiments, the seed region block comprises six or more nucleoside units. In some embodiments, the seed region block comprises seven or more nucleoside units. In some embodiments, the post-seed region block is an Sp block, and the sugar moieties each comprise a 2'-F modification. In some embodiments, the post-seed region block is an Sp block, the internucleotide linkages are each modified internucleotide linkages, and the sugar moieties each comprise a 2'-F modification. In some embodiments, the post-seed region block is an Sp block, the internucleotide linkages are each phosphorothioate linkages, and the sugar moieties each comprise a 2'-F modification. In some embodiments, the post-seed region block comprises four or more nucleoside units. In some embodiments, the post-seed region block comprises five or more nucleoside units. In some embodiments, the post-seed region block comprises six or more nucleoside units. In some embodiments, the post-seed region block comprises seven or more nucleoside units. In some embodiments, the seed region and / or the post-seed region may comprise a block. In some embodiments, the seed region and / or the post-seed region comprise a stereochemical block.

[0051] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) have a common base sequence; 2) contain one or more modified sugar moieties and modified internucleotide linkages.

[0052] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single stranded RNA interference, the first plurality of oligonucleotides comprising: 1) having a common base sequence complementary to a target sequence in the transcription product; 2) contain one or more modified sugar moieties and modified internucleotide linkages.

[0053] In some embodiments, the reference condition is the absence of the composition. In some embodiments, the reference condition is the presence of the reference composition. Examples of reference compositions comprising a plurality of oligonucleotides for reference are described in detail in the present disclosure. In some embodiments, the reference plurality of oligonucleotides has different structural elements (chemical modifications, stereochemistry, etc.) compared to the first plurality of oligonucleotides in the provided composition. In some embodiments, the provided oligonucleotide composition comprising the first plurality of oligonucleotides is chiral controlled in that the first plurality of oligonucleotides comprises one or more chiral controlled internucleotide linkages. In some embodiments, the provided oligonucleotide composition comprising the first plurality of oligonucleotides is chiral controlled in that the first plurality of oligonucleotides comprises 1-20 chiral controlled internucleotide linkages. In some embodiments, the first plurality of oligonucleotides comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 chiral controlled internucleotide linkages. In some embodiments, the reference composition is a stereoirregular preparation of oligonucleotides with the same chemical modification. In some embodiments, the reference composition is a mixture of stereoisomers, while the provided composition is a single-stranded RNAi agent of one stereoisomer. In some embodiments, the reference oligonucleotides have the same base sequence as the first oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same chemical modification as the first oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same sugar modification as the first oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same base modification as the first oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same internucleotide bond modification as the first oligonucleotides in the provided composition.In some embodiments, the reference oligonucleotides have the same base sequence and the same chemical modifications as the first plurality of oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same stereochemistry as the first plurality of oligonucleotides in the provided composition, but different chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.).

[0054] In some embodiments, the disclosure provides a composition comprising a PNPLA3 oligonucleotide, the oligonucleotide is complementary or substantially complementary to a target RNA sequence and has a length of about 15 to about 49 total nucleotides, and the oligonucleotide comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0055] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a single stranded RNAi agent, the single stranded RNAi agent being complementary or substantially complementary to a target RNA sequence, having a length of from about 15 to about 49 total nucleotides, and capable of inducing target-specific RNA interference, the single stranded RNAi agent comprising at least one unnatural base, sugar, and / or internucleotide linkage.

[0056] In some embodiments, the length is between 15 and 49, between about 17 and about 49, between 17 and 49, between about 19 and about 29, between 19 and 29, between about 19 and about 25, between 19 and 25, between about 19 and about 23, or between 19 and 23 total nucleotides.

[0057] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) having a common base sequence that is complementary or substantially complementary to a target sequence in the transcription product; 2) contains one or more modified sugar moieties and modified internucleotide linkages; The oligonucleotide composition is characterized in that, when contacted with a transcript, it results in improved knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0058] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single stranded RNA interference, the first plurality of oligonucleotides comprising: 1) having a common base sequence complementary to a target sequence in the transcription product; 2) contains one or more modified sugar moieties and modified internucleotide linkages; The oligonucleotide composition is characterized in that, when it is contacted with a transcript in an RNA interference system, it improves RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0059] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification The oligonucleotide is of a specific oligonucleotide type defined by:

[0060] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single stranded RNA interference, the first plurality of oligonucleotides comprising: 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification The oligonucleotide is of a specific oligonucleotide type defined by:

[0061] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides of a PNPLA3 oligonucleotide type, the oligonucleotide type comprising: 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification is defined by The composition is chiral controlled in that it is enriched for a particular oligonucleotide type relative to a substantially racemic preparation of an oligonucleotide having the same base sequence; The oligonucleotide composition is characterized in that, when contacted with a transcript, it results in improved knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0062] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides of a PNPLA3 oligonucleotide type, the oligonucleotide type comprising: 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification is defined by The composition is chiral controlled in that it is enriched for a particular oligonucleotide type relative to a substantially racemic preparation of an oligonucleotide having the same base sequence; The oligonucleotide composition is characterized in that, when it is contacted with a transcript in an RNA interference system, it improves RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0063] In some embodiments, the oligonucleotides provided have any of the formats shown in FIG. 1 or have any structural elements shown in any of the formats shown in FIG.

[0064] In some embodiments, a provided single stranded RNAi agent has any of the formats shown in FIG. 1 or has any structural element shown in any of the formats shown in FIG.

[0065] In particular, the present disclosure presents data showing that various oligonucleotides of the disclosed format have the ability to induce a decrease in the expression and / or level of a target gene or its gene product when targeted to any of several different sequences in any of several different genes.In some embodiments, the present disclosure presents data showing that various RNAi agents of the disclosed format have the ability to induce RNA interference to any of many different sequences in any of many different genes.

[0066] In some embodiments, PNPLA3 oligonucleotides having any of the structures described and / or shown herein are capable of inducing RNA interference. In some embodiments, PNPLA3 oligonucleotides having any of the structures described and / or shown herein are capable of inducing RNase H-mediated knockdown. In some embodiments, PNPLA3 oligonucleotides having any of the structures described and / or shown herein are capable of inducing RNA interference and / or RNase H-mediated knockdown. In some embodiments, PNPLA3 oligonucleotides include any structural element of any oligonucleotide described herein or any format described herein or shown in FIG. 1. In some embodiments, PNPLA3 oligonucleotides include any structural element of any oligonucleotide described herein or any format described herein or shown in FIG. 1 and are capable of inducing RNA interference. In some embodiments, PNPLA3 oligonucleotides include any structural element of any oligonucleotide described herein or any format described herein or shown in FIG. 1 and are capable of inducing RNase H-mediated knockdown. In some embodiments, the PNPLA3 oligonucleotide comprises any structural element of any oligonucleotide described herein, or any format described herein or shown in FIG. 1, and has the ability to induce RNA interference and / or RNase H-mediated knockdown.

[0067] In some embodiments, the RNAi agent comprises any one or more of a 5'-end structure, a 5'-end region, a seed region, a post-seed region, and a 3'-end region, and an optional additional chemical moiety. In some embodiments, the seed region is any seed region described herein or known in the art. In some embodiments, the post-seed region can be any region between the seed region and the 3'-end region described herein or known in the art. In some embodiments, the 3'-end region can be any 3'-end region described herein or known in the art. In some embodiments, the optional additional chemical moiety can be any optional additional chemical moiety described herein or known in the art. Any individual 5'-end structure, 5'-end region, seed region, post-seed region, 3'-end region, and optional additional chemical moiety described herein or known in the art can be independently combined with any other 5'-end structure, 5'-end region, seed region, post-seed region, 3'-end region, and optional additional chemical moiety described herein or known in the art. In some embodiments, by way of non-limiting example, the region of the single stranded RNAi agent is a 5' end structure, a 5' end region, a seed region, a post-seed region, a portion of the seed region, a portion of the post-seed region, or a 3' end dinucleotide.

[0068] In some embodiments, the base sequence of the oligonucleotide provided consists of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the oligonucleotide provided comprises the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the oligonucleotide provided comprises a sequence comprising a sequence of 15 consecutive bases of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the oligonucleotide provided comprises a sequence comprising a sequence of 20 consecutive bases with up to 5 mismatches of the base sequence of any oligonucleotide disclosed herein.

[0069] In some embodiments, the oligonucleotides provided are capable of inducing a decrease in expression and / or level of a target gene or its gene product. In some embodiments, the oligonucleotides provided are capable of inducing single-stranded RNAi interference. In some embodiments, the oligonucleotides provided are capable of inducing RNase H-mediated knockdown. In some embodiments, the oligonucleotides provided are capable of inducing single-stranded RNA interference and RNase H-mediated knockdown. In some embodiments, the oligonucleotides comprise a sequence that targets any transcript or gene targeted by the oligonucleotides disclosed herein.

[0070] In some embodiments, provided oligonucleotides target PNPLA3.

[0071] In some embodiments, the provided oligonucleotides can be used to reduce or inhibit the activity, level, and / or expression of the PNPLA3 gene or its gene product. In some embodiments, the provided oligonucleotides can be used to reduce or inhibit the activity, level, and / or expression of the gene or its gene product, where aberrant or excessive activity, level, and / or expression of the gene or its gene product, deleterious mutations in the gene or its gene product, or abnormal tissue or intercellular or subcellular distribution of the gene or its gene product are associated with, cause, and / or associated with a disorder. In some embodiments, the provided oligonucleotides can be used to treat and / or manufacture a medicament for treating a disorder that is associated with, causes, and / or associated with aberrant or excessive activity, level, and / or expression of the gene or its gene product, or abnormal distribution of the gene or its gene product.

[0072] In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein that have the ability to target PNPLA3 and are useful for treating a PNPLA3-associated disorder and / or for the manufacture of a treatment for a PNPLA3-associated disorder.

[0073] In some embodiments, the PNPLA3 oligonucleotide capable of targeting a gene comprises a base sequence that is a part of the base sequence of a target gene, or comprises a base sequence that is complementary or substantially complementary to a part of the base sequence of a target gene.In some embodiments, the length of the part is at least 15 bases.In some embodiments, the base sequence of single-stranded RNAi agent can comprise or consist of a base sequence that has a specific number of maximum mismatches with a specific base sequence.

[0074] In some embodiments, a mismatch is a difference between base sequences or lengths when two sequences are maximally aligned and compared. As a non-limiting example, a mismatch is counted if there is a difference between a base at a particular position in one sequence and a base at the corresponding position in the other sequence. Thus, for example, a mismatch is counted if a particular base (e.g., A) is present at a position in one sequence and a different base (e.g., G, C, or U) is present at the corresponding position in the other sequence. For example, a mismatch is counted if a base (e.g., A) is present at a position in one sequence and no base is present at the corresponding position in the other (e.g., the position is an abasic nucleotide that contains a phosphate-sugar backbone but no base) or if the position is skipped. A single-stranded nick in either sequence (or in the sense or antisense strand) may not be counted as a mismatch, for example, if one sequence contains the sequence 5'-AG-3' but the other sequence contains the sequence 5'-AG-3' with a single-stranded nick between the A and G, it will not be counted as a mismatch. Base modifications are generally not considered mismatches, for example, if one sequence contains a C and the other sequence contains a modified C (e.g., 5mC) at the same position, it may not be counted as a mismatch. In some embodiments, for purposes of counting mismatches, a T substituted with a U, or vice versa, is not considered a mismatch.

[0075] In some embodiments, the PNPLA3 oligonucleotide is complementary or completely complementary or 100% complementary to the target sequence (e.g., RNA such as mRNA), which means that the base sequence of the oligonucleotide does not have any mismatch with the sequence that is completely complementary to the target sequence (e.g., base-pairs via Watson-Crick base pairing). Without wishing to be bound by any particular theory, the present disclosure specifies that for single-stranded RNAi agents, it is not necessary that the 5'-terminal nucleotide portion or the 3'-terminal dinucleotide base-pairs with the target. These may be mismatches. Furthermore, antisense oligonucleotides or single-stranded RNAi agents may have a small number of internal mismatches, but still induce the expression and / or level reduction of the target gene or its gene product, and / or NaseH-mediated knockdown and / or RNA interference. If the first base sequence of the PNPLA3 oligonucleotide (e.g., antisense oligonucleotide or single-stranded RNAi agent) has a small number of mismatches with the reference base sequence that is 100% complementary to the target sequence, the first base sequence is substantially complementary to the target sequence. In some embodiments, the PNPLA3 oligonucleotide (e.g., antisense oligonucleotide or single-stranded RNAi agent) can have a base sequence that is complementary or substantially complementary to the target sequence. In some embodiments, the complementarity is determined based on Watson-Crick base pairing (guanine-cytosine and adenine-thymine / uracil), where guanine, cytosine, adenine, thymine, uracil can be arbitrarily and independently modified but maintain their pairing hydrogen bond pattern that is seen in the unmodified case. In some embodiments, a sequence that is complementary to another sequence includes at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 bases.

[0076] In some embodiments, the PNPLA3 oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common pattern of backbone linkages. In some embodiments, the common pattern of backbone linkages comprises at least 10 modified internucleotide linkages.

[0077] In some embodiments, the common pattern of backbone linkages comprises at least 10 phosphorothioate linkages. In some embodiments, the PNPLA3 oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises at least one internucleotide linkage in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least one internucleotide linkage that is phosphorothioate in the Sp configuration. In some embodiments, the oligonucleotides in the provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, the provided compositions are chiral controlled PNPLA3 oligonucleotide compositions in that the compositions comprise predetermined levels of oligonucleotides of individual oligonucleotide types, where the PNPLA3 oligonucleotide types are defined by: 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modifications.

[0078] As discussed above and understood in the art, in some embodiments, the base sequence of a PNPLA3 oligonucleotide can refer to the identity and / or modification state of the nucleoside residues in the oligonucleotide (e.g., the state of the sugar and / or base components compared to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or can refer to the hybridization properties of such residues (i.e., the ability to hybridize with a particular complementary residue).

[0079] In some embodiments, a particular oligonucleotide type may be defined by: 1A) base identity, 1B) Base modification pattern, 1C) Glycosylation pattern, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modifications. Thus, in some embodiments, particular oligonucleotide types may share the same bases but differ in their patterns of base modifications and / or sugar modifications, hi some embodiments, particular oligonucleotide types may share the same bases and patterns of base modifications (including, for example, the absence of base modifications), but may differ in their patterns of sugar modifications.

[0080] In some embodiments, a particular oligonucleotide type is chemically identical in that it has the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same backbone linkage pattern (e.g., natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the stereochemical (Rp / Sp) pattern of chiral internucleotide linkages), and the same backbone phosphorus modification pattern (e.g., the modification pattern on the internucleotide phosphorus atom (-S-, etc., and -L-R1 of Formula I).

[0081] In particular, the disclosure provides oligonucleotide compositions and techniques for optimizing properties (e.g., improving single-stranded RNA interference, RNase H-mediated knockdown, etc.). In some embodiments, the disclosure provides methods for attenuating immune responses associated with administration of oligonucleotides and compositions thereof (i.e., administering an oligonucleotide composition such that an undesirable immune response to the oligonucleotide in the composition is reduced, e.g., compared to that observed with a reference nucleotide composition of comparable or identical nucleotide sequence). In some embodiments, the disclosure provides methods for increasing binding of oligonucleotides and compositions thereof to a particular protein. In some embodiments, the disclosure provides methods for increasing binding of oligonucleotides and compositions thereof to a particular protein. In some embodiments, the disclosure provides methods for enhancing delivery of oligonucleotides and compositions thereof. Among other things, the present disclosure includes the recognition that optimal delivery of an oligonucleotide to its target requires, in some embodiments, a balance between binding of the oligonucleotide to a particular protein so that the oligonucleotide may be transported to a desired location, and proper release of the oligonucleotide from a particular protein so that the oligonucleotide may exert its desired function (e.g., hybridization to its target, cleavage of its target, inhibition of translation, modulation of transcript processing, etc.) As exemplified herein, the present disclosure recognizes that improved oligonucleotide properties can be achieved through, among other things, chemical modifications and / or stereochemistry.

[0082] In some embodiments, the disclosure provides a method for treating or preventing a disease, the method comprising administering to a subject a PNPLA3 oligonucleotide composition described herein.

[0083] In some embodiments, the disease is one that can be cured, ameliorated, or a new beneficial function introduced by knocking down a target nucleic acid via single-stranded RNA interference after administration of a provided composition.

[0084] In some embodiments, the consensus sequence comprises a sequence selected from Table 1 A. In some embodiments, the consensus sequence is a sequence selected from Table 1 A. In some embodiments, the pattern of backbone chiral centers is selected from those set forth in Table 1A.

[0085] In some embodiments, the disclosure provides a method comprising administering a composition comprising a first plurality of oligonucleotides, the composition exhibiting improved delivery compared to a reference composition comprising a plurality of oligonucleotides, each of the plurality of oligonucleotides in the reference composition having a similar common base sequence, but the plurality of oligonucleotides: the stereochemical structure of each of the oligonucleotides in the plurality of references differs from one another; and / or At least some of the oligonucleotides in the plurality of oligonucleotides of the reference have a structure that is different from the structure exhibited by the plurality of oligonucleotides of the composition. They are structurally different from the first plurality of oligonucleotides in that they

[0086] In some embodiments, the disclosure provides a method of administering a PNPLA3 oligonucleotide composition having the ability to induce a decrease in expression and / or levels of a target gene or its gene product, the method comprising administering a first plurality of oligonucleotides having a common nucleotide sequence, the improvement including: administration of the PNPLA3 oligonucleotides constituting the first plurality of oligonucleotides is characterized by improved delivery as compared to a reference oligonucleotide composition of the same common nucleotide sequence; Includes.

[0087] In some embodiments, the disclosure provides a method of administering a PNPLA3 oligonucleotide composition capable of inducing single stranded RNA interference, the method comprising administering a first plurality of oligonucleotides having a common nucleotide sequence, the improvement including: administration of the PNPLA3 oligonucleotides constituting the first plurality of oligonucleotides is characterized by improved delivery as compared to a reference oligonucleotide composition of the same common nucleotide sequence; Includes.

[0088] In some embodiments, the present disclosure provides a single stranded RNAi agent of a PNPLA3 oligonucleotide selected from any of the tables (including but not limited to Table 1A) or otherwise disclosed herein. In some embodiments, the present disclosure provides a single stranded RNAi agent of a PNPLA3 oligonucleotide selected from any of the tables (including but not limited to Table 1A) or otherwise disclosed herein, wherein the oligonucleotide is conjugated to a lipid moiety.

[0089] In some embodiments, the provided oligonucleotide comprises a lipid moiety. In some embodiments, the lipid moiety is incorporated by complexation with a lipid. In some embodiments, the lipid moiety is a fatty acid. In some embodiments, the PNPLA3 oligonucleotide is complexed to a fatty acid. In some embodiments, the provided single-stranded RNAi agent further comprises a lipid. In some embodiments, the provided single-stranded RNAi agent comprises a lipid moiety complexed to the 9th or 11th nucleotide (counting from the 5' end). In some embodiments, the PNPLA3 oligonucleotide is complexed to a fatty acid at the base. In some embodiments, the provided single-stranded RNAi agent comprises a lipid moiety. In some embodiments, the provided single-stranded RNAi agent comprises a lipid moiety complexed to the 9th or 11th nucleotide (counting from the 5' end).

[0090] In some embodiments, the single stranded RNAi agent is any one of the preceding compositions, further comprising one or more additional components.

[0091] In some embodiments, provided oligonucleotides have the ability to degrade target transcripts (eg, RNA) via both RNase H and RNAi mechanisms.

[0092] In some embodiments, conjugating a lipid moiety to a PNPLA3 oligonucleotide improves at least one property of the oligonucleotide. In some embodiments, the improved property includes increased activity (e.g., increased ability to induce a decrease in expression and / or level of a target gene or its gene product, and / or to induce single-stranded RNA interference, and / or to induce RNase H-mediated knockdown) and / or improved tissue distribution. In some embodiments, the tissue is muscle tissue. In some embodiments, the tissue is skeletal muscle, gastrocnemius muscle, triceps muscle, heart, or diaphragm. In some embodiments, the improved property includes decreased hTLR9 agonist activity. In some embodiments, the improved property includes hTLR9 antagonist activity. In some embodiments, the improved property includes increased hTLR9 antagonist activity.

[0093] In general, the properties of the oligonucleotide compositions described herein can be assessed using any suitable assay.

[0094] One of skill in the art would readily be able to recognize and / or develop a suitable assay for a particular oligonucleotide composition.

[0095] definition As used herein, the following definitions apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001.

[0096] As used herein, "a" or "an" may mean one or more. When used in the claims, the words "a" or "an," when used with the word "comprising," may mean one or more. As used herein, "another" may mean at least a second or more.

[0097] The term "about" refers to a relative term indicating an approximation of ±10%, in one embodiment ±5%, and in another embodiment ±2% of the nominal value to which the term refers. In the field of this disclosure, this degree of approximation is reasonable unless it is clearly stated that the value requires a narrower range.

[0098] Aliphatic: As used herein, "aliphatic" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated units (not aromatic), or a combination thereof. In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms, and in still other embodiments, an aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0099] Alkenyl: As used herein, "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds.

[0100] Alkyl: As used herein, the term "alkyl" has its usual meaning in the art and may include saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight chain alkyl or branched chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C20 for straight chain, C2-C20 for branched chain), or about 1-10 carbon atoms. In some embodiments, a cycloalkyl ring has about 3-10 carbon atoms in its ring structure, where the ring is monocyclic, bicyclic, or polycyclic, or about 5, 6, or 7 carbons in the ring structure. In some embodiments, an alkyl group may be a lower alkyl group, where the lower alkyl group contains 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyl).

[0101] Alkynyl: The term "alkynyl" as used herein, refers to an alkyl group, as defined herein, having one or more triple bonds.

[0102] Antisense: As used herein, the term "antisense" refers to an oligonucleotide or other nucleic acid characteristic that has a base sequence that is complementary or substantially complementary to a target nucleic acid and has the ability to hybridize to the target nucleic acid. In some embodiments, the target nucleic acid is a target gene mRNA. In some embodiments, hybridization is necessary for or results in an action, such as reducing the level, expression or activity of the target nucleic acid or its gene product. As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide that is complementary to a target nucleic acid. In some embodiments, the antisense oligonucleotide has the ability to induce a reduction in the level, expression or activity of the target nucleic acid or its gene product. In some embodiments, the antisense oligonucleotide has the ability to induce a reduction in the level, expression or activity of the target nucleic acid or its gene product through a mechanism involving RNase H, steric hindrance and / or RNA interference.

[0103] Approximately: As used herein, the term "approximately" or "about" when in reference to a number is generally taken to include numbers within a range of 5%, 10%, 15%, or 20% in either direction (greater or less) of that number unless otherwise stated or otherwise evident from the context (unless such number is a value that may be less than 0% or greater than 100%). In some embodiments, use of the term "about" when in reference to dosage means ±5 / mg / kg / day.

[0104] Aryl: As used herein, the term "aryl," used alone or as part of a larger moiety in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5-30 ring members, with at least one ring in the system being aromatic. In some embodiments, the aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of 5-14 ring members, with at least one ring in the system being aromatic, with each ring in the system containing 3-7 ring members. In some embodiments, the aryl group is a biaryl group. "Aryl" may be used interchangeably with "aryl ring." In some embodiments of the present disclosure, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0105] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide is a portion that contains consecutive amino acids, or a collection of consecutive amino acids, both of which are characteristic of the protein or polypeptide. Each such consecutive stretch will generally contain at least two amino acids. Generally, a characteristic portion is one that shares at least one functional characteristic with the related intact protein, in addition to the sequence identity specified above.

[0106] Characteristic structural element: The term "characteristic structural element" or "structural element" refers to a distinct structural element present in all members of a family of polypeptides, small molecules, or nucleic acids, and can therefore be used by those skilled in the art to define the members of the family. In some embodiments, the structural elements of single-stranded RNAi agents include, but are not limited to, 5'-end structure, 5'-end region, 5'-nucleotide portion, seed region, post-seed region, 3'-end region, 3'-end dinucleotide, 3'-cap, modification pattern, stereochemical pattern in the backbone, additional chemical moieties, etc.

[0107] Equivalent: The term "equivalent" is used herein to describe two (or more) settings of conditions or circumstances that are sufficiently similar to one another to allow for a comparison of the results obtained or the phenomena observed. In some embodiments, equivalent settings of conditions or circumstances are characterized by a number of substantially identical characteristics and one or a small number of altered characteristics. One of skill in the art will recognize that settings of conditions are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to reasonably justify concluding that differences in the results obtained and the phenomena observed under the different settings of conditions or circumstances are caused by or indicative of changes in those altered characteristics.

[0108] Alicyclic: The terms "alicyclic," "carbocyclic," "carbocyclyl," "carbocyclic radical," and "carbocycle" are used interchangeably and as used herein refer to a saturated or partially unsaturated, but non-aromatic, cycloaliphatic monocyclic, bicyclic, or polycyclic ring system, as described herein, having 3 to 30 ring members unless otherwise specified. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, alicyclic groups have 3 to 6 carbons. In some embodiments, alicyclic groups are saturated and are cycloalkyl. The term "alicyclic" can also include an aliphatic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, an alicyclic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, an alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to a C ring that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the remainder of the molecule. 3 -C 6 Monocyclic hydrocarbons, or C 8 -C 10 Bicyclic hydrocarbons or C 8 -C 10 Polycyclic hydrocarbons, or C, which are fully saturated or contain one or more units of unsaturation, but are not aromatic, and have a single point of attachment to the rest of the molecule. 9 -C 16 Refers to polycyclic hydrocarbons.

[0109] Heteroaliphatic: The term "heteroaliphatic" is given its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, the heteroaliphatic group includes any of C, CH, CH 2 , and C.H. 3is independently substituted with one or more heteroatoms (including oxidized and / or substituted forms thereof). In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0110] Heteroalkyl: As used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, and the like.

[0111] Heteroaryl: As used herein, the terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy", refer to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, in which at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. Heteroaryl groups are groups having, in some embodiments, 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), in some embodiments 5, 6, 9, or 10 ring atoms. In some embodiments, heteroaryl groups have 6, 10, or 14 pi electrons shared in the cyclic arrangement, and have 1 to 5 heteroatoms in addition to the carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl. As used herein, the terms "heteroaryl" and "heteroaralkyl" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups may be monocyclic, bicyclic, or polycyclic."Heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, where the alkyl and heteroaryl portions independently may be optionally substituted.

[0112] Heteroatom: As used herein, the term "heteroatom" refers to an atom that is neither carbon nor hydrogen. In some embodiments, the heteroatom is oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon, any basic nitrogen or quaternized form of substitutable nitrogen of a heterocyclic ring (e.g., N in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR+ (in N-substituted pyrrolidinyl), etc.).

[0113] Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical", and "heterocyclic ring" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic ring moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, the heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, having one or more, preferably 1-4 heteroatoms as defined above, in addition to carbon atoms. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, and nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic group", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups may be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, where the alkyl and heterocyclyl moieties may be independently substituted.

[0114] Lower alkyl: The term "lower alkyl" refers to any alkyl group having a C 1-4 Examples of lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0115] Lower haloalkyl: The term “lower haloalkyl” refers to a C substituted with one or more halogen atoms. 1-4 "alkyl" refers to a linear or branched alkyl group of the formula:

[0116] Optionally substituted: As described herein, for example, oligonucleotides of the present disclosure may contain optionally substituted and / or substituted moieties. In general, "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at every position. In some embodiments, an optionally substituted group is not substituted. The combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of a stable or chemically feasible compound. As used herein, the term "stable" refers to a compound that is substantially unaltered when exposed to conditions that allow for the production, detection, and, in some embodiments, their recovery, purification, and use for one or more purposes disclosed herein.

[0117] Suitable monovalent substituents for a substitutable atom (e.g., the appropriate carbon atom) are independently: halogen; 2 ) 0-4 R o ;-(CH 2 ) 0-4 OR o ;-O(CH 2 ) 0-4 R o, -O-(CH 2 ) 0-4 C(O)OR°;-(CH 2 ) 0-4 CH(OR o ) 2 Optionally substituted with R° -(CH 2 ) 0-4 Ph; optionally substituted with R° -(CH 2 ) 0-4 O(CH 2 ) 0-1 Ph; -CH=CH, optionally substituted with R°; -(CH 2 ) 0-4 O(CH 2 ) 0-1 -Pyridyl; -NO 2 ;-CN;-N 3 ;-(CH 2 ) 0-4 N(R o ) 2 ;-(CH 2 ) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH 2 ) 0-4 N(R o )C(O)NR o 2 ;-N(R o )C(S)NR o 2 ;-(CH 2 ) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2 ;-N(R o )N(R o )C(O)OR o ;-(CH 2 ) 0-4 C(O)R o ;-C(S)R o ;-(CH 2) 0-4 C(O)OR o ;-(CH 2 ) 0-4 C(O)SR o ;-(CH 2 ) 0-4 C(O)OSiR o 3 ;-(CH 2 ) 0-4 OC(O)R o ;-OC(O)(CH 2 ) 0-4 SR,-SC(S)SR°;-(CH 2 ) 0-4 SC(O)R o ;-(CH 2 ) 0-4 C(O)NR o 2 ;-C(S)NR o 2 ;-C(S)SR°;-SC(S)SR°,-(CH 2 ) 0-4 OC(O)NR o 2 ;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH 2 C(O)R o ;-C(NOR o )R o ;-(CH 2 ) 0-4 SSR o ;-(CH 2 ) 0-4 S(O) 2 R o ;-(CH 2 ) 0-4 S(O) 2 OR o ;-(CH 2 ) 0-4 OS(O) 2 R o ;-S(O) 2 NR o 2 ;-(CH 2 ) 0-4 S(O)R o ;-N(R o )S(O) 2 NRo 2 ;-N(R o )S(O) 2 R o ;-N(OR o )R o ;-C(NH)NR o 2 ;-Si(R) 3 ;-OSi(R) 3 ;-B(R) 2 ;-OB(R) 2 ;-OB(OR) 2 ;-P(R) 2 ;-P(OR) 2 ;-OP(R) 2 ;-OP(OR) 2 ;-P(O)(R) 2 ;-P(O)(OR) 2 ;-OP(O)(R) 2 ;-OP(O)(OR) 2 ;-OP(O)(OR)(SR);-SP(O)(R) 2 ;-SP(O)(OR) 2 ;-N(R)P(O)(R) 2 ;-N(R)P(O)(OR) 2 ;-P(R) 2 [B(R) 3 ];-P(OR) 2 [B(R) 3 ];-OP(R) 2 [B(R) 3 ];-OP(OR) 2 [B(R) 3 ];-(C 1-4 Linear or branched alkylene)ON(R o ) 2 ; or -(C 1-4 Linear or branched alkylene)C(O)ON(R o ) 2 , (where each R o are optionally substituted as defined below and independently represent hydrogen, C 1-20 C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon and phosphorus 1-20 , heteroaliphatic, -CH 2 -(C 6-14 Aryl), -O(CH2 ) 0-1 (aryl), (C 6-14 Aryl), -CH 2 -(5-14 membered heteroaryl ring), a 5-20 membered monocyclic, bicyclic or polycyclic, saturated, partially unsaturated or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, regardless of the above definition, two independently occurring o together with their intervening atoms form a 5-20 membered, monocyclic, bicyclic or polycyclic, saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be optionally substituted as defined below.

[0118] Suitable R o The above monovalent substituents (or the ring formed by two independent occurrences of R° taken together with the intervening atoms) are independently selected from halogen, -(CH 2 ) 0-2 R ● , -(Halo R ● ), -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR ● , -(CH 2 ) 0-2 CH(OR ● ) 2 ;-O(Halo R ● ), -CN, -N 3 , -(CH 2 ) 0-2 C(O)R ● , -(CH 2 ) 0-2 C(O)OH, -(CH 2 ) 0-2 C(O)OR ● , -(CH 2 ) 0-2 S.R. ● , -(CH 2 ) 0-2 SH, -(CH 2 ) 0-2 NH 2 , -(CH 2 )0-2 NHR ● , -(CH 2 ) 0-2 NR ● 2 , -NO 2 , -SiR ● 3 , -OSiR ● 3 , -C(O)SR ● 、 -(C 1-4 Linear or branched alkylene)C(O)OR ● , or -SSR ● , (where each R ● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, and a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include ═O and ═S.

[0119] Suitable divalent substituents (e.g., at the appropriate carbon atoms) are independently: =O, =S, =NNR * 2 , =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O) 2 R * , =NR * , =NOR * , -O(C(R * 2 )) 2-3 O-, or -S(C(R * 2 )) 2-3 S-, (wherein each R * is hydrogen, optionally substituted as defined below 1-6A preferred divalent substituent attached to adjacent substitutable carbons of an "optionally substituted" group is -O(CR * 2 ) 2-3 O-, (wherein each R * is hydrogen, optionally substituted as defined below 1-6 aliphatic and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0120] R * Suitable substituents on the aliphatic groups are independently halogen, -R ● , -(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH 2 , -NHR ● , -NR ● 2 , or -NO 2 where each R ● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0121] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to include rings with multiple sites of unsaturation, but is not intended to include any of the aryl or heteroaryl moieties as defined herein.

[0122] RNA interference: The term "RNA interference" or "RNAi" as used herein refers to a post-transcriptional target gene silencing process involving RISC (RNA-induced silencing complex). The process of RNAi reportedly occurs naturally when longer dsRNA is cleaved by ribonuclease III (Dicer) and converted into shorter fragments called siRNAs. Naturally occurring siRNAs (small interfering RNAs) are typically about 21-23 nucleotides in length, have about 19 base pair duplexes and two single-stranded overhangs, and are typically RNA. These RNA segments reportedly then induce degradation of the target nucleic acid, such as mRNA or pre-mRNA (pre-mRNA). Dicer is also reportedly involved in excising 21- and 22-nucleotide small RNAs (stRNAs) from precursor RNAs with conserved structures involved in translational control. Hutvagner et al. 2001, Science, 293, 834. It is known to those skilled in the art that RNAi can be mediated by single-stranded or double-stranded oligonucleotides that contain sequences that are complementary or substantially complementary to target sequences (e.g., in target mRNA). Thus, in some embodiments of the present disclosure, single-stranded oligonucleotides described herein can act as RNAi agents, and in some embodiments, double-stranded oligonucleotides described herein can act as RNAi agents. In some embodiments, the RNAi response involves an endonuclease complex commonly referred to as RNA-induced silencing complex (RISC), where RISC induces cleavage of single-stranded mRNAs that are complementary to the antisense strand of siRNA. In some embodiments, RISC induces cleavage of target RNAs that are complementary to the oligonucleotides provided that can function as single-stranded RNAi agents. In some embodiments, cleavage of target RNA occurs in the middle of the region that is complementary to the antisense strand of siRNA double-stranded RNAi agents or siRNA single-stranded RNAi agents. In some embodiments, RNA interference is induced by single-stranded oligonucleotides that act as single-stranded RNAi agents that can induce RNA interference in a mechanism involving the RISC pathway.

[0123] RNAi agent: As used herein, the terms "RNAi agent", "iRNA agent", and the like refer to a PNPLA3 oligonucleotide that, when administered to a system in which or expressing a target gene product (e.g., a transcript of a target gene, such as a pre-mRNA or mRNA), reduces the level and / or activity (e.g., translation) of the target gene product. In some embodiments, the RNAi agent may be or include a single-stranded or double-stranded oligonucleotide. In some embodiments, the RNAi agent may have a structure recognized in the art as an siRNA (short inhibitory RNA), shRNA (short or small hairpin RNA), dsRNA (double-stranded RNA), microRNA, and the like. In some embodiments, the RNAi agent may specifically bind to an RNA target (e.g., a transcript of a target gene). In some embodiments, the RNAi agent, upon binding to its target, is incorporated into RISC (RNA-induced silencing complex). In some embodiments, the RNAi agent induces degradation of its target and / or inhibits translation of its target, which in some embodiments occurs through a mechanism involving the RISC (RNA-induced silencing complex) pathway. In some embodiments, the RNAi agent is a PNPLA3 oligonucleotide that activates the RISC complex / pathway. In some embodiments, the RNAi agent comprises an antisense strand sequence. In some embodiments, the RNAi agent comprises only one oligonucleotide strand (e.g., a single-stranded oligonucleotide). In some embodiments, a single-stranded RNAi agent oligonucleotide may be or comprise a sense or antisense strand sequence, as described by Sioud 2005 J.Mol.Biol.348:1079-1090. In some embodiments, the RNAi agent is a compound that has the ability to induce RNA interference. In some embodiments, the RNAi agent may have a structure or format found in a "canonical" siRNA structure. In some embodiments, the RNAi agent may have a structure that differs from a "canonical" siRNA structure.To name just a few examples, in some embodiments, the RNAi agent may be longer or shorter than the canonical one, may have blunt ends, and / or may include one or more modifications, mismatches, gaps, and / or nucleotide replacements. In some embodiments, the RNAi agent includes a 3' end cap as described in the present disclosure. Without wishing to be bound by any particular theory, applicants propose that in some embodiments, the 3' end cap may enable both of the following two functions: (1) achieving RNA interference, and (2) increasing the duration of activity and / or biological half-life (which may be achieved, for example, by increasing binding to the PAZ domain of Dicer and / or the PAZ domain of one or more Ago proteins, and / or reducing or preventing degradation of the RNAi agent (e.g., by nucleases, such as in serum or intestinal fluids). In some embodiments, the RNAi agent of the present disclosure targets (e.g., binds, anneals, etc. to) a target mRNA. In some embodiments, when an RNAi agent is exposed to its target, the activity, level, and / or expression of the target is decreased, e.g., the target is "knocked down" or "knocked out." Specifically, in some embodiments, when a disease, disorder, and / or condition is characterized by overexpression and / or increased activity of a target gene, administering the RNAi agent to a cell, tissue, or subject is sufficient to restore activity to normal levels or to knock down the target gene sufficiently to reduce activity to a level that can alleviate, ameliorate, mitigate, suppress, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or features of the disease, disorder, and / or disease state, disorder state, and / or condition. In some embodiments, the RNAi agent is double-stranded, comprising an antisense strand, which is a single-stranded RNAi agent as described herein, in combination with a sense strand, which in combination with the sense strand is capable of inducing RNA interference.

[0124] Single-stranded RNA interference: As used herein, the expression "single-stranded RNAi" or "single-stranded RNA interference" or the like refers to the process or method of gene silencing that is at least partially induced by administering a single-stranded RNAi agent to the system (e.g., cell, tissue, organ, subject, etc.) that the agent should induce RNAi in, and requires RISC pathway.These terms may be used in certain cases herein to distinguish from "double-stranded RNAi" or "double-stranded RNA interference", in which a double-stranded RNAi agent is administered to a system and can be further processed, for example, one of its two strands is incorporated into RISC, resulting in, for example, translation suppression, target RNA cleavage, etc.

[0125] Single-stranded RNAi agent: As used herein, the term "single-stranded RNAi agent" refers to the single-stranded oligonucleotide that can induce single-stranded RNA interference (RNAi or iRNA) or gene silencing through RISC pathway.Single-stranded RNAi agent can comprise one or more single-stranded nucleotide polymers.

[0126] Subject: As used herein, the term "subject" or "subject" refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; others; etc.) and plants. In some embodiments, the subject may be afflicted with and / or susceptible to a disease, disorder, and / or condition.

[0127] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting all or nearly all of the extent or degree of a feature or characteristic of interest. A base sequence that is substantially complementary to a second sequence is not identical to the second sequence, but is mostly or nearly identical to the second sequence. Furthermore, those skilled in the art of biology will understand that biological and chemical phenomena rarely go to completion and / or proceed to completion, or achieve or avoid absolute consequences. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0128] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0129] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who is at a higher risk of developing the disease, disorder, and / or condition than individuals in the general community. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0130] Systemic: As used herein, the terms "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" have their art-recognized meaning of referring to administration of a compound or composition so that it enters the entire body of the recipient.

[0131] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0132] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose. In some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0133] Treat: As used herein, the terms "treat", "treatment" or "treating" refer to any method used to partially or completely alleviate, ameliorate, reduce, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathology associated with the disease, disorder, and / or condition.

[0134] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0135] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning of referring to an entity having a structure and / or activity that is found in nature in a "normal" state or situation (as opposed to mutant, diseased, altered, etc.). One of skill in the art will recognize that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0136] Nucleic acid: As used herein, the term "nucleic acid" includes any nucleotide and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, either RNA or DNA analogs made from modified nucleotides and / or modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. The terms encompass polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or N- or C-glycosides of modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotide linkages. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both a ribose moiety and a deoxyribose moiety, and nucleic acids containing a ribose moiety and a modified ribose moiety. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing from 2 to about 10,000 nucleotide monomer units and the prefix oligo- refers to a nucleic acid containing from 2 to about 200 nucleotide monomer units.

[0137] Nucleotide: As used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide consisting of a heterocyclic base, a sugar, and one or more internucleotide bonds. Natural bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but it should be understood that natural and unnatural base analogs are also included. Natural sugars are the pentoses deoxyribose (forming DNA) or ribose (forming RNA), but it should be understood that natural and unnatural sugar analogs are also included. Nucleotides are linked via internucleotide bonds to form nucleic acids, or polynucleotides. Many internucleotide bonds are known in the art, such as, but not limited to, phosphate, phosphorothioate, boranophosphate, etc. Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, boranophosphate, methylphosphonate, phosphonoacetate, thiophosphonoacetate, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, natural nucleotides include naturally occurring bases, sugars, and internucleotide bonds. As used herein, the term "nucleotide" also encompasses structural analogs (such as modified nucleotides and nucleotide analogs) that are used in place of natural or naturally occurring nucleotides.

[0138] Modified Nucleotides: The term "modified nucleotides" includes any chemical moiety that is structurally different from a naturally occurring nucleotide but is capable of performing at least one function of a naturally occurring nucleotide. In some embodiments, modified nucleotides include modifications to the sugar, base, and / or internucleotide linkage. In some embodiments, modified nucleotides include modified sugars, modified nucleobases, and / or modified internucleotide linkages. In some embodiments, modified nucleotides are capable of performing at least one function of a nucleotide, such as forming subunits of a polymer that are capable of base pairing with a nucleic acid that includes at least a complementary base sequence.

[0139] Analog: The term "analog" refers to any functional analogue whose chemical moiety is structurally different from a reference chemical moiety or a reference class of moieties, but has the ability to perform at least one function of such reference chemical moiety or reference class of moieties.Non-limiting examples include nucleotide analogues that are structurally different from nucleotides but perform at least one function of nucleotides, nucleobase analogues that are structurally different from nucleobases but perform at least one function of nucleobases, etc.

[0140] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar.

[0141] Modified nucleosides: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a naturally occurring nucleoside, but containing a chemical modification that makes the moiety different from a naturally occurring nucleoside. Non-limiting examples of modified nucleosides include those containing modifications to the base and / or sugar. Non-limiting examples of modified nucleosides include those having 2' modifications to the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (those lacking a nucleobase). In some embodiments, modified nucleosides are capable of performing at least one function of a nucleoside, such as forming a portion of a polymer capable of base pairing with a nucleic acid containing at least a complementary base sequence.

[0142] Nucleoside Analog: The term "nucleoside analog" refers to a chemical moiety that is chemically different from a naturally occurring nucleoside, but is capable of performing at least one function of a nucleoside. In some embodiments, a nucleoside analog comprises a sugar analog and / or a nucleobase analog. In some embodiments, a modified nucleoside is capable of performing at least one function of a nucleoside, such as forming a portion of a polymer that is capable of base pairing with a nucleic acid containing a complementary base sequence.

[0143] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in a closed and / or open state. In some embodiments, a sugar is a monosaccharide. In some embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs that are used in place of traditional sugar molecules, such as glycols, polymers of which form the backbone of the nucleic acid analog glycol nucleic acid (GNA). As used herein, the term "sugar" also encompasses structural analogs that are used in place of natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars.

[0144] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic state, or some other physicochemical property of a sugar.

[0145] Nucleic acid base: The term "nucleic acid base" refers to a portion of a nucleic acid that participates in hydrogen bonds that bind one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common natural nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the natural nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the natural nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase," e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, modified nucleobase mimics mimic the spatial arrangement, electronic state, or some other physicochemical properties of nucleobases, and retain the hydrogen bond properties that bind one nucleic acid strand to another in a sequence-specific manner.In some embodiments, modified nucleobases can pair with all five natural bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of oligonucleotide duplexes.As used herein, the term "nucleobase" also encompasses structural analogs (such as modified nucleobases and nucleobase analogs) that are used in place of natural or naturally occurring nucleotides.

[0146] Modified nucleobase: The term "modified nucleobase", "modified base" and the like refer to a chemical moiety that is chemically different from a nucleobase but has the ability to perform at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase that includes a modification. In some embodiments, a modified nucleobase has the ability to perform at least one function of a nucleobase, such as forming a part of a polymer that has the ability to base pair with a nucleic acid that includes at least a complementary base sequence.

[0147] 3' end cap: The term "3' end cap" refers to a non-nucleotide chemical moiety attached to the 3' end of a PNPLA3 oligonucleotide (e.g., an RNAi agent). In some embodiments, the 3' end dinucleotide may be replaced by a 3' end cap. In some embodiments, the 3' end cap of a PNPLA3 oligonucleotide performs at least one of the following functions: achieving RNA interference induced by the oligonucleotide, protecting the oligonucleotide from degradation or reducing the amount or rate of degradation of the oligonucleotide (e.g., by nucleases), reducing off-target effects of the sense strand, or enhancing the activity, duration, or efficiency of RNA interference induced by the oligonucleotide. When a 3' end cap is described as a "non-nucleotide", it is intended that the 3' end cap is linked to the remaining sugar moiety of the PNPLA3 oligonucleotide, rather than to a nucleotide or oligonucleotide moiety, and that this linkage is performed in the same manner as if the 3' end cap were a part of the PNPLA3 oligonucleotide chain. Certain examples of 3' end caps are described herein. One of skill in the art will understand that other 3' end caps known in the art can be utilized in accordance with the present disclosure.

[0148] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group may then be unmasked by removal of the blocking group. In some embodiments, a blocking group is a protecting group.

[0149] Moiety: The term "moiety" refers to a specific segment of a functional group of a molecule. A chemical moiety is a recognized chemical entity that is incorporated into or attached to a molecule.

[0150] Solid Support: The term "solid support" refers to any support that allows for nucleic acid synthesis. In some embodiments, the term refers to glass or a polymer that is insoluble in the medium utilized in the reaction steps performed to synthesize nucleic acids and that has been derivatized to contain reactive groups. In some embodiments, the solid support is Highly Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). In some embodiments, the solid support is Controlled Pore Glass (CPG). In some embodiments, the solid support is a hybrid support of Controlled Pore Glass (CPG) and Highly Cross-linked Polystyrene (HCP).

[0151] Linker or Linking Moiety: The terms "linker", "linking moiety", and the like refer to any chemical moiety that links one chemical moiety to another. In some embodiments, a linker is a moiety that links one oligonucleotide to another in a multimer. In some embodiments, a linker is a moiety that is optionally positioned between a terminal nucleoside and a solid support, or between a terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0152] Gene: As used herein, the terms "gene", "recombinant gene", and "gene construct" refer to a DNA molecule or a portion of a DNA molecule that codes for a protein or a portion thereof. In addition to the DNA molecule may contain an open reading frame that codes for a protein (as an exon sequence), it may further contain intron sequences. As used herein, the term "intron" refers to a DNA sequence present in a given gene that is not translated into protein and that is found between exons in some, but not all, cases. It may be desirable to regulate the activity or expression of a gene by operably linking it to (or by the gene may contain) one or more promoters, enhancers, repressors, and / or other regulatory sequences, as is well known in the art.

[0153] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes a recombinant polynucleotide synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.

[0154] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, which may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0155] An oligonucleotide may be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" encompasses single-stranded oligonucleotides. A single-stranded oligonucleotide may have a double-stranded region (formed by two portions of a single-stranded oligonucleotide), and a double-stranded oligonucleotide (comprising two oligonucleotide strands) may have a single-stranded region, such as in a region where the two oligonucleotide strands are not complementary to each other. In some embodiments, the oligonucleotide has the ability to induce a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the oligonucleotide has the ability to induce a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the oligonucleotide has the ability to induce a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. Examples of oligonucleotides include, but are not limited to, structural genes, genes including regulatory and termination regions, self-replicating systems such as viral or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNA, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0156] Double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as RNAi agents or iRNA agents.In some embodiments, these RNA interference-inducing oligonucleotides are associated with a cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC).In many embodiments, double-stranded RNAi agents are long enough that they can be cleaved by endogenous molecules, such as Dicer, to generate smaller oligonucleotides that can enter RISC machinery and participate in RISC-mediated cleavage and / or translational repression of target sequences, such as target mRNA sequences.

[0157] The oligonucleotides of the present disclosure may be of various lengths. In certain embodiments, the oligonucleotides may range from about 2 to about 200 nucleotides in length. In various related embodiments, the single-stranded, double-stranded, and triple-stranded oligonucleotides may range from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides in length. In some embodiments, the PNPLA3 oligonucleotides are about 10 to about 40 nucleotides in length. In some embodiments, the PNPLA3 oligonucleotides are about 9 to about 39 nucleotides in length. In some embodiments, the oligonucleotides are at least 4 nucleotides in length. In some embodiments, the oligonucleotides are at least 5 nucleotides in length. In some embodiments, the oligonucleotides are at least 6 nucleotides in length. In some embodiments, the oligonucleotides are at least 7 nucleotides in length. In some embodiments, the oligonucleotides are at least 8 nucleotides in length. In some embodiments, the oligonucleotides are at least 9 nucleotides in length. In some embodiments, the oligonucleotides are at least 10 nucleotides in length. In some embodiments, the oligonucleotides are at least 11 nucleotides in length. In some embodiments, the oligonucleotide is at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a complementary duplex at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a complementary duplex at least 21 nucleotides in length. In some embodiments, each nucleotide counted in length independently comprises an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil.

[0158] Internucleotide bond: As used herein, the term "internucleotide bond" generally refers to a bond that links the nucleoside units of a PNPLA3 oligonucleotide or nucleic acid. In some embodiments, the internucleotide bond is a phosphodiester bond (natural phosphate bond) found in naturally occurring DNA and RNA molecules. In some embodiments, the term "internucleotide bond" includes modified internucleotide bond. In some embodiments, the internucleotide bond is a "modified internucleotide bond" in which each oxygen atom of the phosphodiester bond is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties include, but are not limited to, =S, =Se, =NR', -SR', -SeR', -N(R'). 2 , B(R') 3、 In some embodiments, the internucleotide linkage is selected from -S-, -Se-, and -N(R')-, where each R' is as defined and described in the present disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage ( [ka] ), or a modified phosphorothioate triester linkage.

[0159] One of skill in the art will appreciate that internucleotide linkages can exist as anions or cations at a given pH, depending on the presence of acidic or basic moieties in the linkage.

[0160] In some embodiments, "All-(Rp)" or "All-(Sp)" is used to indicate that all chiral phosphorus atoms of an oligonucleotide have the same Rp or Sp configuration, respectively.

[0161] Oligonucleotide type: As used herein, the term "oligonucleotide type" refers to an oligonucleotide that is an oligonucleotide having a particular base sequence, a backbone linkage pattern (i.e., the pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, etc.), a backbone chiral center pattern (i.e., the pattern of bond phosphorus stereochemistry (Rp / Sp)), and a pattern of backbone phosphorus modifications (e.g., "-XLR" in Formula I). 1 "PNPLA3" oligonucleotides having a pattern of "groups" (such as a cyclic nucleotide sequence, ...

[0162] Those skilled in the art will recognize that the synthesis method of the present disclosure provides a degree of control during synthesis of a PNPLA3 oligonucleotide chain, whereby each nucleotide unit of the oligonucleotide chain can be pre-designed and / or pre-selected to have a specific stereochemistry at the binding phosphorus and / or a specific modification at the binding phosphorus, and / or a specific base, and / or a specific sugar. In some embodiments, the PNPLA3 oligonucleotide chain is pre-designed and / or pre-selected to have a specific combination of stereocenters at the binding phosphorus. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or pre-determined to have a specific combination of modifications at the binding phosphorus. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or pre-selected to have a specific combination of bases. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or pre-selected to have a specific combination of one or more of the structural characteristics described above. In some embodiments, the present disclosure provides a composition comprising or consisting of a plurality of oligonucleotide molecules (e.g., a chiral controlled oligonucleotide composition). In some embodiments, all such molecules are the same type of molecule (i.e., all such molecules are structurally identical to each other). However, in many embodiments, provided compositions include multiple oligonucleotides of different types, typically in predetermined relative amounts.

[0163] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical assignment of the chiral linkage phosphorus in the chiral internucleotide linkage in the PNPLA3 oligonucleotide. In some embodiments, the control is achieved through the chiral elements that are absent from the sugar and base moieties of the PNPLA3 oligonucleotide. For example, in some embodiments, the control is achieved using one or more chiral auxiliary groups during oligonucleotide preparation, as exemplified in the present disclosure, and such chiral auxiliary groups are often part of the chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will recognize that conventional oligonucleotide synthesis without the use of chiral auxiliary groups does not allow for stereochemical control at the chiral internucleotide linkage when the chiral internucleotide linkage is formed using the conventional oligonucleotide synthesis. In some embodiments, the stereochemical assignment of each chiral linkage phosphorus in the chiral internucleotide linkage in the PNPLA3 oligonucleotide is controlled.

[0164] Chirality Controlled Oligonucleotide Composition: As used herein, the terms "chirality controlled oligonucleotide composition", "chirality controlled nucleic acid composition", and the like refer to a composition comprising multiple oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, where the multiple oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chirality controlled internucleotide linkages), and the level of the multiple oligonucleotides (or nucleic acids) in the composition has been predetermined (e.g., via preparation of the chirality controlled oligonucleotide to form one or more chiral internucleotide linkages). In some embodiments, about 1% to 100% (e.g., about 5% to 100%, about 10% to 100%, about 20% to 100%, about 30% to 100%, about 40% to 100%, about 50% to 100%, about 60% to 100%, about 70% to 100%, about 80% to 100%, about 90% to 100%, about 95% to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, About 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said plurality of oligonucleotides.In some embodiments, about 1% to 100% (e.g., about 5% to 100%, about 10% to 100%, about 20% to 100%, about 30% to 100%, about 40% to 100%, about 50% to 100%, about 60% to 100%, about 70% to 100%, about 80 to 100%, about 90 to 100%, about 95 to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91% or more) of all oligonucleotides sharing a common base sequence in a chiral controlled oligonucleotide composition. , about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said plurality of oligonucleotides.In some embodiments, between about 1% and 100% (e.g., between about 5% and 100%, between about 10% and 100%, between about 20% and 100%, between about 30% and 100%, between about 40% and 100%, between about 50% and 100%, between about 60% and 100%, between about 70% and 100%, between about 80% and 100%, between about 90% and 100%, between about 95% and 100%, between about 50% and 90%, or between about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said oligonucleotides.In some embodiments, the predetermined level is between about 1% and 100% (e.g., between about 5% and 100%, between about 10% and 100%, between about 20% and 100%, between about 30% and 100%, between about 40% and 100%, between about 50% and 100%, between about 60% and 65% of all oligonucleotides in the composition, or all oligonucleotides in the composition that share a common base sequence (e.g., of a plurality of oligonucleotides, or of a single PNPLA3 oligonucleotide type), all oligonucleotides in the composition, or all oligonucleotides in the composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, all oligonucleotides in the composition, or all oligonucleotides in the composition that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotide linkage types, and / or a common pattern of internucleotide linkage modifications. about 60% to 100%, about 70% to 100%, about 80% to 100%, about 90% to 100%, about 95% to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said oligonucleotides.In some embodiments, the plurality of oligonucleotides is 1-50 (e.g., about 1-10, about 1-20, about 5-10, about 5-20, about 10-15, about 10-20, about 10-25, about 10-30, about or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20, or at least at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty) chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides comprises between about 1% and 100% (e.g., between about 5% and 100%, between about 10% and 100%, between about 20% and 100%, between about 30% and 100%, between about 40% and 100%, between about 50% and 100%, between about 60% and 100%, between about 70% and 100%, between about 80% and 100%, between about 90% and 100%, between about 95% and 100%, between about 50% and 90%, between about 5%, between about 10%, between about 15%, between about 20%, between about 25%, between about 30%, between about 35%, between about 40%, between about 45%, between about 50%, between about 55%, between about 60%, between about 65%, between about 70%, between about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, or at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% share the same stereochemistry. In some embodiments, each chiral internucleotide linkage is a chiral controlled internucleotide linkage and the composition is a completely chiral controlled oligonucleotide composition.In some embodiments, less than all chiral internucleotide linkages are chiral controlled internucleotide linkages, and the composition is a partially chiral controlled oligonucleotide composition. In some embodiments, the chiral controlled oligonucleotide composition comprises a predetermined level of an individual oligonucleotide type or nucleic acid type. For example, in some embodiments, the chiral controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide composition comprises two or more oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, the chiral controlled oligonucleotide composition is a composition of oligonucleotides of one oligonucleotide type, and the composition comprises multiple oligonucleotides of that oligonucleotide type at a predetermined level.

[0165] Chirally pure: As used herein, the term "chirally pure" is used to describe a relative amount of PNPLA3 oligonucleotides, e.g., single-stranded RNAi agents, in which all of the oligonucleotides are present in a single diastereomeric form with respect to the bound phosphorus.

[0166] Chirally homogeneous: As used herein, the term "chirally homogeneous" is used to describe the PNPLA3 oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the bond phosphorus.For example, the PNPLA3 oligonucleotide whose all nucleotide units have the stereochemistry of Rp at the bond phosphorus is chirally homogeneous.Similarly, the PNPLA3 oligonucleotide whose all nucleotide units have the stereochemistry of Sp at the bond phosphorus is chirally homogeneous.

[0167] Predetermined: Predetermined (or pre-determined) means deliberately selected, as opposed to, for example, randomly occurring or realized without control. Upon reading this specification, one skilled in the art will understand that the present disclosure provides techniques that allow for the selection of a particular chemistry and / or stereochemistry to be incorporated into an oligonucleotide composition, and further provides techniques that allow for the controlled preparation of an oligonucleotide composition having such chemistry and / or stereochemistry. Such a provided composition is "predetermined" as described herein. A composition that may contain an oligonucleotide is a composition that happens to be produced through a process that is not controlled to deliberately produce a particular chemistry and / or stereochemistry, and is not a "predetermined" composition. In some embodiments, a predetermined composition is a composition that can be deliberately reproduced (e.g., through repetition of a controlled process). In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute and / or relative amounts (ratios, proportions, etc.) of the plurality of oligonucleotides in the composition are controlled. In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition is achieved through chiral controlled oligonucleotide preparation.

[0168] Linked phosphorus: As defined herein, the phrase "linked phosphorus" is used to indicate that the particular phosphorus atom being referred to is a phosphorus atom present in an internucleotide linkage, which phosphorus atom corresponds to the phosphodiester phosphate atom of the internucleotide linkage present in natural DNA and RNA. In some embodiments, the linked phosphorus atom is in a modified internucleotide linkage, in which each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the linked phosphorus atom is represented by the formula I, L In some embodiments, the binding phosphorus atom is chiral.

[0169] P-modification: As used herein, the term "P-modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P-modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus. In some embodiments, a "P-modification" includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus. 1 where X, L and R 1 Each of is independently as defined and described in this disclosure.

[0170] For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, inside cover of the Handbook of Chemistry and Physics, 67th Ed., 1986-87.

[0171] The methods and structures described herein for the compounds and compositions of the present disclosure also apply to the pharma- ceutically acceptable acid or base addition salts, and all stereoisomers of these compounds and compositions.

[0172] Detailed Description of Certain Embodiments Synthetic oligonucleotides provide useful molecular tools in a variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and new nanomaterial applications. The applications of naturally occurring nucleic acids (e.g., unmodified DNA or unmodified RNA) are limited, for example, by their susceptibility to endonuclease and exonuclease attack. Thus, various synthetic counterparts have been developed to circumvent these shortcomings. These synthetic counterparts include synthetic oligonucleotides that contain chemical modifications (e.g., base, sugar, backbone modifications, etc.) that, among other things, reduce the susceptibility of these molecules to degradation and improve other properties of the oligonucleotides. From a structural point of view, in addition to the fact that modifications of the internucleotide phosphate bonds can introduce chirality, certain properties of the oligonucleotides can be influenced by the arrangement of phosphorus atoms that form the backbone of the oligonucleotide. For example, in vitro studies have shown that the properties of antisense oligonucleotides (e.g., binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, etc.) are influenced, among other things, by the chirality of the backbone phosphorus atoms.

[0173] In particular, the present disclosure includes the recognition that structural elements of an oligonucleotide can significantly affect its properties and activity (e.g., stability, specificity, selectivity, activity for reducing the level of a target gene product (transcript and / or protein), etc.), such as chemical modifications (e.g., sugar modifications, base modifications, and / or internucleotide linkage modifications) or patterns thereof, conjugation to lipids or other moieties, and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or patterns thereof). In some embodiments, the properties of an oligonucleotide can be tailored by optimizing the chemical modifications (base modifications, sugar modifications, and / or internucleotide linkage modifications), chemical modification patterns, stereochemistry, and / or stereochemical patterns.

[0174] In some embodiments, the present disclosure shows that oligonucleotide compositions comprising oligonucleotides with controlled structural elements (controlled chemical modifications and / or controlled backbone stereochemical patterns) provide unexpected properties and activities (including, but not limited to, those described herein). In some embodiments, compositions comprising oligonucleotides with chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.) or patterns thereof have improved properties and activities. Non-limiting examples of such improved properties include induction of reduced expression and / or levels of a target gene or its gene product, and / or induction of RNA interference, and / or induction of RNase H-mediated knockdown. In some embodiments, the present disclosure provides techniques (e.g., oligonucleotides, compositions, methods, etc.) for single-stranded RNAi. In some embodiments, the oligonucleotides provided are ssRNAi agents.

[0175] In some embodiments, RNA interference is reportedly a post-transcriptional targeted gene silencing technique in which an RNAi agent is used to target an RNA (e.g., a gene transcript such as a messenger RNA (mRNA)) that contains a sequence complementary to the RNAi agent, causing cleavage mediated by the RISC (RNA-induced silencing complex) pathway. In nature, a form of RNAi reportedly occurs when long dsRNA (double-stranded RNA) (e.g., exogenous dsRNA introduced into mammalian cells) is cleaved by ribonuclease III (Dicer) and converted into shorter fragments called siRNAs. siRNAs (small interfering RNAs or short inhibitory RNAs) typically have a length of about 21-23 nucleotides and contain a duplex of about 19 base pairs. The smaller RNA segments then reportedly mediate degradation of the target mRNA. The RNAi response also reportedly features an endonuclease complex commonly referred to as the RNA-induced silencing complex (RISC), which induces cleavage of single-stranded mRNA complementary to the antisense strand of the siRNA. Cleavage of the target RNA reportedly occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. The use of an RNAi agent against a target transcript reportedly reduces gene activity, levels, and / or expression, e.g., "knocks down" or "knocks out" a target gene or sequence. Artificial siRNAs are useful for both therapeutic and experimental applications.

[0176] In one embodiment, the RNA interference agent comprises a single stranded RNA that interacts with a target RNA sequence to induce cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into plant and invertebrate cells is reportedly converted into siRNAs through degradation by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease III-like enzyme, reportedly processes dsRNA to convert it into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then reportedly incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). One or more endonucleases in the RISC bind to the appropriate target mRNA, cleaving the target and inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present disclosure relates to single-stranded RNA that promotes the formation of a RISC complex to cause silencing of a target gene.

[0177] In some embodiments, suitable RNAi agents can be selected by any process known in the art or that may occur to one of skill in the art in accordance with the present disclosure. For example, the selection criteria may include one or more of the following steps: initial analysis of the target gene sequence and design of the RNAi agent (which may take into account sequence similarity across species (human, cynomolgus monkey, mouse, etc.) and dissimilarity to other (non-target) genes); Screening in vitro (e.g., at 10 nM in cells expressing the target transcript), determining EC50 or IC50 in cells, determining the viability of cells treated with the RNAi agent (in some embodiments, it is hoped that the RNAi agent against the target does not inhibit the viability of such cells), testing with human PBMCs (peripheral blood mononuclear cells) (e.g., testing levels of TNF-alpha to estimate immunogenicity, where immunostimulatory sequences are typically less desirable), testing in human whole blood assays (where fresh human blood is treated with the RNAi agent and levels of cytokines / chemokines [e.g., TNF-alpha (tumor necrosis factor-alpha) and / or MCP1 (monocyte chemotactic protein 1)] are determined, where immunostimulatory sequences are typically less desirable), determining gene knockdown in vivo using cells or tumors in test animals, and optimizing specific modifications of the RNAi agent.

[0178] The so-called canonical siRNA structure is reportedly a double-stranded RNA molecule, with each strand being approximately 21 nucleotides in length. The two strands are reportedly an antisense (or "guide") strand (which recognizes and binds to a complementary sequence in the target transcript) and a sense (or "passenger") strand (which is complementary to the antisense strand). The sense and antisense strands are reportedly largely complementary, and typically form two 3' overhangs of two nucleotides at each end.

[0179] Although canonical siRNA structures are reportedly double-stranded, RNAi agents can also be single-stranded. In some embodiments, a single-stranded RNAi agent corresponds to the antisense strand of a double-stranded siRNA, and such a single-stranded RNAi agent does not include a corresponding passenger strand.

[0180] However, it has been reported that not all structural elements tested for single-stranded RNAi agents are effective, and some structural elements introduced into PNPLA3 oligonucleotides can reportedly interfere with single-stranded RNA interference activity.

[0181] In some embodiments, the present disclosure provides oligonucleotides and compositions useful as RNAi agents. In some embodiments, the present disclosure provides oligonucleotides and compositions useful as single-stranded RNAi agents. The present disclosure provides, among other things, single-stranded oligonucleotides of novel structure capable of inducing RNA interference. Without wishing to be bound by any particular theory, the present disclosure specifies that single-stranded RNAi agents have advantages over double-stranded RNAi agents. For example, single-stranded RNAi agents have lower cost of ownership since only one strand needs to be constructed. Additionally or alternatively, only one strand (the antisense strand) is administered to target the target transcript. The source of off-target effects induced by dsRNA is the incorporation of the sense strand into RISC, which binds to and knocks down undesired targets (Jackson et al. 2003 Nat. Biotech. 21:635-637), whereas single-stranded RNAi agents may induce fewer off-target effects compared to their double-stranded counterparts. Furthermore, some single-stranded RNAi agents, including some disclosed herein, can target specific sequences that have not previously been successfully targeted using double-stranded RNAi agents (e.g., some single-stranded RNAi agents can reduce the levels of a sequence and / or the levels of the sequence's products (transcripts and / or proteins) more significantly than double-stranded RNAi agents).The present disclosure provides, among other things, novel formats (modifications, stereochemistry, combinations thereof, etc.) of oligonucleotides capable of inducing single-stranded RNA interference.

[0182] Oligonucleotides In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of the target gene or its gene product. In some embodiments, the provided oligonucleotides can induce a decrease in the level of the target product. In some embodiments, the provided oligonucleotides can reduce the level of the transcript of the target gene. In some embodiments, the provided oligonucleotides can reduce the level of the mRNA of the target gene. In some embodiments, the provided oligonucleotides can reduce the level of the protein encoded by the target gene. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of the target gene or its gene product via RNA interference. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of the target gene or its gene product via RNA interference or biochemical mechanisms that do not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of the target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or levels of a target gene or its gene product by steric blocking of translation after binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing, and / or by exon inclusion or exclusion.In some embodiments, oligonucleotides are provided in accordance with the present disclosure that include one or more structural elements described herein or known in the art, such as base sequence, modification, stereochemistry, internucleotide linkage pattern, backbone linkage pattern, backbone chiral center pattern, backbone phosphorus modification pattern, additional chemical moieties (including, but not limited to, one or more targeting moieties, lipid moieties, and / or carbohydrate moieties), seed region, post-seed region, 5' terminal structure, 5' terminal region, 5' nucleotide moiety, 3' terminal region, 3' terminal dinucleotide, 3' terminal cap, etc. In some embodiments, the seed region of a PNPLA3 oligonucleotide is or includes the 2nd to 8th nucleotides, the 2nd to 7th nucleotides, the 2nd to 6th nucleotides, the 3rd to 8th nucleotides, the 3rd to 7th nucleotides, the 3rd to 7th nucleotides, or the 4th to 8th nucleotides or the 4th to 7th nucleotides, counting from the 5' end, and the post-seed region of the oligonucleotide is the region immediately adjacent to the 3' side of the seed region and is interposed between the seed region and the 3' terminal region.

[0183] In some embodiments, a provided composition comprises a PNPLA3 oligonucleotide. In some embodiments, a provided composition comprises one or more lipid moieties, one or more carbohydrate moieties (other than the sugar moieties of the nucleoside units that form the oligonucleotide chain with internucleotide linkages, unless otherwise specified), and / or one or more targeting components.

[0184] In some embodiments, different alleles of the same sequence (e.g., gene) exist where the presence and / or activity of a particular allele (and / or one or more products thereof (e.g., RNA and / or protein products)) (disease-associated alleles) is associated (e.g., correlated) with the presence, onset, and / or severity of one or more diseases and / or conditions, and such different alleles are not so associated, or are associated to a lesser extent (e.g., exhibit a less significant or statistically insignificant correlation). In some such embodiments, the oligonucleotides and methods thereof described herein may preferentially or specifically target the associated allele(s) compared to one or more less / unassociated allele(s).

[0185] In some embodiments, the target sequence is a sequence to which the PNPLA3 oligonucleotide described herein binds. In many embodiments, the target sequence is identical to or an exact complement of the sequence of the provided oligonucleotide or the sequence of consecutive residues present therein (e.g., the provided oligonucleotide comprises a target binding sequence that is identical to or an exact complement of the target sequence). In some embodiments, a small number of differences / mismatches between (the relevant portion of) the PNPLA3 oligonucleotide and its target sequence are tolerated. In many embodiments, the target sequence is present in a target gene. In many embodiments, the target sequence is present in a transcription product (e.g., mRNA and / or pre-mRNA) produced from the target gene.

[0186] In some embodiments, the target sequence comprises one or more allelic sites (i.e., positions where allelic variation occurs within the target gene). In some such embodiments, the provided oligonucleotides bind preferentially or specifically to one allele compared to one or more other alleles. In some embodiments, the target binding sequence is identical to the target sequence of one allele or is an exact complement of the target sequence of one allele. In some embodiments, the target binding sequence is identical to the target sequence of one allele. In some embodiments, the target binding sequence is an exact complement of the target sequence of one allele. In some embodiments, the provided oligonucleotides bind preferentially to disease-associated alleles. In some embodiments, the provided oligonucleotides bind preferentially to disease-associated alleles and comprise a target binding sequence that is identical to the target sequence of the disease-associated allele but not to the other allele(s) or a target binding sequence that is an exact complement of the target sequence of the disease-associated allele but not to the other allele(s). For example, in some embodiments, the PNPLA3 oligonucleotides provided herein (or their target binding sequence portions) have a sequence identical to the target sequence of a specific allele version, or a sequence that is the exact complement of the target sequence of a specific allele version. In some embodiments, the target sequence is the sequence of a specific allele. In some embodiments, the PNPLA3 oligonucleotides provided herein (or their target binding sequence portions) have a sequence identical to the allele site of a disease-associated allele, or a sequence that is the exact complement of the allele site of a disease-associated allele.

[0187] As those skilled in the art will understand, according to the present disclosure, target sequence can include various allele sites.In some embodiments, target sequence includes SNP.In some embodiments, target sequence includes mutation.In some embodiments, SNP is SNP of PNPLA3.

[0188] A variety of linkers, lipid moieties, carbohydrate moieties, and targeting moieties can be utilized in accordance with the present disclosure, including many known in the art. In some embodiments, the lipid moiety is the targeting moiety. In some embodiments, the carbohydrate moiety is the targeting moiety. In some embodiments, the targeting moiety is the lipid moiety. In some embodiments, the targeting moiety is the carbohydrate moiety. As will be readily understood by those skilled in the art, a variety of linkers, including those described herein, can be utilized in accordance with the present disclosure to link two moieties, for example, to link a lipid / carbohydrate / targeting component to a PNPLA3 oligonucleotide moiety. As will be readily understood by those skilled in the art, the linkers described for linking two moieties can also be used to link other moieties, for example, a linker for linking a lipid moiety to a PNPLA3 oligonucleotide moiety can also be used to link a carbohydrate moiety or a targeting moiety to a PNPLA3 oligonucleotide moiety, and vice versa.

[0189] In some embodiments, the present disclosure provides chiral controlled oligonucleotides and oligonucleotide compositions.For example, in some embodiments, the provided compositions contain a predetermined level of one or more individual oligonucleotide types, where PNPLA3 oligonucleotide types are defined by: 1) base sequence; 2) backbone bond pattern; 3) backbone chiral center pattern; and 4) backbone P-modification pattern.In some embodiments, a particular oligonucleotide type may be defined by 1A) base identity; 1B) base modification pattern; 1C) sugar modification pattern; 2) backbone bond pattern; 3) backbone chiral center pattern; and 4) backbone P-modification pattern.In some embodiments, oligonucleotides of the same oligonucleotide type are identical. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides, the compositions comprising a predetermined level of a plurality of oligonucleotides, the plurality of oligonucleotides sharing a common base sequence and comprising the same configuration of linking phosphorus in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 chiral internucleotide linkages (chiral controlled internucleotide linkages).

[0190] In some embodiments, provided oligonucleotides include 2-30 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 5-30 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 10-30 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 1 chiral controlled internucleotide linkage. In some embodiments, provided oligonucleotides include 2 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 3 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 4 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 5 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 6 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 7 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 8 chiral controlled internucleotide linkages. In some embodiments, provided oligonucleotides include 9 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided comprises 10 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided comprises 11 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided comprises 12 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided comprises 13 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided comprises 14 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided has 15 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided has 16 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided has 17 chiral controlled internucleotide linkages.In some embodiments, the oligonucleotide provided has 18 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided has 19 chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide provided has 20 chiral controlled internucleotide linkages.

[0191] In some embodiments, the oligonucleotide provided is a unimer. In some embodiments, the oligonucleotide provided is a P-modified unimer. In some embodiments, the oligonucleotide provided is a stereounimer. In some embodiments, the oligonucleotide provided is a stereounimer with an Rp configuration. In some embodiments, the oligonucleotide provided is a stereounimer with an Sp configuration.

[0192] In some embodiments, the oligonucleotide provided is an altomer. In some embodiments, the oligonucleotide provided is a P-modified altomer. In some embodiments, the oligonucleotide provided is a stereo altomer.

[0193] In some embodiments, the oligonucleotides provided are blockmirs. In some embodiments, the oligonucleotides provided are P-modified blockmirs. In some embodiments, the oligonucleotides provided are stereoblockmirs.

[0194] In some embodiments, the oligonucleotides provided are gapmers.

[0195] In some embodiments, the oligonucleotide provided is a skipmer.

[0196] In some embodiments, the oligonucleotide provided is a hemimer. In some embodiments, the hemimer is a PNPLA3 oligonucleotide in which the 5' or 3' terminal region has a sequence that possesses a structural feature that the remainder of the oligonucleotide does not have. In some embodiments, the 5' or 3' terminal region has or comprises 2-20 nucleotides. In some embodiments, the structural feature is a base modification. In some embodiments, the structural feature is a sugar modification. In some embodiments, the structural feature is a P-modification. In some embodiments, the structural feature is the stereochemistry of the chiral internucleotide linkage. In some embodiments, the structural feature is or comprises a base modification, a sugar modification, a P-modification, or the stereochemistry of the chiral internucleotide linkage, or a combination thereof. In some embodiments, the hemimer is a PNPLA3 oligonucleotide in which each of the sugar moieties in the 5' terminal region share a common modification. In some embodiments, the hemimer is a PNPLA3 oligonucleotide in which each of the sugar moieties in the 3' terminal region share a common modification. In some embodiments, the common sugar modification in the 5'- or 3'-terminal region is not shared by any of the other sugar moieties in the oligonucleotide. In some embodiments, an example of a hemimer is a PNPLA3 oligonucleotide that contains a sequence of substituted or unsubstituted 2'-O-alkyl sugar-modified nucleosides, bicyclic sugar-modified nucleosides, β-D-ribonucleosides or β-D-deoxyribonucleosides (e.g., 2'-MOE-modified nucleosides and LNA™ or ENA™ bicyclic sugar-modified nucleosides) in one terminal region and a sequence of nucleosides with different sugar moieties (e.g., substituted or unsubstituted 2'-O-alkyl sugar-modified nucleosides, bicyclic sugar-modified nucleosides or natural nucleosides) in the other terminal region. In some embodiments, the provided oligonucleotides are combinations of one or more of unimers, altmers, blockmers, gapmers, hemimers and skipmers. In some embodiments, the oligonucleotides provided are a combination of one or more of unimers, altmers, blockmers, gapmers, and skipmers.For example, in some embodiments, the oligonucleotides provided are both altmers and gapmers. In some embodiments, the oligonucleotides provided are both gapmers and skipmers. Numerous alternative pattern combinations are available, limited only by the commercial and / or synthetic availability of the building blocks required to synthesize the oligonucleotides provided according to the methods of the present invention, as will be recognized by those skilled in the art of chemistry and synthesis. In some embodiments, the hemimer structure provides beneficial advantages. In some embodiments, the oligonucleotides provided are 5'-hemimers that contain a modified sugar moiety at the 5'-terminal sequence. In some embodiments, the oligonucleotides provided are 5'-hemimers that contain a modified 2'-sugar moiety at the 5'-terminal sequence.

[0197] In some embodiments, the provided oligonucleotide comprises one or more optionally substituted nucleotides. In some embodiments, the provided oligonucleotide comprises one or more modified nucleotides. In some embodiments, the provided oligonucleotide comprises one or more optionally substituted nucleosides. In some embodiments, the provided oligonucleotide comprises one or more modified nucleosides. In some embodiments, the provided oligonucleotide comprises one or more optionally substituted LNA.

[0198] In some embodiments, the oligonucleotide provided comprises one or more optionally substituted nucleobases.In some embodiments, the oligonucleotide provided comprises one or more optionally substituted natural nucleobases.In some embodiments, the oligonucleotide provided comprises one or more optionally substituted modified nucleobases.In some embodiments, the oligonucleotide provided comprises one or more 5-methylcytidine; 5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxylcytosine.In some embodiments, the oligonucleotide provided comprises one or more 5-methylcytidine.

[0199] In some embodiments, each base (BA) is independently an optionally substituted or protected adenine, cytosine, guanosine, thymine, or uracil nucleobase. As will be understood by those skilled in the art, a variety of protected nucleobases can be utilized in accordance with the present disclosure, including those widely known in the art (e.g., those used in oligonucleotide preparations (e.g., the protected nucleobases described in WO / 2010 / 064146, WO / 2011 / 005761, WO / 2013 / 012758, WO / 2014 / 010250, US2013 / 0178612, WO / 2014 / 012081, WO / 2015 / 107425, WO2017 / 015555, and WO2017 / 062862, each of which is incorporated herein by reference).

[0200] In some embodiments, provided oligonucleotides comprise one or more optionally substituted sugars. In some embodiments, provided oligonucleotides comprise one or more optionally substituted sugars present in naturally occurring DNA and RNA. In some embodiments, provided oligonucleotides comprise one or more optionally substituted ribose or deoxyribose. In some embodiments, provided oligonucleotides contain one or more optionally substituted ribose or deoxyribose, where one or more of the hydroxyl groups of the ribose or deoxyribose moiety are optionally, and independently, halogen, -N(R') 2 , -OR', or -SR', where each R' is independently as defined above and described herein. In some embodiments, the provided oligonucleotides contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently a halogen, -N(R') 2, -OR' or -SR', where each R' is independently as defined above and described herein. In some embodiments, oligonucleotides are provided that contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with a halogen. In some embodiments, oligonucleotides are provided that contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with one or more -F. halogens. In some embodiments, oligonucleotides are provided that contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with -OR', where each R' is independently as defined above and described herein. In some embodiments, provided oligonucleotides contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with -OR', where each R' is independently an optionally substituted C 1 -C 6 In some embodiments, the oligonucleotides provided contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with -OR', where each R' is independently an optionally substituted C 1 -C 6 In some embodiments, the oligonucleotides provided contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with -OMe. In some embodiments, the oligonucleotides provided contain one or more optionally substituted deoxyribose, where the 2' position of the deoxyribose is optionally and independently substituted with -O-methoxyethyl.

[0201] In some embodiments, the oligonucleotide provided is a hybridized oligonucleotide strand. In certain embodiments, the oligonucleotide provided is a partially hybridized oligonucleotide strand. In certain embodiments, the oligonucleotide provided is a fully hybridized oligonucleotide strand. In certain embodiments, the oligonucleotide provided is a double-stranded oligonucleotide. In certain embodiments, the oligonucleotide provided is a triple-stranded oligonucleotide (e.g., triplex).

[0202] In some embodiments, any one of the structures comprising the PNPLA3 oligonucleotides described in WO2012 / 030683 can be modified according to the methods of the present disclosure to produce chiral controlled compositions thereof. For example, in some embodiments, the chiral controlled compositions control the stereochemistry of any one or more of the chiral binding phosphorus atoms by optionally incorporating one or more P-modifications described in WO2012 / 030683 or the present disclosure. For example, in some embodiments, a particular nucleotide unit of the PNPLA3 oligonucleotide described in WO2012 / 030683 is preselected to obtain with chiral control of the binding phosphorus of the nucleotide unit and / or is P-modified with chiral control of the binding phosphorus of the nucleotide unit.

[0203] In some embodiments, the provided oligonucleotides comprise nucleic acid analogs, such as, for example, GNA, LNA, PNA, TNA, F-HNA (F-THP or 3'-fluorotetrahydropyran), MNA (mannitol nucleic acid, e.g., Leumann 2002 Bioorg. Med. Chem. 10:841-854), ANA (anitol nucleic acid), and morpholino.

[0204] In some embodiments, provided oligonucleotides are characterized as having the ability to directly or indirectly increase or decrease the activity of a protein, or inhibit or promote protein expression. In some embodiments, provided oligonucleotides are characterized in that they are useful in controlling cell proliferation, viral replication, and / or any other cell signaling process.

[0205] In some embodiments, the 5'-end and / or 3'-end of the provided oligonucleotide is modified. In some embodiments, the 5'-end and / or 3'-end of the provided oligonucleotide is modified with a terminal cap moiety. Examples of such modifications, including terminal cap moieties, are detailed herein and in the art, for example, those described in U.S. Patent Application Publication US2009 / 0023675A1.

[0206] In some embodiments, oligonucleotides of the PNPLA3 oligonucleotide type characterized by 1) a common base sequence and length, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone chiral centers have the same chemical structure, e.g., they have the same base sequence, the same pattern of nucleotide modifications, the same backbone linkage pattern (i.e., the pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, etc.), the same pattern of backbone chiral centers (i.e., the pattern of bond phosphorus stereochemistry (Rp / Sp)), and the same pattern of backbone phosphorus modifications (e.g., the "-XLR" of Formula I). 1 " group pattern).

[0207] Single-stranded RNAi agents and antisense oligonucleotides In some embodiments, the present disclosure provides oligonucleotides. In some embodiments, the present disclosure provides oligonucleotides that reduce the expression and / or level of target genes or their gene products. Those skilled in the art will understand after reading this disclosure that the provided oligonucleotides can act as RNAi agents in some embodiments. Alternatively, or in addition, in some embodiments, the provided oligonucleotides can act via RNase H-dependent mechanisms and / or other biochemical mechanisms that do not involve RNA interference.

[0208] In particular, the present disclosure defines certain structural attributes that may be particularly desirable and / or effective for PNPLA3 oligonucleotides. In particular, the present disclosure defines certain structural attributes that may be particularly desirable and / or effective for PNPLA3 oligonucleotides that act as RNAi agents. In some embodiments, the present disclosure defines certain structural attributes that may be particularly desirable and / or effective for PNPLA3 oligonucleotides that act via RNase H-dependent mechanisms and / or other biochemical mechanisms. In some embodiments, the present disclosure defines certain structural attributes that may be particularly desirable and / or effective for single-stranded ssRNAi agents (ssRNAi or ssRNAi agents), and in some such embodiments, such structural attributes may differ from the structural attributes that are particularly desirable and / or effective for the corresponding strand of a double-stranded RNAi agent (dsRNAi or dsRNAi agent), as further described herein below. In some embodiments, the provided oligonucleotide is a single-stranded RNAi agent (e.g., one that can be incorporated into RISC and / or induce or enhance RISC-mediated targeting). In some embodiments, the oligonucleotides provided are antisense oligonucleotides (e.g., those that are incorporated into RNase H and / or that induce or enhance RNase-H mediated cleavage of a target and / or that may function via a different biochemical mechanism).

[0209] In some embodiments (including some single stranded oligonucleotide embodiments), oligonucleotides that act as RNAi agents may have one or more structural attributes and / or functional properties that differ from oligonucleotides that act via RNase H-dependent mechanisms. In some embodiments, PNPLA3 oligonucleotides can induce a decrease in expression and / or levels of a target gene or its gene product by steric blocking of translation after binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing, and / or by exon inclusion or exon exclusion (e.g., skipping). In some embodiments, PNPLA3 oligonucleotides can function or perform a significant proportion (e.g., 10-100%, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or more than 90%) of their function without relying on RNA interference or RISC.

[0210] In some embodiments, the oligonucleotides provided are antisense oligonucleotides (ASOs) that induce cleavage of the target RNA that is mediated by RNase H and not by RISC (RNA interference silencing complex).

[0211] In some embodiments, the oligonucleotides provided are single stranded RNAi (ssRNAi) agents that induce cleavage of target mRNAs mediated by RISC (RNA interference silencing complex) and not by RNase H enzyme. In some embodiments, the PNPLA3 oligonucleotides can function or a significant proportion (e.g., 10-100%, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or more than 90%) of their function without dependence on RNase H.

[0212] Double-stranded RNAi agents can also use RISC and induce cleavage of target mRNA without the use of RNase H enzyme. In some embodiments, single-stranded RNAi agents differ from double-stranded RNAi agents in that ssRNAi agents only contain a single oligonucleotide strand and generally do not contain a significant length of double-stranded region, whereas dsRNAi agents contain a significant length of double-stranded region (e.g., at least about 15 bp, or about 19 bp in "orthodox" siRNA). In some embodiments, dsRNAi contains two separate complementary strands (not covalently linked) that form a double-stranded region (e.g., in "orthodox" siRNA), or contains a long single strand that contains two complementary sequences that form a double-stranded region together (e.g., in shRNA or short hairpin RNA). In some embodiments of dsRNAi, the passenger strand has a single-stranded nick and forms two strands. In some embodiments, the present disclosure indicates that the sequence and / or structural elements (chemical modifications, stereochemistry, etc.) required for a single-stranded RNAi agent to be effective may differ from those required for a double-stranded RNAi agent to be effective.

[0213] In particular, the present disclosure includes the recognition that certain designs (e.g., sequences and / or structural elements) that may be suitable for double-stranded RNAi agents may not be suitable for single-stranded RNAi agents (including single-stranded RNAi agents of the provided forms described herein), and vice versa. In some embodiments, the present disclosure provides effective ssRNAi designs. In some embodiments, the present disclosure shows that certain base sequences, when combined with structural elements (such as modifications, stereochemistry, additional chemical moieties, or moieties) in accordance with the present disclosure, can result in oligonucleotides with unexpectedly high activity, e.g., such oligonucleotides have unexpectedly high activity when administered as ssRNAi agents, especially when compared to oligonucleotides that contain the same sequence but are double-stranded when administered as dsRNAi agents. In some embodiments, the present disclosure shows that certain base sequences, when combined with structural elements (such as modifications, stereochemistry, additional chemical moieties, or moieties) in accordance with the present disclosure, can result in oligonucleotides with unexpectedly high activity (e.g., the ability to reduce the expression and / or levels of a target gene or its gene product).

[0214] Structural and functional differences between single-stranded RNAi (ssRNAi) agents, double-stranded RNAi (dsRNAi) agents, and RNase H-dependent antisense oligonucleotides (ASOs) In some embodiments, single-stranded RNAi (ssRNAi) agents, double-stranded RNAi (dsRNAi) agents, and RNase H-dependent antisense oligonucleotides (ASOs) all cleave the target RNA and / or reduce the expression and / or level of a target gene or its gene product after the agent or oligonucleotide (or a portion thereof) binds to a complementary (or substantially complementary) target RNA (e.g., mRNA or pre-mRNA). In some embodiments, RNAi agents, whether double-stranded or single-stranded, utilize the RISC or RNA interference silencing complex (including the Ago-2 (Argonaute-2) enzyme). In some embodiments, RNase H-dependent antisense oligonucleotides are single-stranded and utilize a different enzyme, RNase H. RNase H is reportedly a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. See U.S. Pat. No. 7,919,472. See also Saetrom (2004 Bioinformatics 20:3055-3063), Kretschmer-Kazemi Far et al. (2003 Nucleic Acids 31:4417-4424), Bertrand et al. (2002) Biochem. Biophys. Res. Comm. 296:1000-1004), Vickers et al. (2003 J. Biol. Chem. 278:7108). In some embodiments, oligonucleotides capable of inducing RNase H-mediated knockdown include, but are not limited to, those that consist of or contain a region of contiguous 2'-deoxynucleotide units that do not contain a 2'-modification. In some embodiments, oligonucleotides capable of inducing RNase H-mediated knockdown are gap-spread oligonucleotides, or gapmers. In some embodiments, a gapmer comprises an internal region that contains a number of nucleotides that support cleavage by RNase H and is located between an external region having a number of nucleotides that are chemically distinct from the nucleosides of the internal region.In some embodiments, a gapmer comprises a stretch of 2'-deoxynucleotides without a 2'-modification flanked by or adjacent to one or two wings. In some embodiments, the gap induces cleavage of the corresponding RNA target by RNase H. In some embodiments, the wings do not induce cleavage by RNase H or serve as a substrate for cleavage by RNase H. The wings can be of different lengths (including but not limited to, 1-8 nt) and can contain a variety of modifications or analogs (including but not limited to, 2'-modifications, including but not limited to, 2'-OMe and 2'-MOE). See, for non-limiting examples, U.S. Patent Nos. 9,550,988, 7,919,472, 5,013,830, 5,149,797, 5,220,007, 5,256,775, 5,366,878, 5,403,711, 5,491,133, 5,565,350, 5,623,065, 5,652,355, 5,652,356, and 5,700,922. In some embodiments, the presence of one or more of such modifications or analogs may correlate with altered (e.g., increased, decreased, or altered) cleavage of the target by RNase H.

[0215] In some embodiments, even the antisense strand of a double-stranded RNAi agent is structurally different from an RNase H-dependent antisense oligonucleotide. In some embodiments, RNase H-dependent antisense oligonucleotides and siRNA oligonucleotides appear to have opposite characteristics, both in terms of 5'-end structure and overall duplex stability.

[0216] Double-stranded RNAi agents can reportedly occur naturally in cells by the Dicer enzyme, which cleaves longer RNA molecules (such as double-stranded RNA from invading viruses) into dsRNA. The canonical structure of a dsRNA agent includes two RNA strands, each about 19-23 nt in length, which anneal to form a double-stranded region of about 19-21 bp and two 3' dinucleotide overhangs. For double-stranded RNAi agents, the antisense strand reportedly is incorporated into RISC after the sense strand is unwound from the duplex. Apart from the natural separation of double-stranded RNAi agents into antisense and sense strands, single-stranded RNAi agents have not been reported to occur naturally in human cells.

[0217] In particular, the present disclosure provides the teaching that single-stranded RNAi agent is not simply the isolated antisense strand of double-stranded RNAi agent in many cases, for example, in that the effectiveness of the antisense strand of effective dsRNAi agent can be much lower compared to the dsRNAi agent, and when ssRNAi agent is constructed as a dsRNAi agent (for example, by annealing with sense strand), it can be much lower in effectiveness compared to the ssRNAi agent.In some embodiments, double-stranded RNAi agent and single-stranded RNAi agent are different in many significant ways.The structural parameters of double-stranded RNAi agent are not necessarily reflected in single-stranded RNAi agent.

[0218] In some embodiments, the present disclosure teaches that the target sequence suitable for double-stranded RNAi agent may not be suitable for single-stranded RNAi agent, and vice versa.For example, at least in some cases, the single-stranded version of double-stranded RNAi agent may be ineffective.As a non-limiting example, some ssRNAi agents are constructed with sequences derived from dsRNAi are shown in table 46A.These ssRNAi based on dsRNAi are generally less effective compared to corresponding dsRNAi.

[0219] In some embodiments, double-stranded and single-stranded RNAi agents also differ in their sensitivity to the incorporation of chiral-controlled internucleotide bonds. For example, Matranga et al. (2005 Cell 123:607-620) reported that the introduction of a single Sp internucleotide bond (e.g., a single Sp PS) into the sense strand of a double-stranded RNAi agent significantly reduces RISC assembly and RNA interference activity. In contrast, in some embodiments, the data presented herein surprisingly shows that the incorporation of an Sp internucleotide bond (e.g., Sp PS) can perform two functions for a single-stranded RNAi agent: (a) it improves stability against nucleases, and (b) it does not interfere with RNA interference activity. Many of the exemplary oligonucleotides can serve as effective single-stranded RNAi agents that contain one or more chiral-controlled internucleotide bonds (e.g., Sp internucleotide bonds or Sp PS (phosphorothioate) are presented herein).

[0220] Alternatively or additionally, double-stranded RNAi agents and single-stranded RNAi agents may differ in immunogenicity. In some embodiments, some single-stranded RNAi agents are reportedly more immunogenic than double-stranded RNAi agents. Sioud J.Mol.Biol.(2005)348,1079-1090. In some embodiments, some double-stranded RNAi agents are reportedly not induced immune response, while the corresponding single-stranded RNAi agents induced immune response. In some embodiments, the present disclosure provides oligonucleotides with low immunogenicity. In some embodiments, such oligonucleotides can be utilized as ssRNAi reagents.

[0221] In particular, the present disclosure includes the recognition that certain conventional designs of single-stranded RNAi agents, including sequences, from double-stranded RNAi agents often fail to produce effective single-stranded RNAi agents. In some embodiments, the present disclosure surprisingly shows that effective ssRNAi agents can be obtained from ssRNAi agents derived from sequences of effective RNase H-dependent ASOs (see Table 46A).

[0222] In some embodiments, the disclosure provides oligonucleotides that can be utilized as effective RNase-H dependent ASOs, which include a region of 2'-deoxynucleotides that have no 2'-modifications and are complementary or substantially complementary to an RNA sequence or a portion thereof. In some embodiments, the region can be, for example, a core sequence of about 10 nt flanked on one or both sides by wings that are chemically distinct from the core and can include, by way of non-limiting example, 2'-modifications or internucleotide linkage modifications.

[0223] Oligonucleotides In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides can induce a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after binding to the target gene mRNA, and / or altering or disrupting mRNA splicing, and / or exon inclusion or exon exclusion.

[0224] In some embodiments, the oligonucleotides provided have a structural element or format described herein or a portion thereof.

[0225] In some embodiments, the provided oligonucleotides capable of inducing a decrease in the expression and / or level of a target gene or its gene product have structural elements or formats described herein or portions thereof.

[0226] In some embodiments, the provided oligonucleotides capable of inducing a decrease in expression and / or levels of a target gene or its gene product have the format of any oligonucleotide disclosed herein, such as those shown in Table 1A or in a figure or table, or elsewhere disclosed herein.

[0227] In some embodiments, the oligonucleotide provided has any of the formats shown in FIG.

[0228] This disclosure presents data showing that various oligonucleotides in various formats have the ability to target any of several different sequences in several different genes in several different species to induce a decrease in the expression and / or levels of a target gene or its gene product. Although not shown, additional data has been obtained that supports the efficacy of ssRNAi agents in the disclosed formats.

[0229] In some embodiments, oligonucleotides that are provided that have the ability to induce RNase H-mediated knockdown have structural elements or formats described herein or portions thereof.

[0230] In some embodiments, the oligonucleotides provided that are capable of inducing RNase H-mediated knockdown have the format of any oligonucleotide disclosed herein, such as those shown in Table 1A or in a figure or table, or otherwise disclosed herein.

[0231] In some embodiments, the oligonucleotide provided has any of the formats shown in FIG.

[0232] This disclosure presents data showing that a variety of oligonucleotides in a variety of formats have the ability to induce RNase H-mediated knockdown in a number of different genes in a number of different species, at any of a number of different sequences. Although not shown, additional data has been obtained that supports the efficacy of ssRNAi agents in the disclosed format.

[0233] In some embodiments, provided oligonucleotides capable of inducing single stranded RNA interference have structural elements or formats described herein or portions thereof.

[0234] In some embodiments, the provided oligonucleotides capable of inducing single-stranded RNA interference have the format of any oligonucleotide disclosed herein, such as those shown in Table 1A or in a figure or table, or elsewhere disclosed herein.

[0235] In some embodiments, a provided single stranded RNAi agent has any of the formats shown in FIG.

[0236] This disclosure presents data showing that various RNAi agents in various formats have the ability to induce RNA interference in any of multiple different genes, against any of multiple different sequences.Although not shown, additional data has been obtained that supports the effectiveness of the ssRNAi agents in the disclosed formats.

[0237] In some embodiments, the target of RNAi is a transcript. In some embodiments, the transcript is a pre-mRNA. In some embodiments, the transcript is a mature RNA. In some embodiments, the transcript is an mRNA. In some embodiments, the transcript comprises a mutation. In some embodiments, the mutation is a frameshift. In some embodiments, the transcript comprises a premature termination codon. In some embodiments, the target of RNAi is an RNA that is not an mRNA. In some embodiments, the target of RNAi is a non-coding RNA. In some embodiments, the target of RNAi is a long non-coding RNA. In some embodiments, the provided oligonucleotides in the provided compositions, e.g., the first plurality of oligonucleotides, comprise a base modification, a sugar modification, and / or an internucleotide linkage modification. In some embodiments, the provided oligonucleotides comprise a base modification and a sugar modification. In some embodiments, the provided oligonucleotides comprise a base modification and an internucleotide linkage modification. In some embodiments, the provided oligonucleotides comprise a sugar modification and an internucleotide linkage modification. In some embodiments, the provided compositions comprise a base modification, a sugar modification, and an internucleotide linkage modification. Examples of chemical modifications, such as base modifications, sugar modifications, internucleotide linkage modifications, etc., are widely known in the art and include, but are not limited to, the modifications described in this disclosure. In some embodiments, the modified base is a substituted A, T, C, G, or U. In some embodiments, the sugar modification is a 2'-modification. In some embodiments, the 2'-modification is a 2-F modification. In some embodiments, the 2'-modification is a 2'-OR 1 In some embodiments, the 2'-modification is 2'-OR 1 where R 1is optionally substituted alkyl. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring. In some embodiments, the modified sugar moiety is a bridged bicyclic or polycyclic ring having 5-20 ring atoms, where one or more ring atoms are optionally and independently heteroatoms. Examples of ring structures are widely known in the art, such as those found in BNAs, LNAs, and the like. In some embodiments, the oligonucleotides provided include both one or more modified internucleotide linkages and one or more natural phosphate linkages. In some embodiments, oligonucleotides including both modified internucleotide linkages and natural phosphate linkages and compositions thereof provide improved properties, such as activity. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the modified internucleotide linkage is a substituted phosphorothioate linkage.

[0238] In particular, the present disclosure encompasses the recognition that stereoirregular oligonucleotide preparations contain multiple distinct chemical entities that differ from each other, for example, in the stereochemical configuration of individual backbone chiral centers within an oligonucleotide chain. Without control of the stereochemistry of backbone chiral centers, stereoirregular oligonucleotide preparations provide uncontrolled compositions that contain undefined levels of oligonucleotide stereoisomers. Although these stereoisomers may have the same base sequence, these stereoisomers are different chemical entities due to at least their different backbone stereochemistry, and may have different properties, such as activity, as shown herein. In particular, the present disclosure provides new compositions that are or contain a specific stereoisomer of the subject oligonucleotide. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, length, backbone bond pattern, and backbone chiral center pattern. As understood in the art, in some embodiments, the base sequence can refer to the identity and / or modification state of the nucleoside residues in the PNPLA3 oligonucleotide (e.g., the state of the sugar and / or base components compared to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization properties of such residues (i.e., the ability to hybridize with a specific complementary residue). In some embodiments, the present disclosure provides PNPLA3 oligonucleotide compositions that include predetermined levels of oligonucleotides of individual oligonucleotide types that are chemically identical, e.g., they have the same base sequence, the same pattern of nucleoside modifications (modifications to the sugar and base moieties, if present), the same pattern of backbone chiral centers, and the same pattern of backbone phosphorus modifications. The present disclosure particularly indicates that individual stereoisomers of a particular oligonucleotide may exhibit different stability and / or activity from each other.In some embodiments, the suitable improvement achieved by including and / or arranging a specific chiral structure in the PNPLA3 oligonucleotide may be comparable to or superior to that achieved by using a specific backbone linkage, residue modification, etc. (e.g., by using a type of modified phosphate [e.g., phosphorothioate, substituted phosphorothioate, etc.], sugar modification [e.g., 2'-modification, etc.], and / or base modification [e.g., methylation, etc.]). In particular, the present disclosure recognizes that in some embodiments, the properties (e.g., activity, etc.) of the PNPLA3 oligonucleotide can be adjusted by optimizing the pattern of backbone chiral centers, optionally in combination with adjusting / optimizing one or more other properties of the oligonucleotide (e.g., linkage pattern, nucleoside modification pattern, etc.). As illustrated by various examples of the present disclosure, the chiral-controlled oligonucleotide compositions provided can exhibit improved properties, such as improved single-stranded RNA interference activity, RNase H-mediated knockdown, improved delivery, etc.

[0239] In some embodiments, the properties of the oligonucleotide can be adjusted by optimizing the stereochemistry (pattern of backbone chiral centers) and the chemical modifications (modifications of the bases, sugars and / or internucleotide linkages) or patterns thereof.

[0240] In some embodiments, a common pattern of backbone chiral centers (e.g., a pattern of backbone chiral centers in a single stranded RNAi agent) comprises the pattern OSOSO, OSSSO, OSSSOS, SOSO, SOSO, SOSOS, SOSOSO, SOSOSOSO, SOSSSO, SSOSSSOSS, SSSOSOSSS, SSSSOSOSSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSS, or RRR, where S represents a phosphorothioate in the Sp configuration, O represents a phosphodiester, and R represents a phosphorothioate in the Rp configuration.

[0241] In some embodiments, the non-chiral center is a phosphodiester bond. In some embodiments, the chiral center of the Sp configuration is a phosphorothioate bond. In some embodiments, the non-chiral center is a phosphodiester bond. In some embodiments, the chiral center of the Sp configuration is a phosphorothioate bond.

[0242] In some embodiments, the oligonucleotides provided comprise any stereochemical pattern described herein. In some embodiments, the oligonucleotides provided comprise any stereochemical pattern described herein and are capable of inducing RNA interference. In some embodiments, the oligonucleotides provided comprise any stereochemical pattern described herein and are capable of inducing RNase H-mediated knockdown. In some embodiments, the oligonucleotides provided comprise any stereochemical pattern described herein and are capable of inducing RNA interference and RNase H-mediated knockdown. In some embodiments, the oligonucleotides provided comprise any stereochemical pattern described herein and are capable of inducing RNA interference and RNase H-mediated knockdown, the stereochemical pattern being present in the seed region and / or the post-seed region. In some embodiments, the oligonucleotides provided comprise any stereochemical pattern described herein and are capable of inducing RNA interference and RNase H-mediated knockdown, the stereochemical pattern being present in the seed region and / or the post-seed region.

[0243] In some embodiments, the oligonucleotides provided comprise any of the modifications or modification patterns described herein. In some embodiments, the oligonucleotides provided comprise any of the modifications or modification patterns described herein and are capable of inducing RNA interference. In some embodiments, the oligonucleotides provided comprise any of the modification patterns described herein and are capable of inducing RNase H mediated knockdown. In some embodiments, the oligonucleotides provided comprise any of the modification patterns described herein and are capable of inducing RNA interference and RNase H mediated knockdown. In some embodiments, the oligonucleotides provided comprise any of the modification patterns described herein and are capable of inducing RNA interference and RNase H mediated knockdown, the modification patterns being present in the seed and / or post seed regions. In some embodiments, the oligonucleotides provided comprise any of the modification patterns described herein and are capable of inducing RNA interference and RNase H mediated knockdown, the modification patterns being present in the seed and / or post seed regions. In some embodiments, the modification or modification pattern is a modification or modification pattern at the 2' position of the sugar. In some embodiments, the modification or modification pattern is a sugar modification or modification pattern, for example, at the 2' position of the sugar, including but not limited to, 2'-deoxy, 2'-F, 2'-OMe, 2'-MOE, and 2'-OR1 (wherein R1 is an optionally substituted C1-6 alkyl).

[0244] In some embodiments, the present disclosure shows that 2'-F modification can improve, among other things, single-stranded RNA interference. In some embodiments, the present disclosure shows that Sp internucleotide bond can improve the stability of oligonucleotides, especially when located at the 5'-end and 3'-end. In some embodiments, the present disclosure shows that natural phosphate bond and / or Rp internucleotide bond can improve the removal of oligonucleotides from the system. As will be understood by those skilled in the art, various assays known in the art can be used to evaluate such properties according to the present disclosure.

[0245] In some embodiments, provided oligonucleotides capable of inducing single-stranded RNA interference comprise one or more modified sugar moieties, hi some embodiments, 5% or more of the sugar moieties of the provided oligonucleotides are modified.

[0246] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides have the same base sequence; The first plurality of oligonucleotides contains one or more modified sugar moieties, or contains one or more natural phosphate linkages and one or more modified internucleotide linkages.

[0247] In some embodiments, the first plurality of oligonucleotides comprises one or more modified sugar moieties. In some embodiments, provided oligonucleotides comprise one or more modified sugar moieties.

[0248] In some embodiments, the compositions provided alter single-stranded RNA interference of the transcript, resulting in the suppression of undesired targets and / or biological functions. In some embodiments, in such cases, the compositions provided can also induce cleavage of the transcript after hybridization.

[0249] In some embodiments, each of the first plurality of oligonucleotides comprises one or more modified sugar moieties and / or one or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises no more than about 95% unmodified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises no more than about 50% unmodified sugar moieties. In some embodiments, each of the first plurality of oligonucleotides comprises no more than about 5% unmodified sugar moieties. In some embodiments, each sugar moiety of the first plurality of oligonucleotides is independently modified.

[0250] In some embodiments, each of the first plurality of oligonucleotides comprises two or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises three or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises four or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises five or more modified internucleotide linkages. In some embodiments, each of the first plurality of oligonucleotides comprises ten or more modified internucleotide linkages.

[0251] In some embodiments, the percentage of natural phosphate bonds in each of the first plurality of oligonucleotides is about 30% or less. In some embodiments, the percentage of natural phosphate bonds in each of the first plurality of oligonucleotides is about 20% or less. In some embodiments, the percentage of natural phosphate bonds in each of the first plurality of oligonucleotides is about 10% or less. In some embodiments, the percentage of natural phosphate bonds in each of the first plurality of oligonucleotides is about 5% or less.

[0252] In some embodiments, the oligonucleotides provided are enriched in one or more isotopes. In some embodiments, the oligonucleotides provided are labeled, e.g., with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the oligonucleotides provided in the compositions provided, e.g., the first plurality of oligonucleotides, contain base modifications, sugar modifications, and / or internucleotide linkage modifications, and the oligonucleotides are enriched in deuterium. In some embodiments, the oligonucleotides provided are labeled with deuterium (-) at one or more positions. 1 H is- 2 In some embodiments, one or more of the PNPLA3 oligonucleotides or any moieties conjugated to the oligonucleotide (e.g., targeting moiety, lipid moiety, etc.) 1 H 2Replaced with H. Such oligonucleotides can be used in any of the compositions or methods described herein.

[0253] The present invention includes all pharma- ceutically acceptable isotopically labeled compounds in which one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

[0254] Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen ( 2 H and 3 H), carbon isotopes ( 11 C. 13 C, and 14 C), chlorine isotopes ( 36 Cl), fluorine isotopes ( 18 F), isotopes of iodine ( 123 I, 124 I, and 125 I, etc.), nitrogen isotopes ( 13 N and 15 N), oxygen isotopes ( 15 O. 17 O, and 18 O), phosphorus isotopes ( 32 P), and sulfur isotopes ( 35 S, etc.

[0255] Certain isotopically labeled compounds of formula (I) (e.g., those incorporating a radioactive isotope) are useful in drug testing and / or substrate tissue distribution studies. 3 H) and carbon-14 (i.e. 14 C) is particularly useful for this purpose given its ease of incorporation and the availability of means for its detection.

[0256] Heavy isotopes (deuterium (i.e. 2Replacement with ribozymes such as ribozymes (H) may be preferred in some circumstances because certain therapeutic advantages may result from increased metabolic stability (e.g., longer in vivo half-life) or reduced dosage requirements.

[0257] Positron-emitting isotopes ( 11 C. 18 F, 15 O, and 13 N) may be useful in positron emission tomography (PET) studies to examine receptor occupancy of substrates.

[0258] Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the accompanying Examples and Preparative Examples, using appropriate isotopically labeled reagents in place of previously utilized non-labeled reagents.

[0259] The compounds of the present invention may contain asymmetric or chiral centers and therefore may exist in different stereoisomeric forms.Unless otherwise specified, it is intended that all stereoisomers of the compounds of the present invention and mixtures thereof, including racemic mixtures, form part of the present invention.In addition, the present invention encompasses all geometric and positional isomers.For example, if a compound of the present invention incorporates a double bond or a fused ring, both the cis and trans forms and mixtures thereof are included within the scope of the present invention.

[0260] The chiral compounds of the invention (and their chiral precursors) can be obtained in enantiomerically enriched form by using chromatography, typically high performance liquid chromatography (HPLC) or supercritical fluid chromatography (SFC), on a chiral stationary phase resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing 0-50%, typically 2-20%, isopropanol, and 0-5% of an alkylamine, typically 0.1% diethylamine (DEA) or isopropylamine. The eluent is concentrated to give the enriched mixture.

[0261] Diastereomeric mixtures can be separated into individual diastereomers based on their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with a suitable optically active compound (e.g., a chiral alcohol or a chiral auxiliary such as Mosher's acid chloride), separating the diastereomers, and converting the individual diastereomers into the corresponding pure enantiomers (e.g., by hydrolysis). Enantiomers can also be separated by using chiral HPLC columns. Alternatively, specific stereoisomers can be synthesized by using optically active starting materials, by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one stereoisomer into the other stereoisomer by asymmetric transformation.

[0262] In some embodiments, control of structural elements of the oligonucleotide can have a significant effect on the desired biological effect, such as chemical modifications (e.g., sugar modifications, base modifications, and / or internucleotide linkage modifications) or patterns, alterations in stereochemistry (e.g., the stereochemistry of backbone chiral internucleotide linkages), replacement of atoms with isotopes of the same element, and / or conjugation to additional chemical moieties (e.g., lipid moieties, targeting moieties, etc.), etc. In some embodiments, the desired biological effect is enhanced by more than two-fold.

[0263] In some embodiments, the desired biological effect is induction of a decrease in expression and / or level of a target gene or its gene product. In some embodiments, the desired biological effect is improvement of single-stranded RNA interference. In some embodiments, the desired biological effect is improvement of RNase H-mediated knockdown. In some embodiments, the desired biological effect is improvement of single-stranded RNA interference and / or RNase H-mediated knockdown.

[0264] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) having a common base sequence complementary to a target sequence in the transcription product; 2) contain one or more modified sugar moieties and modified internucleotide linkages.

[0265] In some embodiments, the provided oligonucleotide compositions are characterized in that when they are contacted with a transcript in a single-stranded RNA interference system, they improve RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0266] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single stranded RNA interference, the PNPLA3 oligonucleotide type being: 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification is defined by The composition is chiral controlled in that the composition is enriched for a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence; The oligonucleotide composition is characterized in that when it is contacted with a transcript in a single-stranded RNA interference system, it improves RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0267] In some embodiments, each consecutive nucleoside unit is independently positioned after and / or before a modified internucleotide linkage. In some embodiments, each consecutive nucleoside unit is independently positioned after and / or before a phosphorothioate linkage. In some embodiments, each consecutive nucleoside unit is independently positioned after and / or before a chiral controlled modified internucleotide linkage. In some embodiments, each consecutive nucleoside unit is independently positioned after and / or before a chiral controlled phosphorothioate linkage. In some embodiments, the modified internucleotide linkage has the structure of Formula I. In some embodiments, the modified internucleotide linkage has the structure of Formula Ia.

[0268] In some embodiments, the disclosure provides a single stranded RNAi agent comprising a predetermined level of a first plurality of oligonucleotides, the first plurality of oligonucleotides have the same base sequence; The first plurality of oligonucleotides comprises a seed region comprising 2, 3, 4, 5, 6, 7 or more consecutive Sp modified internucleotide linkages and a post-seed region comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive Sp modified internucleotide linkages.

[0269] In some embodiments, the seed region comprises two or more consecutive Sp modified internucleotide linkages.

[0270] In some embodiments, the modified internucleotide linkage has the structure of Formula I. In some embodiments, the modified internucleotide linkage has the structure of Formula Ia.

[0271] As described herein, in some embodiments, the provided oligonucleotide compositions are characterized in that, when contacted with a transcript in a single-stranded RNA interference system, they result in improved RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0272] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) Common base sequence and length, 2) common patterns of skeletal bonds, and 3) Common patterns of skeletal chiral centers and the composition is a substantially pure preparation of a single oligonucleotide, in that a given level of oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0273] In some embodiments, the common base sequence and length may be referred to as the shared base sequence. In some embodiments, oligonucleotides with a common base sequence may have the same pattern of nucleoside modifications, such as sugar modifications, base modifications, etc. In some embodiments, the pattern of nucleoside modifications may be represented by a combination of position and modification. In some embodiments, the pattern of backbone linkages includes the position and type (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.) of each internucleotide linkage. The pattern of backbone chiral centers of PNPLA3 oligonucleotides can be specified by the combination of 5' to 3' bond phosphorus stereochemistry (Rp / Sp). As exemplified above, the position of the non-chiral linkages can be known, for example, from the pattern of backbone linkages.

[0274] As will be understood by those skilled in the art, stereoirregular or racemic preparations of oligonucleotides are often prepared by non-stereoselective and / or low stereoselective coupling of nucleotide monomers without using any chiral auxiliary, chiral modification reagent and / or chiral catalyst. In some embodiments, in a substantially racemic (or chirally uncontrolled) preparation of oligonucleotides, all or most of the coupling steps are not chirally controlled in that the coupling steps are not specifically performed to provide enhanced stereoselectivity. An example of a substantially racemic preparation of oligonucleotides is the preparation of phosphorothioate oligonucleotides by sulfurizing the phosphite triester from the commonly used phosphoramidite oligonucleotide synthesis with either tetraethylthiuram disulfide (TETD) or 3H-1,2-bensodithiol-3-one 1,1-dioxide (BDTD), a process known in the art. In some embodiments, a substantially racemic preparation of oligonucleotides provides a substantially racemic oligonucleotide composition (or a chiral non-controlled oligonucleotide composition).

[0275] As will be appreciated by one of skill in the art, in some embodiments, the diastereoselectivity of coupling or conjugation can be assessed via the diastereoselectivity of dimer formation under identical or comparable conditions, where the dimers have the same 5'- and 3'-nucleosides and internucleotide linkages.

[0276] In some embodiments, the present disclosure provides a chiral controlled oligonucleotide composition of a first plurality of oligonucleotides, the composition being chiral controlled in that it is enriched for oligonucleotides of a single oligonucleotide type compared to a substantially racemic preparation of the same oligonucleotide. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide composition of a first plurality of oligonucleotides, the composition being enriched for oligonucleotides of a single oligonucleotide type compared to a substantially racemic preparation of the same oligonucleotide, the oligonucleotides of the single oligonucleotide type being 1) Common base sequence and length, 2) common patterns of skeletal bonds, and 3) Common patterns of skeletal chiral centers Share.

[0277] In some embodiments, the disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single stranded RNA interference, the oligonucleotides comprising: 1) Common base sequence and length, 2) common patterns of skeletal bonds, and 3) Common patterns of skeletal chiral centers and wherein the specific oligonucleotide type is characterized by The compositions are chiral controlled in that they are enriched for oligonucleotides of a particular oligonucleotide type as compared to a substantially racemic preparation of the oligonucleotide having the same base sequence and length.

[0278] In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have the same structure.

[0279] In some embodiments, oligonucleotides of a PNPLA3 oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides of a PNPLA3 oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides of a PNPLA3 oligonucleotide type have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides of a PNPLA3 oligonucleotide type are identical.

[0280] In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of sugar modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of base modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers have a common pattern of backbone phosphorus modifications and a common pattern of nucleoside modifications. In some embodiments, oligonucleotides having a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers are identical.

[0281] In some embodiments, the oligonucleotides in the provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, the common base sequence is a base sequence of a PNPLA3 oligonucleotide type. In some embodiments, the provided compositions are chiral controlled PNPLA3 oligonucleotide compositions in that the composition comprises a first plurality of oligonucleotides of an individual oligonucleotide type at a predetermined level, the PNPLA3 oligonucleotide type being 1) Nucleotide sequence, 2) skeletal bond pattern, 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification is defined as follows:

[0282] As discussed above and understood in the art, in some embodiments, the base sequence of a PNPLA3 oligonucleotide can refer to the identity and / or modification state of the nucleoside residues in the oligonucleotide (e.g., the state of the sugar and / or base components compared to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or can refer to the hybridization properties of such residues (i.e., the ability to hybridize with a particular complementary residue).

[0283] In some embodiments, a particular oligonucleotide type has the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same backbone linkage pattern (e.g., the pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the stereochemical (Rp / Sp) pattern of chiral internucleotide linkages), and the same pattern of backbone phosphorus modifications (e.g., the pattern of modifications on the internucleotide phosphorus atom, e.g., the -S of Formula I). - , and -LR 1 ) are chemically identical.

[0284] In particular, the present disclosure recognizes that combining structural elements of oligonucleotides (e.g., the pattern of chemical modifications, the pattern of backbone linkages, the pattern of backbone chiral centers, and / or the pattern of backbone phosphorus modifications) can result in surprising improvements in properties (such as biological activity).

[0285] In some embodiments, the chirally controlled (and / or stereochemically pure) preparations provided are RNAi agent oligonucleotides.

[0286] In some embodiments, the chiral controlled (and / or stereochemically pure) preparations provided are preparations of oligonucleotides that contain one or more modified backbone bonds, bases, and / or sugars.

[0287] In some embodiments, the compositions provided include oligonucleotides that include one or more residues that are modified at the sugar moiety. In some embodiments, the compositions provided include oligonucleotides that include one or more residues that are modified at the 2' position of the sugar moiety (referred to herein as "2'-modified"). Examples of such modifications are described above and herein, and include, but are not limited to, 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, S-cEt, and the like. In some embodiments, the compositions provided include oligonucleotides that include one or more 2'-modified residues. For example, in some embodiments, the oligonucleotides provided include one or more 2'-O-methoxyethyl (2'-MOE) modified residues. In some embodiments, the compositions provided include oligonucleotides that do not include any 2'-modifications. In some embodiments, the compositions provided are oligonucleotides that do not include any 2'-MOE residues. That is, in some embodiments, the oligonucleotides provided are not MOE modified. Additional examples of sugar modifications are described herein.

[0288] In some embodiments, the one or more is one. In some embodiments, the one or more is two. In some embodiments, the one or more is three. In some embodiments, the one or more is four. In some embodiments, the one or more is five. In some embodiments, the one or more is six. In some embodiments, the one or more is seven. In some embodiments, the one or more is eight. In some embodiments, the one or more is nine. In some embodiments, the one or more is ten. In some embodiments, the one or more is at least one. In some embodiments, the one or more is at least two. In some embodiments, the one or more is at least three. In some embodiments, the one or more is at least four. In some embodiments, the one or more is at least five. In some embodiments, the one or more is at least six. In some embodiments, the one or more is at least seven. In some embodiments, the one or more is at least eight. In some embodiments, the one or more is at least nine. In some embodiments, one or more is at least ten.

[0289] In some embodiments, the sugar moiety that does not have a 2'-modification is a sugar moiety present in a natural DNA nucleoside.

[0290] One of skill in the art will appreciate that various regions of a target transcript may be targeted by the provided compositions and methods. In some embodiments, the base sequence of the provided oligonucleotide comprises an intron sequence. In some embodiments, the base sequence of the provided oligonucleotide comprises an exon sequence. In some embodiments, the base sequence of the provided oligonucleotide comprises an intron sequence and an exon sequence.

[0291] As will be understood by those skilled in the art, the oligonucleotides and compositions provided can specifically target a large number of nucleic acid polymers.For example, in some embodiments, the oligonucleotides and compositions provided can target the transcription product of a nucleic acid sequence, where the common base sequence of the oligonucleotide (e.g., the base sequence of a certain PNPLA3 oligonucleotide type) is or comprises the sequence complementary to the sequence of the transcription product. In some embodiments, the common base sequence comprises the sequence complementary to the target sequence. In some embodiments, the common base sequence is the sequence complementary to the target sequence. In some embodiments, the common base sequence is or comprises the sequence 100% complementary to the target sequence. In some embodiments, the common base sequence comprises the sequence 100% complementary to the target sequence. In some embodiments, the common base sequence is the sequence 100% complementary to the target sequence.

[0292] I

[0293] In some embodiments, the common base sequence is or comprises a sequence complementary to the distinctive sequence element. In some embodiments, the common base sequence comprises a sequence complementary to the distinctive sequence element. In some embodiments, the common base sequence is a sequence complementary to the distinctive sequence element. In some embodiments, the common base sequence is or comprises a sequence 100% complementary to the distinctive sequence element. In some embodiments, the common base sequence comprises a sequence 100% complementary to the distinctive sequence element. In some embodiments, the common base sequence is a sequence 100% complementary to the distinctive sequence element. In some embodiments, the distinctive sequence element is, by way of non-limiting example, a seed region, a post-seed region, or a portion of the seed region, or a portion of the post-seed region, or a 3' terminal dinucleotide.

[0294] In some embodiments, the characteristic sequence element comprises or is a mutation. In some embodiments, the characteristic sequence element comprises a mutation. In some embodiments, the characteristic sequence element is a mutation. In some embodiments, the characteristic sequence element comprises or is a point mutation. In some embodiments, the characteristic sequence element comprises a point mutation. In some embodiments, the characteristic sequence element is a point mutation. In some embodiments, the characteristic sequence element comprises or is a SNP. In some embodiments, the characteristic sequence element comprises a SNP. In some embodiments, the characteristic sequence element is a SNP.

[0295] In some embodiments, the consensus base sequence is 100% matched to the target sequence, and the target sequence is not 100% matched to a similar sequence of the target sequence.

[0296] For example, in some embodiments, the consensus sequence matches a mutation in a disease-causing copy or allele of a target nucleic acid sequence but does not match a non-disease-causing copy or allele at the mutation site, hi some other embodiments, the consensus sequence matches a SNP in a disease-causing allele of a target nucleic acid sequence but does not match a non-disease-causing allele at the corresponding site.

[0297] In particular, the present disclosure recognizes that base sequence can affect the properties of an oligonucleotide. In some embodiments, when an oligonucleotide having a certain base sequence is used to inhibit a target via a pathway that includes, for example, RNase H, the base sequence can affect the cleavage pattern of the target. For example, structurally similar (all phosphorothioate linkages, all stereochemistries) oligonucleotides with different sequences can have different cleavage patterns.

[0298] In some embodiments, the consensus sequence is a sequence that includes a SNP.

[0299] As will be understood by those skilled in the art, the oligonucleotide compositions and methods provided have various uses known to those skilled in the art.The evaluation methods of the compositions provided, as well as their properties and uses, are also widely known and practiced by those skilled in the art.Examples of properties, uses, and / or methods include, but are not limited to, those described in WO / 2014 / 012081 and WO / 2015 / 107425.

[0300] In some embodiments, the chiral internucleotide linkage has the structure of Formula I. In some embodiments, the chiral internucleotide linkage is phosphorothioate. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of the provided compositions has the structure of Formula I. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of the provided compositions is phosphorothioate.

[0301] In some embodiments, the oligonucleotides of the present disclosure comprise one or more modified sugar moieties. In some embodiments, the oligonucleotides of the present disclosure comprise one or more modified base moieties. Various modifications can be introduced into the oligonucleotides and / or moieties as known to those of skill in the art and as described herein. For example, in some embodiments, the modifications are those described in US9006198, WO2014 / 012081, and WO / 2015 / 107425, each of which sugar and base modifications are incorporated herein by reference.

[0302] In some embodiments, the sugar modification is a 2'-modification. Commonly used 2'-modifications include, but are not limited to, 2'-OR 1 In the formula, R 1is not hydrogen. In some embodiments, the modification is 2'-OR, where R is an optionally substituted aliphatic. In some embodiments, the modification is 2'-OMe. In some embodiments, the modification is 2'-O-MOE. In some embodiments, the present disclosure demonstrates that the inclusion and / or arrangement of certain chirally pure internucleotide linkages can result in stability improvements comparable to or superior to those achieved through the use of modified backbone linkages, bases, and / or sugars. In some embodiments, the provided single oligonucleotide of the provided compositions has no modification on the sugar. In some embodiments, the provided single oligonucleotide of the provided compositions has no modification on the 2'-position of the sugar (i.e., the two groups at the 2'-position are either -H / -H or -H / -OH). In some embodiments, the provided single oligonucleotide of the provided compositions has no 2'-MOE modifications.

[0303] In some embodiments, the 2'-modification is -OL- or -L-, which links the 2'-carbon of the sugar moiety to another carbon of the sugar moiety. In some embodiments, the 2'-modification is -OL- or -L-, which links the 2'-carbon of the sugar moiety to the 4'-carbon of the sugar moiety. In some embodiments, the 2'-modification is S-cEt. In some embodiments, the modified sugar moiety is an LNA moiety.

[0304] In some embodiments, the 2'-modification is -F. In some embodiments, the 2'-modification is FANA. In some embodiments, the 2'-modification is FRNA.

[0305] In some embodiments, the sugar modification is a 5'-modification, such as R-5'-Me, S-5'-Me, and the like.

[0306] In some embodiments, the sugar modification changes the size of the sugar ring, hi some embodiments, the sugar modification is a sugar moiety in FHNA.

[0307] In some embodiments, sugar modifications replace the sugar moiety with another cyclic or acyclic moiety, examples of such moieties are widely known in the art and include, but are not limited to, those used in morpholinos (optionally with their phosphorodiamidate linkages), glycol nucleic acids, and the like.

[0308] In some embodiments, the ssRNAi agent is or comprises a PNPLA3 oligonucleotide selected from the group consisting of any ssRNAi in any format described herein in Figure 1 or elsewhere. One of skill in the art will understand upon reading this specification that the present disclosure does not expressly exclude the possibility that any of the oligonucleotides described herein designated as ssRNAi agents may additionally or alternatively function via another mechanism (e.g., as an antisense oligonucleotide, mediate knockdown via an RNase H mechanism, sterically hinder translation, or any other biochemical mechanism).

[0309] In some embodiments, the antisense oligonucleotide (ASO) is or comprises a PNPLA3 oligonucleotide selected from the group consisting of any oligonucleotide in any format described in Figure 2. Those skilled in the art will understand upon reading this specification that the present disclosure does not expressly exclude the possibility that any of the oligonucleotides described herein designated as antisense oligonucleotides (ASOs) may additionally or alternatively function through another mechanism (e.g., as ssRNAi utilizing RISC). The present disclosure also specifies that various ASOs may function through different mechanisms (e.g., those that utilize RNase H, those that sterically block translation or other post-transcriptional processes, those that change the conformation of the target nucleic acid, etc.).

[0310] In some embodiments, the hybrid oligonucleotide is or comprises a PNPLA3 oligonucleotide selected from the group consisting of WV-2111, WV-2113, WV-2114, WV-2148, WV-2149, WV-2152, WV-2153, WV-2156, WV-2157, WV-2387, WV-3069, WV-7523, WV-7524, WV-7525, WV-7526, WV-7527, WV-7528, and any oligonucleotide of any of formats S40-S42 of FIG. 1L, or any oligonucleotide of any of formats 30-32, formats 66-69, or formats 101-103 of FIG. 1. Those of skill in the art will understand upon reading this specification that the present disclosure does not expressly exclude the possibility that any of the oligonucleotides described herein that are designated as hybrid oligonucleotides may additionally or alternatively function via another mechanism (e.g., as an antisense oligonucleotide, mediate knockdown via an RNase H mechanism, sterically hinder translation, or any other biochemical mechanism).

[0311] Chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or interfering with mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the provided oligonucleotides are chiral controlled.

[0312] The present disclosure provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high crude purity or high diastereomeric purity. In some embodiments, the present disclosure provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high crude purity. In some embodiments, the present disclosure provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high diastereomeric purity.

[0313] In some embodiments, the single-stranded RNAi agent is a substantially pure preparation of a PNPLA3 oligonucleotide type, in that if oligonucleotides that are not of a particular oligonucleotide type are present in the composition, such oligonucleotides are impurities resulting from the preparation process of that oligonucleotide type (optionally after a particular purification procedure).

[0314] In some embodiments, the disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with respect to the chiral binding phosphorus. In some embodiments, the disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with the structure of formula I. In some embodiments, the disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with respect to the chiral binding phosphorus and comprising one or more phosphodiester linkages. In some embodiments, the disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with the structure of formula I and one or more phosphodiester linkages. In some embodiments, the disclosure provides oligonucleotides comprising one or more diastereomerically pure internucleotide linkages with the structure of formula Ic and one or more phosphodiester linkages. In some embodiments, such oligonucleotides are prepared by using stereoselective oligonucleotide synthesis as described in the present application to form pre-designed diastereomerically pure internucleotide linkages with respect to the chiral binding phosphorus. Exemplary internucleotide linkages include those with the structure of formula I, and exemplary internucleotide linkages are further described below.

[0315] In some embodiments, the present disclosure provides chiral controlled oligonucleotides, wherein at least two of the individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P-modifications.

[0316] Internucleotide bond In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or interfering with mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the provided oligonucleotides include any internucleotide linkage described herein or known in the art.

[0317] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown may contain any internucleotide linkage described herein or known in the art.

[0318] A non-limiting example of an internucleotide bond or an unmodified internucleotide bond is a phosphodiester, and a non-limiting example of a modified internucleotide bond is one in which one or more oxygens of a phosphodiester are replaced by, for example, sulfur (as found in phosphorothioates), H, alkyl, or another moiety or element that is not oxygen. A non-limiting example of an internucleotide bond is a moiety that does not contain phosphorus but serves to link two sugars. A non-limiting example of an internucleotide bond is a moiety that does not contain phosphorus but serves to link two sugars in the backbone of a PNPLA3 oligonucleotide. Non-limiting examples of nucleotides, modified nucleotides, nucleotide analogs, internucleotide bonds, modified internucleotide bonds, bases, modified bases, and base analogs, sugars, modified sugars, and sugar analogs, as well as nucleosides, modified nucleosides, and nucleoside analogs are further disclosed herein.

[0319] In certain embodiments, the internucleotide linkage has the structure of Formula I: [ka] wherein each variable is as defined and described below. In some embodiments, the linkage of formula I is chiral. In some embodiments, the disclosure provides chiral controlled oligonucleotides comprising one or more modified internucleotide linkages of formula I. In some embodiments, the disclosure provides chiral controlled oligonucleotides comprising one or more modified internucleotide linkages of formula I, and wherein each internucleotide linkage of formula I within the oligonucleotide has a P-modification that is different from each other. In some embodiments, the disclosure provides chiral controlled oligonucleotides comprising one or more modified internucleotide linkages of formula I, and wherein each internucleotide linkage of formula I within the oligonucleotide has a -XLR that is different from each other. 1In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising one or more modified internucleotide linkages of formula I, where each individual internucleotide linkage of formula I within the oligonucleotide has a different X from each other. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising one or more modified internucleotide linkages of formula I, where each individual internucleotide linkage of formula I within the oligonucleotide has a different -LR 1 In some embodiments, the chiral controlled oligonucleotide is a PNPLA3 oligonucleotide in a provided composition that is of a particular oligonucleotide type. In some embodiments, the chiral controlled oligonucleotide is a PNPLA3 oligonucleotide in a provided composition that has a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern. In some embodiments, the chiral controlled oligonucleotide is a PNPLA3 oligonucleotide in a chiral controlled composition that is a composition of a particular oligonucleotide type, and the chiral controlled oligonucleotide is an oligonucleotide of that type. In some embodiments, the chiral controlled oligonucleotide is a PNPLA3 oligonucleotide in a provided composition that includes a predetermined level of a plurality of oligonucleotides, and the plurality of oligonucleotides share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers, and the chiral controlled oligonucleotides share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0320] In some embodiments, the chiral controlled oligonucleotides contain different internucleotide phosphorus linkages.

[0321] In some embodiments, the phosphorothioate triester linkage contains, e.g., is used to control the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester linkage does not include a chiral auxiliary. In some embodiments, the phosphorothioate triester linkage is intentionally maintained until and / or during administration to a subject.

[0322] In some embodiments, the chiral controlled oligonucleotide is attached to a solid support. In some embodiments, the chiral controlled oligonucleotide is cleaved from the solid support.

[0323] In some embodiments, the chiral controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive modified internucleotide linkages, hi some embodiments, the chiral controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive phosphorothioate triester internucleotide linkages.

[0324] In some embodiments, the disclosure provides compositions consisting of or including a plurality of provided oligonucleotides (e.g., chiral controlled oligonucleotide compositions). In some embodiments, such provided oligonucleotides are all of the same type, i.e., all have the same base sequence, backbone linkage pattern (i.e., the pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., the pattern of bond phosphorus stereochemistry (Rp / Sp)), and backbone phosphorus modification pattern (e.g., the "-XLR" of Formula I disclosed herein). 1 In some embodiments, all of the oligonucleotides of the same type are identical. In many embodiments, however, provided compositions include multiple oligonucleotide types, typically in predetermined relative amounts.

[0325] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may contain any internucleotide linkage described herein or known in the art. In some embodiments, the moiety that binds ASPGR (e.g., the GalNAc moiety) is any GalNAc or variant or modification thereof described herein or known in the art. In some embodiments, the PNPLA3 oligonucleotides, the PNPLA3 oligonucleotides that induce RNA interference, the PNPLA3 oligonucleotides that induce RNase H-mediated knockdown, or the PNPLA3 oligonucleotides that induce both RNA interference and RNase H-mediated knockdown may include any internucleotide linkage described herein or known in the art in combination with any other structural elements or modifications described herein, including but not limited to, base sequence or portions thereof, sugars, bases (nucleobases), stereochemistry or patterns thereof, additional chemical moieties (including but not limited to targeting moieties, lipid moieties, carbohydrate moieties, etc.), seed region, post-seed region, 5' terminal structure, 5' terminal region, 5' nucleotide moieties, 3' terminal region, 3' terminal dinucleotide, 3' terminal cap, length, additional chemical moieties (including but not limited to targeting moieties, lipid moieties, GalNAc, etc.), format or any structural elements thereof, and / or any other structural elements or modifications described herein. In some embodiments, the disclosure relates to multimers of any such oligonucleotides.

[0326] In some embodiments, the chiral controlled oligonucleotide comprises one or more modified internucleotide phosphorus linkages, e.g., phosphorothioate or phosphorothioate triester linkages.

[0327] In some embodiments, the modified internucleotide linkage is phosphorothioate. In some embodiments, the modified internucleotide linkage is selected from, for example, those described in: US20110294124, US20120316224, US20140194610, US20150211006, US20150197540, WO2015107425, PCT / US2016 / 043542, and PCT / US2016 / 043598, Whittaker et al. 2008 Tetrahedron Letters 49:6984-6987.

[0328] Non-limiting examples of internucleotide linkages include those reported in the art, including, but not limited to, those described in any of the following: Gryaznov, S.; Chen, J.-KJ Am. Chem. Soc. 1994, 116, 3143; Jones et al. J. Org. Chem. 1993, 58, 2983; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Lauritsen et al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem. Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Petersen et al. 2003 TRENDS Biotech.21:74-81, Schultz et al.1996 Nucleic Acids Res.24:2966, Ts'o et al.Ann.NYAcad.Sci.1988,507,220, and Vasseur et al.J.Am.Chem.Soc.1992,114,4006.

[0329] In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where one or more U's are replaced with T's. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where the sequence has more than 50% identity with the sequence of any oligonucleotide disclosed herein.

[0330] In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides having the sequence of any of the oligonucleotides disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising the sequence found in any of the oligonucleotides disclosed herein, wherein the oligonucleotide has a pattern of backbone linkages, a pattern of backbone chiral centers, and / or a pattern of backbone phosphorus modifications as described herein.

[0331] In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where one or more Ts are replaced with Us. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where the sequence has greater than 50% identity to any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where the sequence has greater than 60% identity to any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where the sequence has greater than 70% identity to any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, where the sequence has greater than 80% identity to any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, which sequence has greater than 90% identity to any oligonucleotide sequence disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any oligonucleotide disclosed herein, which sequence has greater than 95% identity to any oligonucleotide sequence disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides chiral controlled oligonucleotides having a sequence of any oligonucleotide disclosed herein.

[0332] In some embodiments, the present disclosure provides chiral controlled oligonucleotides comprising a sequence found in any of the oligonucleotides disclosed herein, wherein at least Another internucleotide bond is [ka] In some embodiments, the disclosure provides a chiral controlled oligonucleotide comprising the sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage is: [ka] In some embodiments, the present disclosure provides chiral controlled oligonucleotides having a sequence found in any of the oligonucleotides disclosed herein, wherein at least one internucleotide linkage is [ka] In some embodiments, the disclosure provides chiral controlled oligonucleotides having the sequence of any of the oligonucleotides disclosed herein, wherein each internucleotide linkage is: [ka] In some embodiments, the present disclosure provides a chiral controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein each cytosine is optionally and independently replaced by 5-methylcytosine. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein at least one cytosine is optionally and independently replaced by 5-methylcytosine. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein each cytosine is optionally and independently replaced by 5-methylcytosine.

[0333] Bases (nucleobases) In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or interfering with mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the provided oligonucleotides comprise any nucleobase described herein or known in the art.

[0334] In some embodiments, the nucleobase present in the provided oligonucleotide is a natural nucleobase or a modified nucleobase derived from a natural nucleobase. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, each of which has its amino group protected by an acyl protecting group, and other modified nucleobases, such as fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, 8-substituted purine, xanthine, or hypoxanthine (the last two being natural decomposition products). Examples of modified nucleobases are described in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313. In some embodiments, the modified nucleobase is a substituted uracil, thymine, adenine, cytosine or guanine. In some embodiments, the modified nucleobase is a functional replacement, for example, for hydrogen bonding and / or base pairing, of uracil, thymine, adenine, cytosine or guanine. In some embodiments, the nucleobase is optionally a substituted uracil, thymine, adenine, cytosine, 5-methylcytosine or guanine. In some embodiments, the nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine or guanine.

[0335] In some embodiments, the modified base is an optionally substituted adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the modified nucleobase is independently an adenine, cytosine, guanine, thymine, or uracil modified by one or more modifications, which modify: (1) the nucleobase is modified by one or more optionally substituted groups independently selected from acyl, halogen, amino, azido, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof; (2) one or more atoms of the nucleobase are independently replaced with a different atom selected from carbon, nitrogen, or sulfur; (3) one or more double bonds in the nucleobase are independently hydrogenated, or (4) One or more aryl or heteroaryl rings are independently inserted into a nucleobase.

[0336] A variety of additional nucleobases have been described in the art.

[0337] Sugars In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or interfering with mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the provided oligonucleotides comprise any sugar described herein or known in the art.

[0338] In some embodiments, provided oligonucleotides capable of inducing single-stranded RNA interference contain one or more modified sugar moieties, in addition to natural sugar moieties.

[0339] The most common natural nucleotides are composed of a ribose sugar linked to the nucleobases adenosine (A), cytosine (C), guanine (G), and thymine (T) or uracil (U). Also contemplated are modified nucleotides in which the phosphate group or linked phosphate in the nucleotide can be attached to various positions of the sugar or modified sugar. As a non-limiting example, the phosphate group or linked phosphorus can be attached to the 2", 3", 4" or 5" hydroxyl moiety of the sugar or modified sugar. Nucleotides incorporating modified nucleobases as described herein are also contemplated in this context. In some embodiments, nucleotides or modified nucleotides that include an unprotected -OH moiety are used in accordance with the disclosed methods.

[0340] In some embodiments, PNPLA3 oligonucleotides, PNPLA3 oligonucleotides that induce RNA interference, PNPLA3 oligonucleotides that induce RNase H-mediated knockdown, or PNPLA3 oligonucleotides that induce both RNA interference and RNase H-mediated knockdown may contain any base (nucleobase), modified base or base analog described herein or known in the art. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any base described herein or known in the art in combination with any other structural elements or modifications described herein, including, but not limited to, a base sequence or portion thereof, a sugar; an internucleotide linkage; a stereochemistry or pattern thereof; an additional chemical moiety, including, but not limited to, a targeting moiety, a lipid moiety, a GalNAc moiety, etc.; a 5'-terminal structure; a 5'-terminal region; a 5'-nucleotide moiety; a seed region; a post-seed region; a 3'-terminal region; a 3'-terminal dinucleotide; a 3'-terminal cap; a sugar, base or internucleotide linkage modification pattern; a format or any structural elements thereof, and / or any other structural elements or modifications described herein, and in some embodiments, the present disclosure relates to multimers of any such oligonucleotides.

[0341] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may contain any sugar.

[0342] A variety of additional sugars have been described in the art.

[0343] Sequence of PNPLA3 oligonucleotide In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or disrupting mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the oligonucleotides provided can include any base sequence described herein or a portion thereof, where the portion is a span of at least 15 contiguous bases or a span of at least 15 contiguous bases with 1-5 mismatches.

[0344] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any of the base sequences described herein. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any of the base sequences described herein or a portion thereof. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any of the base sequences described herein or a portion thereof, where the portion is a span of 15 consecutive bases or a span of 15 consecutive bases with 1-5 mismatches.

[0345] The sequence of the single stranded RNAi agent has sufficient length and identity to the transcript target to mediate target-specific RNA interference. In some embodiments, the RNAi agent is complementary to a portion of the transcript target sequence.

[0346] The base sequence of single-stranded RNAi agent is complementary to the base sequence of target transcript.As used herein, "target transcript sequence", "target sequence", "target gene" and the like refer to the continuous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of gene, for example, target gene, including the mRNA that is the product of RNA processing of primary transcript.

[0347] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between a strand of a single-stranded RNAi agent and a target sequence, or between an antisense oligonucleotide and a target sequence, as understood from the context in which they are used. A strand of a single-stranded RNAi agent or antisense oligonucleotide or other oligonucleotide is complementary to a strand of a target sequence if each base of the single-stranded RNAi agent, antisense oligonucleotide, or other oligonucleotide, when maximally aligned, can base pair with consecutive bases on the target strand. As a non-limiting example, if a target sequence has, for example, a base sequence of 5'-GCAUAGCGAGCGAGGGAAAAC-3', then a PNPLA3 oligonucleotide having a base sequence of 5'GUUUUCCCUCGCUCGCUAUGC-3' is complementary or fully complementary to the target sequence. Of course, it should be noted that the substitution of U for T or vice versa does not change the amount of complementarity.

[0348] As used herein, a polynucleotide that is "substantially complementary" to a target sequence is largely or nearly complementary, but not 100% complementary. In some embodiments, a substantially complementary sequence (e.g., a single-stranded RNAi agent or a strand of an antisense oligonucleotide) has one, two, three, four or five mismatches compared to a sequence that is 100% complementary to a target sequence. In the case of a single-stranded RNAi agent, the present disclosure notes that in many cases, the 5'-terminal nucleotide (N1) has a mismatch compared to the complement of the target sequence. Similarly, in a single-stranded RNAi agent, the 3'-terminal dinucleotide, if present, may be mismatched compared to the complement of the target sequence. As a non-limiting example, if a target sequence has, for example, a base sequence of 5'-GCAUAGCGAGCGAGGGAAAAC-3', then a single-stranded RNAi agent having a base sequence of 5'TUUUUCCCUCGCUCGCUAUTU-3' is substantially complementary to the target sequence.

[0349] The present disclosure provides a variety of single stranded RNAi agents and antisense oligonucleotides and other oligonucleotides, each having a defined base sequence in Table 1A and elsewhere. In some embodiments, the present disclosure encompasses any oligonucleotide having a base sequence that is, includes, or includes a portion of any of the various single stranded RNAi agents, antisense oligonucleotides and other oligonucleotides disclosed herein. In some embodiments, the disclosure encompasses any oligonucleotide having a base sequence that is, includes, or includes a portion of any of the various single stranded RNAi agents, antisense oligonucleotides, and other oligonucleotides disclosed herein, which has any chemical modification, stereochemistry, format, structural feature described herein (e.g., if the oligonucleotide is a single stranded RNAi agent, a 5' terminal structure, a 5' terminal region, a 5' nucleotide portion, a seed region, a post-seed region, a 3' terminal region, a 3' terminal dinucleotide, a 3' terminal cap, or any structure, pattern, or portion thereof), and / or any other modification (e.g., conjugation to another moiety, e.g., a targeting moiety, a carbohydrate moiety, a GalNAc moiety, a lipid moiety, etc.; and / or multimerization).

[0350] In some embodiments, the PNPLA3 oligonucleotide has a sequence that is, includes, or comprises a portion of any of the oligonucleotides disclosed herein.

[0351] In some embodiments, the disclosure discloses PNPLA3 oligonucleotides of sequences listed herein. In some embodiments, the disclosure discloses PNPLA3 oligonucleotides of sequences listed herein, wherein the oligonucleotides have the ability to induce a decrease in expression and / or levels of a target gene or its gene product. In some embodiments, the PNPLA3 oligonucleotides of sequences listed herein are single-stranded RNAi agents. In some embodiments, the PNPLA3 oligonucleotides of sequences listed herein are antisense oligonucleotides that induce RNase H-mediated knockdown. In some embodiments, the PNPLA3 oligonucleotides of sequences listed herein induce both RNA interference and RNase H-mediated knockdown. In some embodiments, the PNPLA3 oligonucleotides of sequences listed herein include any structure described herein (e.g., any 5'-end structure, 5'-end region, 5'-nucleotide portion, seed region, post-seed region, 3'-end dinucleotide, 3'-end cap, or any portion of any of these structures, or any chemical, stereochemical, additional chemical moiety, etc.). When the oligonucleotide is a ssRNAi agent, the sequence may be preceded by a T (non-limiting examples include 2'-deoxy T, 5'-(R)-Me OH T, 5'-(R)-Me PO T, 5'-(R)-Me PS T, 5'-(R)-Me PH T, 5'-(S)-Me OH T, 5'-(S)-Me PO T, 5'-(S)-Me PS T or 5'-(S)-PH T) or the first nucleobase may be replaced by a T (non-limiting examples include 2'-deoxy T, 5'-(R)-Me OH T, 5'-(R)-Me PO T, 5'-(R)-Me PS T, 5'-(R)-Me PH T, 5'-(S)-Me OH T, 5'-(S)-Me PO T, 5'-(S)-Me PS T or 5'-(S)-PH T). T), and / or a 3' terminal dinucleotide (for example, as non-limiting examples: TT, UU, TU, etc.). In various sequences, U can be replaced with T or vice versa, or sequences can include combinations of U and T.In some embodiments, the PNPLA3 oligonucleotide has a total nucleotide length of about 49, about 45, about 40, about 30, about 35, about 25, about 23 or less. In some embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 total nucleotides with 0-3 mismatches. In some embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 total nucleotides with 0-3 mismatches, where a span with 0 mismatches is complementary and a span with 1 or more mismatches is a non-limiting example of substantial complementarity. In some embodiments where the sequences listed above begin with a U at the 5' end, the U can be deleted and / or replaced with another base. In some embodiments, the present disclosure encompasses any oligonucleotide that has a sequence that is, includes, or includes a portion of the sequence of any oligonucleotide disclosed herein and has a format or portion of a format disclosed herein.

[0352] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any of the base sequences described herein. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any of the base sequences described herein or a portion thereof. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any of the base sequences described herein or a portion thereof, where the portion is a span of 15 consecutive bases or a span of 15 consecutive bases with 1-5 mismatches.In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may comprise any base sequence described herein or a portion thereof in combination with any other structural elements or modifications described herein, including, but not limited to, sugars, bases; internucleotide linkages; stereochemistry or patterns thereof; additional chemical moieties, including, but not limited to, targeting moieties, lipid moieties, GalNAc moieties, etc.; 5'-terminal structures; 5'-terminal regions; 5'-nucleotide moieties; seed regions; post-seed regions; 3'-terminal regions; 3'-terminal dinucleotides; 3'-terminal caps; sugar, base or internucleotide linkage modification patterns; formats or any structural elements thereof, and / or any other structural elements or modifications described herein, and in some embodiments, the present disclosure relates to multimers of any such oligonucleotides.

[0353] Non-limiting examples of oligonucleotides having various base sequences are listed below in Table 1A. Table 1A. Oligonucleotides. PNPLA3 oligonucleotide. [Table 1] TIFF2024099745000008.tif229162TIFF2024099745000009.tif223162 TIFF2024099745000010.tif229162TIFF2024099745000011.tif223162TIFF2024099745000012.tif229162TIFF2024099745000013.tif225162TIFF2024099745000014.tif229162TIFF2024099745000015.tif225162TIFF2024099745000016.tif229162TIFF2024099745000017.tif225162TIFF2024099745000018.tif229162TIFF2024099745000019.tif223162TIFF2024099745000020.tif223162TIFF2024099745000021.tif229162TIFF2024099745000022.tif223162TIFF2024099745000023.tif229162TIFF2024099745000024.tif225162TIFF2024099745000025.tif229162TIFF2024099745000026.tif225162TIFF2024099745000027.tif229162TIFF2024099745000028.tif223162TIFF2024099745000029.tif229162TIFF2024099745000030.tif225162TIFF2024099745000031.tif229162TIFF2024099745000032.tif225162TIFF2024099745000033.tif229162TIFF2024099745000034.tif223162TIFF2024099745000035.tif229162TIFF2024099745000036.tif223162TIFF2024099745000037.tif229162TIFF2024099745000038.tif225162TIFF2024099745000039.tif229162TIFF2024099745000040.tif225162TIFF2024099745000041.tif229162TIFF2024099745000042.tif223162TIFF2024099745000043.tif229162TIFF2024099745000044.tif225162TIFF2024099745000045.tif229162TIFF2024099745000046.tif225162TIFF2024099745000047.tif229162TIFF2024099745000048.tif223162TIFF2024099745000049.tif229162TIFF2024099745000050.tif225162TIFF2024099745000051.tif229162TIFF2024099745000052.tif225162TIFF2024099745000053.tif229162TIFF2024099745000054.tif223162TIFF2024099745000055.tif229162TIFF2024099745000056.tif223162TIFF2024099745000057.tif229162TIFF2024099745000058.tif225162TIFF2024099745000059.tif229162TIFF2024099745000060.tif225162TIFF2024099745000061.tif229162TIFF2024099745000062.tif225162TIFF2024099745000063.tif229162TIFF2024099745000064.tif225162TIFF2024099745000065.tif229162TIFF2024099745000066.tif225162TIFF2024099745000067.tif229162TIFF2024099745000068.tif225162TIFF2024099745000069.tif229162TIFF2024099745000070.tif225162TIFF2024099745000071.tif229162TIFF2024099745000072.tif223162TIFF2024099745000073.tif229162TIFF2024099745000074.tif223162TIFF2024099745000075.tif229162TIFF2024099745000076.tif229162TIFF2024099745000077.tif224162TIFF2024099745 000078.tif222162TIFF2024099745000079.tif226162TIFF2024099745000080.tif220162TIFF2024 099745000081.tif229162TIFF2024099745000082.tif223162TIFF2024099745000083.tif229162TI FF2024099745000084.tif227162TIFF2024099745000085.tif226162TIFF2024099745000086.tif22 9162TIFF2024099745000087.tif223162TIFF2024099745000088.tif229162TIFF202409974500008 9.tif223162TIFF2024099745000090.tif229162TIFF2024099745000091.tif223162TIFF202409974 5000092.tif224162TIFF2024099745000093.tif225162TIFF2024099745000094.tif226162TIFF202 4099745000095.tif227162TIFF2024099745000096.tif229162TIFF2024099745000097.tif223162.

[0354] In some embodiments, the oligonucleotide compositions provided are single stranded RNAi agents listed in Table 1A or described elsewhere herein. In some embodiments, exemplary properties of the oligonucleotides provided have been demonstrated.

[0355] In some embodiments, the oligonucleotide provided has a structure of any of the formats illustrated in FIG.

[0356] This disclosure provides many non-limiting examples of oligonucleotides (e.g., single-stranded RNAi agents) that have the ability to mediate single-stranded RNA interference. Indeed, experimental data (not shown) have shown that various assumed single-stranded RNAi agents have the ability to mediate RNA interference. In some experiments, an in vitro Ago-2 assay is used, including the use of RNA test substrate WV-2372 (targeting a different gene APOC3). The band representing the RNA test substrate does not exist in the presence of oligonucleotides WV-1308 and WV-2420, indicating that these oligonucleotides are single-stranded RNAi agents that have the ability to mediate RNA interference. The remaining lanes are controls: substrate in the absence of negative control ASO WV-2134, substrate in the presence of negative control ASO WV-2134 (no RNA interference), substrate in the absence of test oligonucleotide WV-1308, substrate in the absence of test oligonucleotide WV-2420, substrate alone, WV-2134 added without substrate, and WV-1308 added without substrate. An in vitro Ago-2 assay was also performed using APOC3 mRNA as a test substrate in a 3'RACE assay in Hep3B cells (data not shown). A cleavage product of APOC3 mRNA was detected in the presence of test oligonucleotide WV-3021, which corresponded to cleavage of the mRNA at a site corresponding to the cleavage of WV-3021 between positions 10 and 11. Artificial cleavage products were also detected. Experimental data (not shown) showed that the dual mechanism oligonucleotide WV-2111 has the ability to mediate knockdown by both RNase H and RNA interference. In the experiment, several oligonucleotides have the ability to mediate RNA interference. The RNA test substrate was WV-2372. The experiment showed that the RNA test substrate disappeared in the presence of test oligonucleotides WV-1308, WV-2114, WV-2386, and WV-2387. This indicates that all these oligonucleotides have the ability to function as single-stranded RNAi agents that mediate RNA interference. The remaining lanes are controls.Thus, the experiment shows that oligonucleotides WV-1308, WV-2114, WV-2386 and WV-2387 can all mediate RNA interference. Thus, the experiment shows that some single-stranded RNAi agents (e.g., WV-1308, WV-2420, WV-3021, WV-2111, WV-2114, WV-2386 and WV-2387) can mediate RNA interference. The present disclosure provides many non-limiting examples of oligonucleotides (e.g., single-stranded RNAi agents) that have various sequences, formats, modifications, 5'-terminal regions, seed regions, post-seed regions and 3'-terminal regions and have the ability to mediate single-stranded RNA interference.

[0357] Oligonucleotide format In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of the target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of the target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of the target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of the target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of the target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or interfering with mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the provided oligonucleotides may have any form or part or structural element thereof described herein or known in the art.

[0358] In some embodiments, the PNPLA3 oligonucleotide can have any format or structural element thereof described herein or known in the art.

[0359] In some embodiments, a PNPLA3 oligonucleotide capable of inducing a decrease in the expression and / or level of a target gene or its gene product may have any format or structural element thereof described herein or known in the art.

[0360] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown may have any format or structural elements thereof described herein or known in the art.

[0361] Additional non-limiting examples of various ssRNAi formats are embodied by the various single stranded RNAi agents described herein.

[0362] In some embodiments, the single stranded RNAi provided comprises a 5' end represented by any of the single stranded RNAi formats shown in FIG. 1 or any 5' end of any single stranded RNAi agent or single stranded RNAi format described herein.

[0363] In some embodiments, the single stranded RNAi provided comprises a 5' end structure or region represented by any of the single stranded RNAi formats shown in FIG. 1 or any of the single stranded RNAi agents or single stranded RNAi formats described herein.

[0364] In some embodiments, the single stranded RNAi provided comprises a 5' nucleotide represented by any of the single stranded RNAi formats shown in FIG. 1 or any 5' nucleotide of any single stranded RNAi agent or single stranded RNAi format described herein.

[0365] In some embodiments, the single stranded RNAi provided comprises a 5' nucleoside represented by any of the single stranded RNAi formats shown in FIG. 1 or any 5' nucleoside of any single stranded RNAi agent or single stranded RNAi format described herein.

[0366] In some embodiments, the single stranded RNAi provided comprises a seed region, as represented by any of the single stranded RNAi formats shown in FIG. 1 or any of the seed regions of any of the single stranded RNAi agents or single stranded RNAi formats described herein.

[0367] In some embodiments, the single stranded RNAi provided comprises a post-seed region, as represented by any of the single stranded RNAi formats shown in FIG. 1 or any of the post-seed regions of any single stranded RNAi agent or single stranded RNAi format described herein.

[0368] In some embodiments, the single stranded RNAi provided comprises a post-seed region, or a component thereof, represented by any of the single stranded RNAi formats shown in FIG. 1 or any post-seed region, or component thereof, of any single stranded RNAi agent or single stranded RNAi format described herein.

[0369] In some embodiments, the provided single stranded RNAi comprises a 3' terminal dinucleotide represented by any of the single stranded RNAi formats shown in FIG. 1 or any of the 3' terminal dinucleotides of any single stranded RNAi agent or single stranded RNAi format described herein.

[0370] In some embodiments, the single stranded RNAi provided comprises a seed region having a pattern of internucleotide linkages represented by the pattern of internucleotide linkages of any of the single stranded RNAi formats shown in FIG. 1 or any seed region of any single stranded RNAi agent or single stranded RNAi format described herein.

[0371] In some embodiments, the provided single stranded RNAi comprises a post-seed region having a pattern of internucleotide linkages represented by the pattern of internucleotide linkages of any post-seed region of any single stranded RNAi format shown in FIG. 1 or any single stranded RNAi agent or single stranded RNAi format described herein.

[0372] In some embodiments, the single stranded RNAi provided comprises a post-seed region or component thereof having a pattern of internucleotide linkages represented by the pattern of internucleotide linkages of any post-seed region or component thereof of any single stranded RNAi format shown in FIG. 1 or any single stranded RNAi agent or single stranded RNAi format described herein.

[0373] In some embodiments, the provided single stranded RNAi comprises a 3' terminal dinucleotide having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any 3' terminal dinucleotide of any single stranded RNAi format shown in FIG. 1 or any single stranded RNAi agent or single stranded RNAi format described herein.

[0374] In some embodiments, the provided single stranded RNAi comprises a seed region having a chemical modification pattern represented by the chemical modification pattern of any of the single stranded RNAi formats shown in FIG. 1 or any of the seed regions of any single stranded RNAi agent or single stranded RNAi format described herein.

[0375] In some embodiments, the provided single stranded RNAi comprises a post-seed region having a chemical modification pattern represented by the chemical modification pattern of any of the post-seed regions of any of the single stranded RNAi formats shown in FIG. 1 or any of the single stranded RNAi agents or single stranded RNAi formats described herein.

[0376] In some embodiments, the provided single stranded RNAi comprises a post-seed region or component thereof having a chemical modification pattern represented by the chemical modification pattern of any post-seed region or component thereof of any single stranded RNAi format shown in FIG. 1 or any single stranded RNAi agent or single stranded RNAi format described herein.

[0377] In some embodiments, the provided single stranded RNAi comprises a 3' terminal dinucleotide having a chemical modification pattern represented by the chemical modification pattern of any 3' terminal dinucleotide of any of the single stranded RNAi formats shown in FIG. 1 or any of the single stranded RNAi agents or single stranded RNAi formats described herein.

[0378] In some embodiments, the provided single stranded RNAi comprises a chemical modification represented by any of the single stranded RNAi formats shown in FIG. 1 or any chemical modification of any of the single stranded RNAi agents or single stranded RNAi formats described herein.

[0379] In some embodiments, the provided single stranded RNAi comprises a chemical modification represented by any chemical modification of any single stranded RNAi format shown in FIG. 1 or described herein, where the chemical modification is a phosphate, a linker, or conjugation of a moiety that includes a targeting moiety.

[0380] In some embodiments, the provided single-stranded RNAi comprises a chemical modification represented by any chemical modification of any single-stranded RNAi format shown in Figure 1 or described herein, where the chemical modification is a phosphate, a linker, or a conjugation of a moiety that comprises a targeting moiety, and the targeting moiety comprises a GalNAc moiety. In some embodiments, the GalNAc is a protected GalNAc or a deprotected GalNAc.

[0381] In some embodiments, the PNPLA3 oligonucleotide has the ability to reduce the expression, activity and / or level of a target gene and / or its gene product, and has any of the oligonucleotide formats described herein. In some embodiments, the PNPLA3 oligonucleotide has the ability to reduce the expression, activity and / or level of a target gene and / or its gene product through an RNase H-mediated mechanism or a mechanism involving steric hindrance of translation, and has any of the oligonucleotide formats described herein. In some embodiments, the PNPLA3 oligonucleotide has the ability to reduce the expression, activity and / or level of a target gene and / or its gene product through an RNase H-mediated mechanism or a mechanism involving steric hindrance of translation, and has an asymmetric format. In some embodiments, the PNPLA3 oligonucleotide with an asymmetric format comprises a first wing, a core and a second wing, the core comprises a region of five or more consecutive 2'-deoxynucleotides capable of annealing to a target mRNA to form a structure recognized by RNase H, and the structures of the first and second wings are different. In some embodiments, the first and second wings differ in their pattern of 2'-modifications and / or internucleotide linkages, or the stereochemistry of the internucleotide linkages.

[0382] In some embodiments, the PNPLA3 oligonucleotide has the ability to decrease the expression, activity and / or level of a target gene and / or its gene product and comprises neutral internucleotide bonds (eg, a neutral backbone).

[0383] In some embodiments, the PNPLA3 oligonucleotide comprises a neutral backbone. In some embodiments, the PNPLA3 oligonucleotide comprises an internucleotide linkage that is or comprises a triazole, a neutral triazole, or an alkyne. In some embodiments, the nucleic acid (including but not limited to the PNPLA3 oligonucleotide) comprises an internucleotide linkage that comprises a triazole, a neutral triazole, or an alkyne, where the internucleotide linkage is stereocontrolled and is in the Rp or Sp configuration. In some embodiments, the triazole-containing internucleotide linkage is [ka] In some embodiments, the neutral triazole-containing internucleotide linkage has the formula: [ka] where X is O or S. In some embodiments, the alkyne-containing internucleotide linkage has the formula: [ka] where X is O or S. In some embodiments, the internucleotide linkage comprises a cyclic guanidine. In some embodiments, the internucleotide linkage comprises a cyclic guanidine. [ka] In some embodiments, the neutral internucleotide linkage or the internucleotide linkage containing a cyclic guanidine is stereochemically controlled. In some embodiments, the neutral internucleotide linkage improves the activity, delivery and / or stability of the PNPLA3 oligonucleotide and / or the ability of the PNPLA3 oligonucleotide to undergo endosomal escape.

[0384] As will be appreciated by those skilled in the art, in some cases, [ka] can be used to indicate the binding position ( [ka] As), in some cases, [ka] may be used to indicate a stereoirregular bond.

[0385] Length of PNPLA3 oligonucleotide In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC, including but not limited to RNase H-mediated knockdown of gene expression or steric hindrance. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product by steric blocking of translation after annealing to the target gene mRNA, and / or altering or disrupting mRNA splicing, and / or exon inclusion or exon exclusion. In some embodiments, the oligonucleotides provided can be of any length, where the length of the PNPLA3 oligonucleotide is such that the oligonucleotide has the ability to induce a decrease in expression and / or levels of a target gene or its gene product.

[0386] In some embodiments, the length of the PNPLA3 oligonucleotide is such that the oligonucleotide has the ability to induce a decrease in the expression and / or levels of a target gene or its gene product.

[0387] In some embodiments, the length of RNAi agent is such that RNAi agent has the ability to induce the RNA interference of specific target transcript in a sequence-specific manner.In some embodiments, RNAi agent comprises a sufficient number of nucleic acid bases with sufficient identity to recognize target transcript.In some embodiments, RNAi agent is also suitable length to mediate RNAi interference.

[0388] The target portion of the sequence is at least long enough to serve as a substrate for iRNA-induced cleavage at or near the position. For example, the target sequence is generally about 9-36 nucleotides ("nt") in length, such as about 15-30 nucleotides in length, including all subranges therebetween. Examples of single-stranded RNAi agents of various lengths are shown in Table 1A.

[0389] Figure 1 illustrates non-limiting examples of single stranded RNAi agents having lengths of 19 to 25. Single stranded RNAi agents having any of each of these lengths have been constructed and found to be capable of knocking down target genes. Thus, the single stranded RNAi agents provided can be any of a variety of different lengths.

[0390] Non-limiting examples of formats of ssRNAi agents that are 19 bases in length include formats 20-21 in FIG.

[0391] A non-limiting example of a format for a ssRNAi agent that is 20 bases in length includes format 19 in FIG.

[0392] The 5' end of the PNPLA3 oligonucleotide containing the single stranded RNAi agent In some embodiments, the structure of the 5' end of the PNPLA3 oligonucleotide is such that the oligonucleotide has the ability to induce a decrease in the expression and / or levels of a target gene or its gene product.

[0393] In some embodiments, the structure of the 5' end of the RNAi agent is such that the RNAi agent has the ability to induce RNA interference of a specific target transcript in a sequence-specific manner.

[0394] In some embodiments, the provided oligonucleotides may include any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art. In some embodiments, the provided oligonucleotides capable of inducing RNase H-mediated knockdown may include any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art. In some embodiments, the provided oligonucleotides capable of inducing RNA interference may include any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art. In some embodiments, the provided oligonucleotides capable of inducing RNA interference and RNase H-mediated knockdown may include any 5'-terminal region, 5'-terminal structure, 5'-terminal group, 5'-terminal nucleoside, or 5'-terminal nucleotide described herein or known in the art.

[0395] In particular, the present disclosure recognizes that the 5'-end structure of an oligonucleotide according to the present disclosure, optionally in combination with additional features, can provide unexpected advantages. In some embodiments, the present disclosure provides a 5'-end group (5'-HO-CH of ribose found in natural RNA (or deoxyribose found in natural DNA)) that can surprisingly improve one or more properties and / or activities (e.g., stability, activity, production costs, etc.) of an oligonucleotide. 2 - (corresponding to

[0396] In some embodiments, the 5'-OH group of the provided oligonucleotide is unmodified, i.e., exists as a free -OH. In some embodiments, the 5' terminal group is 5'-HO-CH 2 In particular, the present disclosure demonstrates that, despite reports in the literature that the presence of a 5' phosphate group is required for certain activities, e.g., RNAi activity, provided oligonucleotides having a free 5'-OH group are capable of achieving properties and / or activities (e.g., stability when used as ss-RNAi agents, RNAi activity, etc.) comparable to otherwise identical oligonucleotides that contain a 5' phosphate (or derivative thereof) group.

[0397] In some embodiments, the 5' terminal group does not contain a phosphorus atom. In some embodiments, the 5' terminal group does not contain a phosphate group, or a derivative or bioisoster thereof. In some embodiments, the 5' terminal group does not contain an acidic group. In some embodiments, the 5' terminal group does not contain a carboxyl group. In some embodiments, the 5' terminus does not contain a phosphorus atom or a carboxyl group. In some embodiments, the 5' terminal group is 5'-HO-CH 2 In particular, the present disclosure shows that provided oligonucleotides that lack a 5' phosphate or a derivative or biological equivalent thereof surprisingly have comparable activity to otherwise identical oligonucleotides that lack a 5' phosphate, e.g., the ability to achieve knockdown of mRNA levels of a target gene via the RNAi pathway.

[0398] In some embodiments, the 5' nucleoside unit (including the sugar moiety and nucleobase moiety, but not including the internucleotide linkage between the 5' nucleoside unit and the penultimate 5' nucleoside unit) of the provided oligonucleotide does not contain a phosphate group, or a derivative or biological equivalent thereof. In some embodiments, the 5' nucleoside unit does not contain a phosphorus atom. In some embodiments, the 5' nucleoside does not contain an acidic group. In some embodiments, the 5' nucleoside unit does not contain a -COOH group or a salt form thereof.

[0399] In some embodiments, the 5' terminal group is or includes a phosphate group, or a derivative or bioisoster thereof. In some embodiments, the 5' nucleoside unit includes a 5' group that is a phosphate group, or a derivative or bioisoster thereof. As will be recognized by those of skill in the art, many such groups are known in the art and can be utilized in accordance with the present disclosure.

[0400] In some embodiments, the 5' terminal group is -CH 2 -OP(O)(OH)-(OH) or a salt form thereof. In some embodiments, the 5' nucleoside unit provided is [ka] or a salt form thereof. In some embodiments, the 5' nucleoside unit provided has the structure: [ka] or a salt form thereof. In some embodiments, X is O. In some embodiments, X is S. In some embodiments, R E is -(R)-CH(CH 3 )-OP(O)(OH)-SH or a salt form thereof. In some embodiments, R E is -(R)-CH(CH 3 )-OP(O)(OH)-OH or a salt form thereof. In some embodiments, R E is -(S)-CH(CH 3)-OP(O)(OH)-SH or a salt form thereof. In some embodiments, R E is -(S)-CH(CH 3 )-OP(O)(OH)-OH or a salt form thereof. In some embodiments, the 5' nucleoside unit provided is [ka] or a salt form thereof. In some embodiments, the 5' nucleoside unit provided has the structure: [ka] or a salt form thereof.

[0401] As will be readily appreciated by those skilled in the art, a provided compound, e.g., an oligonucleotide, or a substructure thereof, e.g., the 5'-end structure of an oligonucleotide, an internucleotide bond, etc., may exist partially, or in some cases primarily, as one or more of its salt forms at a certain pH, e.g., physiological pH, due to, e.g., one or more acidic and / or basic moieties therein. In some embodiments, a provided 5' nucleoside unit may exist partially, or in some cases primarily, as one or more of its salt forms. For example, depending on the pH, [ka] teeth, [ka] or combinations thereof. Unless otherwise specified, when a compound or structure provided is recited, all salt forms are included.

[0402] In some embodiments, R E -LP(O)(XR) 2 or a salt form thereof. In some embodiments, R E -LP(O)(XR) 2or a salt form thereof, wherein each X is independently -O-, -S-, or a covalent bond. E -LP(O)(OR) 2 or a salt form thereof. In some embodiments, R E is -LP(O)(OR)(SR) or a salt form thereof. In some embodiments, R E is -LP(O)(OR)(R) or a salt form thereof. In some embodiments, L is a covalent bond or an optionally substituted divalent linear or branched C 1-6 aliphatic, where one or more methylene units are optionally and independently replaced with -O-, -S-, or -N(R')-. E -LR 5s Some implementations In this state, R E In some embodiments, R is -XLR. E teeth, [ka] In some embodiments, R E The X in the middle is -C(R) 2 In some embodiments, X is -. In some embodiments, X is -O-. In some embodiments, X is -S-. In some embodiments, X is -N(R)-. In some embodiments, L is an optionally substituted divalent or multivalent [ka] In some embodiments, L is an optionally substituted [ka] In some embodiments, L comprises a [ka] In some embodiments, R is independently -H, or C 1-10 Alkyl, C 1-10 Allyl and C 6-14aryl. In some embodiments, R is -H. In some embodiments, R E is optionally replaced [ka] In some embodiments, R E teeth, [ka] It is.

[0403] Many phosphate derivatives and / or bioisosteres, as well as 5' nucleoside units, have been described in the literature and can be utilized in accordance with the present disclosure, for example, several such structures are described, for example, in US2016-0194349, US2016-0186175, US20130323836, etc. In some embodiments, the 5' terminal group R E , or 5' nucleoside units are described, for example, in Allerson et al. 2005 J. Med. Chem. 48:901-04; Lima et al. 2012 Cell 150:883-894; Prakash et al. 2015 Nucl. Acids Res. 43:2993-3011; and / or Prakash et al. 2016 Bioorg. Med. Chem. Lett. 26:26:2817-2820 (e.g., T-VP, T-PO, etc.).

[0404] Bridged morpholino and cyclohexenyl nucleotides and nucleosides are described, for example, in U.S. Patent Application Publication No. 2016-0186175 and can be utilized in accordance with the present disclosure.

[0405] Exemplary embodiments of the variables are described broadly in this disclosure. For structures having two or more variables, unless otherwise specified, each variable can independently be any embodiment described herein.

[0406] In some embodiments, a PNPLA3 oligonucleotide capable of inducing a decrease in the expression and / or level of a target gene or its gene product may contain any 5' end described herein or known in the art.

[0407] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNA interference, or the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown may contain any 5' end described herein or known in the art.

[0408] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that induces RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that induces both RNA interference and RNase H-mediated knockdown, may contain any 5' end described herein or known in the art.

[0409] In some embodiments, the 5' end of a provided single stranded RNAi agent comprises a phosphorus-containing moiety (e.g., the 5' end comprises phosphorus). Non-limiting examples of ssRNAi formats in which the 5' end comprises a phosphorus-containing moiety include formats 1-15, 20-21, 23-31, 80-82, 92-95, 97-102, and 104-107 in FIG.

[0410] In some embodiments, the 5' end of a provided single stranded RNAi agent does not include a phosphorus-containing moiety (e.g., the 5' end includ...

Claims

1. A composition comprising an oligonucleotide, (i) the oligonucleotide targets PNPLA3 mRNA; and (ii) The base sequence of the oligonucleotide has greater than 50% identity to TGCCACUGUAGAAAGGCAUTU (SEQ ID NO: 177), and each U can be optionally and independently replaced with T.

2. The composition described in claim 1, wherein the composition is a liquid composition.

3. The composition of claim 1 or 2, wherein the oligonucleotide comprises a targeting portion ("R CD ").

4. The R CD is The composition of claim 3, wherein 5. The composition of claim 4, wherein the R CD is connected to the oligonucleotide via a linker.

6. The linker is , , , , , , or 6. The composition of claim 5 having the structure:

7. R CD is , , , , , or The composition of claim 6, wherein the compound is selected from the group consisting of:

8. A composition described in any one of claims 1 to 7, wherein the oligonucleotide contains at least one internucleotide bond including a phosphorus bond in the Sp configuration.

9. A composition described in any one of claims 1 to 8, wherein the composition is chiral controlled in that it is enriched compared to a substantially racemic preparation of the oligonucleotide.

10. Use in the manufacture of a composition for treating fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic steatohepatitis with liver fibrosis, non-alcoholic steatohepatitis with cirrhosis, or non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in a human, the composition comprising a therapeutically effective amount of a composition according to any one of claims 1 to 9.

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