Oligonucleotide compositions and methods of use thereof

Abstract

The present disclosure provides, inter alia, designed PNPLA3 oligonucleotides, compositions, and methods thereof. In some embodiments, provided oligonucleotide compositions provide improved single-stranded RNA interference and / or RNase H-mediated knockdown. The present disclosure inter alia encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) or patterns thereof, conjugation with additional chemical moieties, and / or stereochemistry [e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages)] and / or patterns thereof, can have significant effects on the properties and activities of oligonucleotides, such as RNA interference (RNAi) activity, stability, delivery, etc. In some embodiments, the present disclosure provides methods of treating diseases using provided oligonucleotide compositions (e.g., in RNA interference and / or RNase H-mediated knockdown).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 514,769, filed on June 2, 2017, and U.S. Provisional Application No. 62 / 670,698, filed on May 11, 2018, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Oligonucleotides targeting PNPLA3 (PNPLA3 oligonucleotides) can be used in various applications, such as therapeutic applications. The use of naturally occurring nucleic acids (such as unmodified DNA or RNA) can be limited, for example, due to their susceptibility to endonucleases and exonucleases. Summary of the invention

[0004] The present disclosure encompasses, inter alia, the recognition that control of structural elements of PNPLA3 oligonucleotides, such as chemical modifications (e.g., modifications of sugars, bases and / or internucleotide linkages) or patterns thereof, changes in stereochemistry (e.g., stereochemistry of 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 asialoglycoprotein receptors or ASGPRs, e.g., GalNAc moieties), etc., can have a significant effect on the properties and / or activities of PNPLA3 oligonucleotides. In some embodiments, the properties and / or activities include, but are not limited to, involvement in directing a decrease in the expression, activity or level of a PNPLA3 gene or its gene product, such as mediated by RNA interference (RNAi interference), single-stranded RNA interference (ssRNAi), RNase H-mediated knockdown, steric hindrance of translation, etc.

[0005] 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. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present invention may be understood more readily by reference to the following detailed description of illustrative embodiments of the invention and the Examples included therein.

[0008] It is to be understood that the present invention is not limited to the specific preparative synthetic methods described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0009] In some embodiments, the present disclosure encompasses the recognition that stereochemistry, particularly the stereochemistry of backbone chiral centers, can unexpectedly improve the properties of PNPLA3 oligonucleotides. In contrast to many previous observations that some structural elements that increase stability can also reduce activity, such as RNA interference, the present disclosure demonstrates that control of stereochemistry can surprisingly increase stability without significantly reducing activity.

[0010] In some embodiments, the present disclosure provides oligonucleotides having certain 5'-terminal structures.

[0011] In some embodiments, the present disclosure provides 5'-terminal structures that can provide oligonucleotides with high biological activity (eg, RNAi activity) when used according to the present disclosure.

[0012] In some embodiments, the present disclosure encompasses the recognition that various additional chemical moieties, such as lipid moieties and / or carbohydrate moieties, when incorporated into an oligonucleotide, can 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 comprise more than one additional chemical moiety. In some embodiments, a PNPLA3 oligonucleotide may comprise two or more additional chemical moieties, wherein the additional chemical moieties are the same or different, or belong to the same class (e.g., targeting moieties, carbohydrate moieties, moieties that bind to ASPGR, lipid moieties, etc.) or do not belong to the same class. In some embodiments, certain additional chemical moieties promote delivery of the oligonucleotide to desired cells, tissues, and / or organs. In some embodiments, certain additional chemical moieties promote internalization of the oligonucleotide and / or increase the stability of the oligonucleotide.

[0013] In some embodiments, the present disclosure provides PNPLA3 oligonucleotide compositions that achieve allele-specific inhibition, wherein transcripts from one allele of a specific target gene are selectively knocked down relative to at least one other allele of the same gene.

[0014] In some embodiments, the present disclosure demonstrates that certain provided structural elements, techniques and / or features are particularly useful for PNPLA3 oligonucleotides (e.g., RNAi agents) that participate in and / or direct RNAi mechanisms. However, in any event, the teachings of the present disclosure are not limited to oligonucleotides that participate or function by any particular mechanism. In some embodiments, the present disclosure relates to any oligonucleotide that can function by any mechanism and comprises any sequence, structure, or format (or portion thereof) described herein. In some embodiments, the present disclosure provides PNPLA3 oligonucleotides that can act by any mechanism and include any sequence, structure or format described herein (or a portion thereof), including but not limited to any 5' terminal structure; 5'-terminal region; first region (including but not limited to seed region); second region (including but not limited to post-seed region); and 3'-terminal region (which can be a 3'-terminal dinucleotide and / or a 3'-terminal cap); optional additional chemical moieties (including but not limited to targeting moieties, carbohydrate moieties, APGR-binding moieties, and lipid moieties); stereochemistry or stereochemistry pattern; modification or modification pattern; internucleotide linkage or internucleotide linkage pattern; modification of sugar or modification pattern of sugar; modification of base or modification pattern of base. In some embodiments, the provided oligonucleotides can participate in (e.g., guide) RNAi mechanisms. In some embodiments, the provided oligonucleotides can participate in RNase H (ribonuclease H) mechanisms. In some embodiments, the provided oligonucleotides can act as translation inhibitors (e.g., can provide steric hindrance of translation). In some embodiments, the provided oligonucleotides can be therapeutic. In some embodiments, the oligonucleotides provided can be used for therapeutic applications, diagnostic applications, research applications and / or nanomaterial applications. In some embodiments, the target is intended to change the expression and / or active specific allele of one or more products (e.g., RNA and / or protein products) relative to the target. In many embodiments, the target allele is a kind of allele associated (e.g., related) with the existence, incidence and / or severity of one or more diseases and / or symptom. Alternatively or in addition, in some embodiments, the target allele is a kind of allele associated with the improvement (e.g., delayed onset, reduced severity, responsiveness to other therapies, etc.) of the level and / or active change of one or more gene products of the target.

[0015] In some embodiments, where the presence and / or activity of a particular allele (disease-associated allele) is associated (e.g., correlated) with the presence, incidence, and / or severity of one or more disorders, diseases, and / or conditions, a different allele of the same gene is present but is not so associated or is less associated (e.g., exhibits less significant or statistically insignificant correlation). In some such embodiments, oligonucleotides and methods thereof as described herein can preferentially or specifically target associated alleles relative to one or more less associated / non-associated alleles, thereby mediating allele-specific inhibition.

[0016] In some embodiments, the PNPLA3 target sequence is a sequence that binds to a PNPLA3 oligonucleotide as described herein. In many embodiments, the target sequence is identical or exactly complementary to a sequence of contiguous residues in a provided oligonucleotide or therein (e.g., the provided oligonucleotide includes a targeted binding sequence that is identical or exactly complementary to the target sequence). In some embodiments, the targeted binding sequence is exactly complementary to a target sequence of a transcript (e.g., pre-mRNA, mRNA, etc.). The targeted binding sequence / target sequence can have a variety of lengths to provide an oligonucleotide with the desired activity and / or properties. In some embodiments, the targeted binding sequence / target sequence comprises 5-50 bases. In some embodiments, a small amount of difference / mismatch between (the relevant portion of) a PNPLA3 oligonucleotide and its target sequence (including, but not limited to, the 5'- and / or 3'-terminal regions of the target and / or oligonucleotide sequence) is tolerated. In many embodiments, the target sequence is present in a transcript (e.g., mRNA and / or pre-mRNA) produced by a target gene.

[0017] In some embodiments, the target sequence includes one or more allelic sites (i.e., the location within the target gene where allelic variation occurs). In some embodiments, the allelic site is a mutation. In some embodiments, the allelic site is a SNP. In some such embodiments, the oligonucleotides provided preferentially or specifically bind to one allele relative to one or more other alleles. In some embodiments, the oligonucleotides provided preferentially bind to disease-associated alleles. For example, in some embodiments, the PNPLA3 oligonucleotides provided herein (or their targeted binding sequence portions) have a sequence that is at least partially identical or exactly complementary to a specific allelic form of the target sequence.

[0018] Unless otherwise indicated, all sequences (including but not limited to base sequences as well as chemical, modified and / or stereochemical patterns) are presented in 5' to 3' order.

[0019] In some embodiments, the present disclosure provides compositions and methods related to PNPLA3 oligonucleotides that are specific for a target and have or comprise 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 at least 15 consecutive nucleotides may be optionally replaced by T or DNA T. In some embodiments, the oligonucleotide is capable of directing ssRNAi.

[0020] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, sugar and / or base modifications.

[0021] 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, wherein each of the base, the sugar, and the internucleotide linkage is independently and optionally naturally occurring or non-naturally occurring. In some embodiments, the nucleoside comprises a base and a sugar, wherein each of the base and the sugar is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2'-deoxy) and RNA (2'-OH) nucleotides; and those nucleotides comprising one or more modifications at 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 may have a structure of Formula I as described in the present disclosure. In some embodiments, the internucleotide linkage is a portion that does not contain phosphorus but is used to connect two natural or non-natural sugars.

[0022] In some embodiments, the present disclosure provides a chirality-controlled PNPLA3 oligonucleotide composition that directs a greater decrease in expression, activity, and / or level of a PNPLA3 gene or its gene product when compared to a reference condition (e.g., in the absence of the composition or in the absence of a reference composition (e.g., a stereo-random composition of oligonucleotides having the same base sequence and chemical modifications)), single-stranded RNA interference, and / or RNase H-mediated knockdown.

[0023] In some embodiments, a PNPLA3 oligonucleotide composition comprising a plurality of oligonucleotides is stereorandom in that the plurality of oligonucleotides do not share a common stereochemistry at any chiral internucleotide linkage. In some embodiments, a PNPLA3 oligonucleotide composition comprising a plurality of oligonucleotides is chirality controlled in that the plurality of oligonucleotides share a common stereochemistry at one or more chiral internucleotide linkages. In some embodiments, the chirality controlled PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides has reduced susceptibility to endonucleases and exonucleases relative to a stereorandom PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides.

[0024] In some embodiments, the composition comprises a multimer of two or more of: a first plurality of PNPLA3 oligonucleotides and / or a second plurality of oligonucleotides, wherein the first and second plurality of oligonucleotides can independently direct knockdown of the same or different targets by RNA interference and / or RNase H-mediated knockdown.

[0025] In some embodiments, a PNPLA3 oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a first plurality of oligonucleotides) is chirality controlled because the plurality of oligonucleotides independently share a common stereochemistry at one or more chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides share a common stereochemical configuration 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 linkages, each of which is independently Rp or Sp. In some embodiments, the plurality of oligonucleotides share a common stereochemical configuration at each chiral internucleotide linkage. In some embodiments, a chiral internucleotide linkage is referred to as a chirality-controlled internucleotide linkage when predetermined levels of the oligonucleotides of the composition share a common stereochemical configuration (independently Rp or Sp).

[0026] In some embodiments, at least 5 internucleotide linkages are chirality controlled; in some embodiments, at least 10 internucleotide linkages are chirality controlled; in some embodiments, at least 15 internucleotide linkages are chirality controlled; in some embodiments, every chiral internucleotide linkage is chirality controlled.

[0027] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of PNPLA3 oligonucleotides that share:

[0028] 1) Common base sequence;

[0029] 2) a common backbone linkage pattern; and

[0030] 3) a common backbone chiral center pattern, the composition is a substantially pure preparation of individual oligonucleotides in that the oligonucleotides in the composition at predetermined levels have a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern.

[0031] In some embodiments, the common backbone chiral center pattern includes at least one internucleotide linkage that includes a chiral center for chirality control.

[0032] In some embodiments, the level of oligonucleotide and / or diastereomeric purity can be determined by analytical methods (eg, chromatography, spectrophotometry, spectroscopy, or any combination thereof).

[0033] The present disclosure encompasses, inter alia, the recognition that stereorandom PNPLA3 oligonucleotide preparations contain multiple different chemical entities that differ from one another, for example, in the stereochemical structure (or stereochemistry) of each backbone chiral center within the oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral center, stereorandom oligonucleotide preparations provide an uncontrolled composition that contains an undefined level of oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and / or chemical modification, they are different chemical entities at least due to their different backbone stereochemistry, and as demonstrated herein, they may have different properties, such as sensitivity to nucleases, activity, distribution, etc. In some embodiments, a specific stereoisomer may be defined, for example, by its base sequence, its length, its backbone linkage pattern, and its backbone chiral center pattern. In some embodiments, the present disclosure demonstrates that the improvements in properties and activities achieved by controlling the stereochemistry within a PNPLA3 oligonucleotide may be comparable to or even better than those achieved by using chemical modifications.

[0034] 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, optionally further comprising an additional chemical moiety.

[0035] In some embodiments, the 5'-terminal region is the entire portion of the PNPLA3 oligonucleotide that is 5' to the seed region. In some embodiments, the 3'-terminal region is the entire portion of the PNPLA3 oligonucleotide that is 3' to the seed region.

[0036] In some embodiments, the 5'-terminal structure is a 5'-terminal group.

[0037] In some embodiments, the 5'-terminal structure comprises a 5'-terminal group.

[0038] In some embodiments, provided oligonucleotides can comprise a 5'-terminal region, a 5'-terminal structure, a 5'-terminal group, a 5'-terminal nucleoside, or a 5'-terminal nucleotide as described herein or known in the art.

[0039] 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.

[0040] In some embodiments, the oligonucleotide provided comprises one or more blocks. In some embodiments, the oligonucleotide provided comprises one or more blocks, wherein the blocks comprise one or more continuous nucleosides and / or nucleotides and / or sugars or bases and / or internucleotide linkages. In some embodiments, the blocks encompass the entire seed region or a portion thereof. In some embodiments, the blocks encompass the entire post-seed region or a portion thereof.

[0041] In some embodiments, provided oligonucleotides are blockers.

[0042] In some embodiments, the oligonucleotides provided are alternating polymers comprising alternating blocks. In some embodiments, block polymers or alternating polymers can be defined by chemical modifications (including presence or absence) (e.g., base modifications, sugar modifications, internucleotide linkage modifications, stereochemistry, etc.) or their patterns.

[0043] In some embodiments, the oligonucleotides provided 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.

[0044] In some embodiments, the PNPLA3 oligonucleotide comprises only two 2'-Fs. In some embodiments, the PNPLA3 oligonucleotide comprises only two 2'-Fs, wherein the two nucleotides are at positions 2 and 14.

[0045] In some embodiments, the PNPLA3 oligonucleotide comprises only two 2'-Fs, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is 2'-deoxy.

[0046] In some embodiments, the PNPLA3 oligonucleotide comprises only two 2'-Fs, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is a 2'-deoxyT.

[0047] In some embodiments, the PNPLA3 oligonucleotide comprises only two 2'-Fs, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is 2'-deoxy, and the 5'-terminal structure is -OH.

[0048] In some embodiments, the PNPLA3 oligonucleotide comprises only two 2'-Fs, wherein the two nucleotides are at positions 2 and 14, and wherein the first nucleotide is a 2'-deoxyT, and the 5'-terminal structure is -OH.

[0049] In some embodiments herein, with respect to PNPLA3 oligonucleotides, "first" (eg, the first nucleotide) refers to the 5' end of the oligonucleotide, and "last" or "terminal" (eg, the last nucleotide or terminal nucleotide) refers to the 3' end.

[0050] In some embodiments, provided oligonucleotides comprise sugars with a particular modification alternating with sugars that are not modified or have a different modification. In some embodiments, sugars with a particular modification occur in one or more blocks.

[0051] In some embodiments, the oligonucleotides provided include one or more blocks, the one or more blocks including a sugar with a specific 2' modification, which alternates with a sugar that is independently not modified or has a different modification. In some embodiments, the oligonucleotides provided include one or more blocks, the one or more blocks including a sugar with a 2'-F modification, which alternates with a sugar that is independently not modified or has a different modification. In some embodiments, the oligonucleotides provided include one or more blocks, the one or more blocks including a sugar with a 2'-OMe modification, which alternates with a sugar that is independently not modified or has a different modification. In some embodiments, the oligonucleotides provided include one or more blocks, the one or more blocks including a sugar with a 2'-OMe modification, which alternates with a sugar with a 2'-F modification.

[0052] In some embodiments, the sugar block has or comprises a 2'-modified pattern of any of the following: ff, fffm, fffmm, fffmmm, fffmmmm, fffmmmmm, fffmmmmmm, fffmmmmmmf, fffmmmmmmff, fffmmmmmmffm, fffmmmmmmffmm, fffmmmmmmffmmf, fffmmmmmmffmmfm, fffmmmmmmffmmfmf, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfmf, fffmmmmmmffmmfmfmfm, fffmmmmmmffmmfmfmfmm, fffmmmmmmffmmfmfmfmmm, ffmmffmm, ffmmmmmmffmmfmfmfmmm, fmfmfmfmfmfmfm, fmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmm, fmfmfmfmfmfmm, fmfmfmfmfmfmmm, fmmffmm, fmmmmmmffmmfmfmfmmm, mff, mffm, mffmf, mffmff, mffmffm, mffmmffmm, mfmfm, mfmfmfmfmfffmfmfmfmmm, mfmfmfmfmfmfmfm, mfmfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmfmfmmm, mfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmm, mfmfmfmfmfmfmm, mfmfmfmfmfmfmmm, mfmfmfmfmfmmm, mfmfmfmfmfmmmfm, mfmfmfmfmfmmmmm, mfmfmfmfmmm, mfmfmfmfmmmfmfm, mfmfmfmfmmmfmmm, mfmfmfmfmmmmmfm, mfmfmfmmm, mfmfmfmmmfmfmfm, mfmfmfmmmfmfmmm, mfmfmfmmmfmfmmmfm, mfmfmfmmmmmfmfm, mfmfmmm, mfmfmmmfmfmfmfm, mfmfmmmfmfmfmmm, mfmfmmmfmfmmmfm, mfmfmmmfmmmfmfm,mfmfmmmmmfmfmfm,mfmmm,mfmmmfmfmfmfmfm,mfmmmfmfmfmfmmm,mfmmmfmfmfmmmfm,mfmmmfmfmmmfmfm,mfmmmfmmmfmfmfm,mfmmmfmmmfmfmfm , mfmmmmmmfmfmfmfm, mmffm, mmffmm, mmffmm, mmffmmf, mmffmmff, mmffmmffm, mmffmmffmm, mmffmmfmfmfmmm, mmm, mmmffmmfmfmfmmm, mmmfmfmf mfmfmfm, mmmfmfmfmfmfmmm, mmmfmfmfmfmmmfm, mmmfmfmfmmmfmfm, mmmfmfmmmfmfmfm, mmmfmmmfmfmfmfm, mmmmffmmfmfmfmmm, mmm, mmmm, mmmm m, mmmmmmffmmfmfmfmmm, mmmmmfmfmfmfmfm, mmmmmm, mmmmmmffmmfmfmfmmm, mfmf, mfmf, mfmfmf, fmfm, fmfmfm, fmfmfmf, dfdf, dfdfdf, dfdfdfd f, fdfd, fdfdfd, fdfdfdfd, dfdfmfmf, dfmfmf, mfdfmf or dfmfdf, wherein m represents 2'-OMe, f represents 2'-F, and d represents no substitution at the 2'-position. In some embodiments, the seed region and / or the post-seed region may comprise a sugar-modified block.

[0053] In some embodiments, the block is a stereochemical block. In some embodiments, the block is an Rp block, because each internucleotide linkage 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, because each internucleotide linkage 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 oligonucleotide provided comprises both Rp blocks and Sp blocks. In some embodiments, the oligonucleotide provided comprises one or more Rp blocks, but does not comprise Sp blocks. In some embodiments, the oligonucleotide provided comprises one or more Sp blocks, but does not comprise Rp blocks. In some embodiments, the oligonucleotide provided comprises one or more PO blocks, wherein each internucleotide linkage of the block is a natural phosphate linkage.

[0054] In some embodiments, the seed region-block is a Sp block, wherein each sugar moiety comprises a 2'-F modification. In some embodiments, the seed region-block is a Sp block, wherein each internucleotide linkage is a modified internucleotide linkage, and each sugar moiety comprises a 2'-F modification. In some embodiments, the seed region-block is a Sp block, wherein each internucleotide linkage is a phosphorothioate linkage, and each sugar moiety comprises a 2'-F modification. In some embodiments, the seed region-block comprises 4 or more nucleoside units. In some embodiments, the nucleoside unit is a nucleoside. In some embodiments, the seed region-block comprises 5 or more nucleoside units. In some embodiments, the seed region-block comprises 6 or more nucleoside units. In some embodiments, the seed region-block comprises 7 or more nucleoside units. In some embodiments, the post-seed region-block is a Sp block, wherein each sugar moiety comprises a 2'-F modification. In some embodiments, the post-seed region-block is a Sp block, wherein each internucleotide linkage is a modified internucleotide linkage, and each sugar moiety comprises a 2'-F modification. In some embodiments, the post-seed region-block is a Sp block, wherein each internucleotide linkage is a phosphorothioate linkage, and each sugar moiety comprises a 2'-F modification. In some embodiments, the post-seed region-block comprises 4 or more nucleoside units. In some embodiments, the post-seed region-block comprises 5 or more nucleoside units. In some embodiments, the post-seed region-block comprises 6 or more nucleoside units. In some embodiments, the post-seed region-block comprises 7 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 comprises a stereochemical block.

[0055] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides:

[0056] 1) have a common base sequence; and

[0057] 2) Contain one or more modified sugar moieties and modified internucleotide linkages.

[0058] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the first plurality of oligonucleotides:

[0059] 1) having a common base sequence that is complementary to a target sequence in a transcript; and

[0060] 2) Contain one or more modified sugar moieties and modified internucleotide linkages.

[0061] In some embodiments, the reference condition is the absence of a composition. In some embodiments, the reference condition is the presence of a reference composition. The exemplary reference composition comprising a plurality of oligonucleotides is fully described in the present disclosure. In some embodiments, compared with the first plurality of oligonucleotides in the composition provided, a plurality of oligonucleotides with reference to different structural elements (chemical modification, stereochemistry, etc.). In some embodiments, the oligonucleotide composition comprising the first plurality of oligonucleotides provided is chiral controlled, because the first plurality of oligonucleotides include one or more chiral controlled internucleotide linkages. In some embodiments, the oligonucleotide composition comprising the first plurality of oligonucleotides provided is chiral controlled, because the first plurality of oligonucleotides include 1-20 chiral controlled internucleotide linkages. In some embodiments, the first plurality of oligonucleotides include 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 stereo-irregular product of an oligonucleotide with the same chemical modification. In some embodiments, the reference composition is a mixture of stereoisomers, and the composition provided is a single-stranded RNAi agent of a stereoisomer. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same base sequence as the first multiple oligonucleotides. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same chemical modification as the first multiple oligonucleotides. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same sugar modification as the first multiple oligonucleotides. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same base modification as the first multiple oligonucleotides. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same internucleotide linkage modification as the first multiple oligonucleotides. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same base sequence and the same chemical modification as the first multiple oligonucleotides. In some embodiments, in the provided compositions, reference multiple oligonucleotides have the same stereochemistry as the first multiple oligonucleotides, but have different chemical modifications, such as base modifications, sugar modifications, internucleotide linkage modifications, etc.

[0062] In some embodiments, the present disclosure provides a composition comprising a PNPLA3 oligonucleotide, wherein the oligonucleotide is complementary or substantially complementary to a target RNA sequence, has a total length of about 15 to about 49 nucleotides, wherein the oligonucleotide comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0063] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a single-stranded RNAi agent, wherein the single-stranded RNAi agent is complementary or substantially complementary to a target RNA sequence, has a total length of about 15 to about 49 nucleotides, and is capable of directing target-specific RNA interference, wherein the single-stranded RNAi agent comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0064] In some embodiments, the length is 15 to 49, about 17 to about 49, 17 to 49, about 19 to about 29, 19 to 29, about 19 to about 25, 19 to 25, about 19 to about 23, or 19 to 23 nucleotides total.

[0065] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides:

[0066] 1) having a common base sequence that is complementary or substantially complementary to a target sequence in a transcript; and

[0067] 2) comprises one or more modified sugar moieties and modified internucleotide linkages,

[0068] The oligonucleotide composition is characterized in that, when contacted with a transcript, knockdown of the transcript is improved compared to knockdown observed under a reference condition selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0069] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the first plurality of oligonucleotides:

[0070] 1) having a common base sequence that is complementary to a target sequence in a transcript; and

[0071] 2) comprises one or more modified sugar moieties and modified internucleotide linkages,

[0072] The oligonucleotide composition is characterized in that, when it is contacted with a transcript in an RNA interference system, RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, wherein the reference conditions are selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0073] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides belongs to a specific oligonucleotide type defined by:

[0074] 1) Base sequence;

[0075] 2) Main chain linkage pattern;

[0076] 3) Main chain chiral center pattern; and

[0077] 4) Main chain phosphorus modification pattern.

[0078] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the first plurality of oligonucleotides belongs to a specific oligonucleotide type defined by:

[0079] 1) Base sequence;

[0080] 2) Main chain linkage pattern;

[0081] 3) Main chain chiral center pattern; and

[0082] 4) Main chain phosphorus modification pattern.

[0083] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides of a PNPLA3 oligonucleotide type, wherein the oligonucleotide type is defined by:

[0084] 1) Base sequence;

[0085] 2) Main chain linkage pattern;

[0086] 3) Main chain chiral center pattern; and

[0087] 4) Main chain phosphorus modification pattern,

[0088] The composition is chirality controlled in that it is enriched in oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence,

[0089] The oligonucleotide composition is characterized in that, when contacted with a transcript, knockdown of the transcript is improved compared to knockdown observed under a reference condition selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0090] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides that are capable of directing single-stranded RNA interference and are of the type of PNPLA3 oligonucleotide defined by:

[0091] 1) Base sequence;

[0092] 2) Main chain linkage pattern;

[0093] 3) Main chain chiral center pattern; and

[0094] 4) Main chain phosphorus modification pattern,

[0095] The composition is chirality controlled in that it is enriched in oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence,

[0096] The oligonucleotide composition is characterized in that, when it is contacted with a transcript in an RNA interference system, RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, wherein the reference conditions are selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0097] In some embodiments, an oligonucleotide is provided having any of the formats shown in FIG. 1 , or any structural element of any of the formats shown in FIG. 1 .

[0098] In some embodiments, a single-stranded RNAi agent is provided having any of the formats shown in FIG. 1 , or any structural element of any of the formats shown in FIG. 1 .

[0099] The data provided by the present disclosure particularly show that various oligonucleotides of the disclosed formats are capable of directing a reduction in the expression and / or level of a target gene or its gene product in any of several different genes when it is targeted to any of several different sequences. In some embodiments, the data provided by the present disclosure show that various RNAi agents of the disclosed formats are capable of directing RNA interference in any of many different genes to any of many different sequences.

[0100] In some embodiments, a PNPLA3 oligonucleotide having any of the structures described and / or shown herein is capable of directing RNA interference. In some embodiments, a PNPLA3 oligonucleotide having any of the structures described and / or shown herein is capable of directing RNase H-mediated knockdown. In some embodiments, a PNPLA3 oligonucleotide having any of the structures described and / or shown herein is capable of directing RNA interference and / or RNase H-mediated knockdown. In some embodiments, a PNPLA3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1. In some embodiments, a PNPLA3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1 and is capable of directing RNA interference. In some embodiments, a PNPLA3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1 and is capable of directing RNase H-mediated knockdown. In some embodiments, the PNPLA3 oligonucleotide comprises any oligonucleotide described herein or any structural element in any format described herein or shown in FIG. 1 , and is capable of directing RNA interference and / or RNase H-mediated knockdown.

[0101] In some embodiments, the RNAi agent comprises one or more of the following: 5'-terminal structure, 5'-terminal region, seed region, post-seed region and 3'-terminal region and optional additional chemical moieties. 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 described herein or known in the art and the 3'-terminal region. In some embodiments, the 3'-terminal region can be any 3'-terminal region described herein or known in the art. In some embodiments, any optional additional chemical moieties can be any optional additional chemical moieties described herein or known in the art. Any single 5'-terminal structure, 5'-terminal region, seed region, post-seed region, 3'-terminal region and optional additional chemical moieties described herein or known in the art can be independently combined with any other 5'-terminal structure, 5'-terminal region, seed region, post-seed region, 3'-terminal region and optional additional chemical moieties described herein or known in the art. In some embodiments, as non-limiting examples, the region of the single-stranded RNAi agent is a 5'-terminal structure, a 5'-terminal region, a seed region, a post-seed region, a portion of a seed region, a portion of a post-seed region, or a 3'-terminal dinucleotide.

[0102] In some embodiments, the base sequence of the oligonucleotide provided is composed 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 the following sequence, and it comprises the sequence of 15 continuous bases of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the oligonucleotide provided comprises the following sequence, and it comprises the sequence of 20 continuous bases (having at most 5 mispairings) of the base sequence of any oligonucleotide disclosed herein.

[0103] In some embodiments, the oligonucleotides provided are capable of directing a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the oligonucleotides provided are capable of directing single-stranded RNAi interference. In some embodiments, the oligonucleotides provided are capable of directing RNA enzyme H mediated knockdown. In some embodiments, the oligonucleotides provided are capable of directing single-stranded RNA interference and RNA enzyme H mediated knockdown. In some embodiments, the oligonucleotides comprise sequences that target any transcript or gene targeted by the oligonucleotides disclosed herein.

[0104] In some embodiments, provided oligonucleotides target PNPLA3.

[0105] In some embodiments, the oligonucleotides provided are useful for reducing or inhibiting the activity, level and / or expression of a PNPLA3 gene or its gene product. In some embodiments, the oligonucleotides provided are useful for reducing or inhibiting the activity, level and / or expression of a gene or its gene product, wherein the abnormal or excessive activity, level and / or expression of a gene or its gene product, a deleterious mutation in a gene or its gene product, or the tissue or cell-to-cell or intracellular distribution of a gene or its gene product is associated with a condition, causes a condition, and / or is associated with a condition. In some embodiments, the oligonucleotides provided are useful for treating a condition and / or preparing a medicament for treating a condition, wherein the condition is associated with, caused by and / or associated with the abnormal or excessive activity, level and / or expression or abnormal distribution of a gene or its gene product.

[0106] In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein, which are capable of targeting PNPLA3 and can be used to treat and / or prepare treatments for PNPLA3-associated disorders.

[0107] In some embodiments, the PNPLA3 oligonucleotide capable of targeting a gene comprises a base sequence that is a portion of the base sequence of the target gene or is complementary or substantially complementary to a portion of the base sequence of the target gene. In some embodiments, the length of a portion is at least 15 bases. In some embodiments, the base sequence of the single-stranded RNAi agent may comprise or consist of a base sequence having a specified maximum number of mismatches with a specified base sequence.

[0108] In some embodiments, mispairing is the difference between base sequence or length when two sequences are compared and compared to the maximum extent.As a non-limiting example, if there is a difference between the base at a specific position in a sequence and the base at a corresponding position in another sequence, it is counted as mispairing.Therefore, for example, if the position in a sequence has a specific base (for example, A), and the corresponding position in another sequence has different bases (for example, G, C or U), it is counted as mispairing.For example, if the position in a sequence has a base (for example, A), and the corresponding position in another sequence does not have a base (for example, the position is a baseless nucleotide comprising a phosphate sugar backbone but without a base) or skips the position, it is also counted as mispairing.Single-stranded nicks in either sequence (or sense strand or antisense strand) may not be counted as mispairing, for example, if a sequence comprises sequence 5'-AG-3', and another sequence comprises sequence 5'-AG-3' and has a single-stranded nick between A and G, it will not be counted as mispairing. 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 the purpose of counting mismatches, substitution of U with T or vice versa is not considered a mismatch.

[0109] In some embodiments, the PNPLA3 oligonucleotide is complementary to or completely or 100% complementary to a target sequence (e.g., RNA, such as mRNA), meaning that the base sequence of the oligonucleotide has no mismatches with a sequence that is completely complementary to the target gene (e.g., base pairs by Watson-Crick base pairing). Without wishing to be bound by any particular theory, the present disclosure indicates that for a single-stranded RNAi agent, the 5'-terminal nucleotide portion or the 3'-terminal dinucleotide does not have to be base paired with the target. These can be mismatches. In addition, an antisense oligonucleotide or single-stranded RNAi agent may have a small number of internal mismatches and still direct a reduction in the expression and / or level of a target gene or its gene product, and / or direct knockdown and / or RNA interference mediated by RNase H. If the first base sequence of a PNPLA3 oligonucleotide (e.g., an antisense oligonucleotide or a single-stranded RNAi agent) has a small number of mismatches with a reference base sequence that is 100% complementary to the target sequence, then the first base sequence is substantially complementary to the target sequence. In some embodiments, a PNPLA3 oligonucleotide (e.g., an antisense oligonucleotide or a single-stranded RNAi agent) may have a base sequence that is complementary or substantially complementary to a target sequence. In some embodiments, complementarity is determined based on Watson-Crick base pairing (guanine-cytosine and adenine-thymine / uracil), wherein guanine, cytosine, adenine, thymine, uracil may be optionally and independently modified, but retain their unmodified pairing hydrogen bonding pattern. In some embodiments, a sequence complementary to another sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 bases.

[0110] In some embodiments, the PNPLA3 oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common backbone linkage pattern. In some embodiments, the common backbone linkage pattern comprises at least 10 modified internucleotide linkages.

[0111] In some embodiments, the common backbone linkage pattern comprises at least 10 phosphorothioate linkages. In some embodiments, the PNPLA3 oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common backbone chiral center pattern. In some embodiments, the common backbone chiral center pattern comprises at least 1 internucleotide linkage of Sp configuration. In some embodiments, the common backbone chiral center pattern comprises at least 1 internucleotide linkage that is a phosphorothioate of Sp configuration. In some embodiments, the oligonucleotides in the provided compositions have a common backbone phosphorus modification pattern. In some embodiments, the provided compositions are chirality controlled PNPLA3 oligonucleotide compositions because the compositions contain oligonucleotides of a predetermined level of a single oligonucleotide type, wherein the PNPLA3 oligonucleotide type is defined by:

[0112] 1) Base sequence;

[0113] 2) Main chain linkage pattern;

[0114] 3) Main chain chiral center pattern; and

[0115] 4) Main chain phosphorus modification pattern.

[0116] As noted above and as understood in the art, in some embodiments, the base sequence of a PNPLA3 oligonucleotide can refer to the identity and / or modification status of the nucleoside residues in the oligonucleotide (e.g., sugar and / or base composition relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues).

[0117] In some embodiments, a particular oligonucleotide type can be defined by:

[0118] 1A) Base identity;

[0119] 1B) base modification pattern;

[0120] 1C) Sugar modification pattern;

[0121] 2) Main chain linkage pattern;

[0122] 3) Main chain chiral center pattern; and

[0123] 4) Main chain phosphorus modification pattern.

[0124] Thus, in some embodiments, oligonucleotides of a particular type may share the same base, but differ in base modification and / or sugar modification patterns. In some embodiments, oligonucleotides of a particular type may share the same base and base modification patterns (including, for example, the absence of base modifications), but differ in sugar modification patterns.

[0125] In some embodiments, oligonucleotides of a particular type are chemically identical in that they have the same base sequence (including length), the same chemical modification pattern of the sugar and base moieties, the same backbone linkage pattern (e.g., a pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same backbone chiral center pattern (e.g., the stereochemical pattern of the chiral internucleotide linkages (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., the modification pattern of the internucleotide phosphorus atoms, such as -S - and -LR of formula I 1 ).

[0126] The present disclosure provides, in particular, oligonucleotide compositions and techniques for optimizing properties (e.g., improved single-stranded RNA interference, RNAse H-mediated knockdown, etc.). In some embodiments, the present disclosure provides methods for reducing the immune response associated with the administration of oligonucleotides and compositions thereof (i.e., the administration of oligonucleotide compositions makes it possible to reduce the adverse immune response to the oligonucleotides in the compositions, for example, relative to the adverse immune response observed with reference compositions of nucleotides having comparable or identical nucleotide sequences). In some embodiments, the present disclosure provides methods for increasing the binding to certain proteins by oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for increasing the binding to certain proteins by oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides methods for enhancing the delivery of oligonucleotides and compositions thereof. The present disclosure particularly encompasses the following understandings: in some embodiments, the optimal delivery of oligonucleotides to their targets involves a balance of oligonucleotide binding to certain proteins, so that the oligonucleotides can be transported to the desired location, and involves the release of oligonucleotides, so that the oligonucleotides can be appropriately released from certain proteins to perform their desired functions, for example, hybridizing with their targets, cutting their targets, inhibiting translation, regulating transcript processing, etc. As exemplified in the present disclosure, the present disclosure recognizes, inter alia, that improvements in oligonucleotide properties may be achieved through chemical modification and / or stereochemistry.

[0127] In some embodiments, the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject a PNPLA3 oligonucleotide composition described herein.

[0128] In some embodiments, the disease is one in which knocking down the target nucleic acid by single-stranded RNA interference can repair, restore, or introduce a new beneficial function following administration of provided compositions.

[0129] In some embodiments, the consensus sequence comprises a sequence selected from Table 1A. In some embodiments, the consensus sequence is a sequence selected from Table 1A. In some embodiments, the backbone chiral center pattern is selected from those described in Table 1A.

[0130] In some embodiments, the disclosure provides a method comprising administering a composition comprising a first plurality of oligonucleotides that exhibits improved delivery compared to a reference composition comprising a plurality of oligonucleotides, each of which also has a common base sequence, but is structurally different from the first plurality of oligonucleotides because:

[0131] The individual oligonucleotides in the reference plurality of oligonucleotides differ from each other in stereochemistry; and / or

[0132] At least some of the oligonucleotides in the reference plurality of oligonucleotides have a structure that is different from the structure represented by the plurality of oligonucleotides of the composition.

[0133] In some embodiments, the present disclosure provides methods of administering a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing a decrease in expression and / or levels of a target gene or its gene product and having a common nucleotide sequence, wherein the improvement comprises:

[0134] Administration of a PNPLA3 oligonucleotide comprising a first plurality of oligonucleotides characterized by improved delivery relative to a reference oligonucleotide composition having the same common nucleotide sequence.

[0135] In some embodiments, the present disclosure provides a method of administering a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference and having a common nucleotide sequence, wherein the improvement comprises:

[0136] Administration of a PNPLA3 oligonucleotide comprising a first plurality of oligonucleotides characterized by improved delivery relative to a reference oligonucleotide composition having the same common nucleotide sequence.

[0137] In some embodiments, the present disclosure provides a single-stranded RNAi agent selected from a PNPLA3 oligonucleotide disclosed in any table (including but not limited to Table 1A) or elsewhere. In some embodiments, the present disclosure provides a single-stranded RNAi agent selected from a PNPLA3 oligonucleotide disclosed in any table (including but not limited to Table 1A) or elsewhere, wherein the oligonucleotide is conjugated to a lipid moiety.

[0138] In some embodiments, the oligonucleotides provided comprise a lipid portion. In some embodiments, the lipid portion is incorporated by conjugation with a lipid. In some embodiments, the lipid portion is a fatty acid. In some embodiments, the PNPLA3 oligonucleotide is conjugated to a fatty acid. In some embodiments, the single-stranded RNAi agent provided further comprises a lipid. In some embodiments, the single-stranded RNAi agent provided comprises a lipid portion conjugated at the 9th or 11th nucleotide (counting from the 5'-end). In some embodiments, the PNPLA3 oligonucleotide is conjugated to a fatty acid at a base. In some embodiments, the single-stranded RNAi agent provided comprises a lipid portion. In some embodiments, the single-stranded RNAi agent provided comprises a lipid portion conjugated at a base at the 9th or 11th nucleotide (counting from the 5'-end).

[0139] In some embodiments, the single-stranded RNAi agent is any of the aforementioned compositions, further comprising one or more additional components.

[0140] In some embodiments, provided oligonucleotides are capable of degrading a target transcript, such as RNA, via both an RNase H mechanism and an RNAi mechanism.

[0141] In some embodiments, the conjugation of a lipid moiety to a PNPLA3 oligonucleotide improves at least one property of the oligonucleotide. In some embodiments, the improved properties include increased activity (e.g., increased ability to direct the expression and / or level of a target gene or its gene product, and / or direct single-stranded RNA interference, and / or direct RNA enzyme 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, triceps, heart, or diaphragm. In some embodiments, the improved properties include reduced hTLR9 agonist activity. In some embodiments, the improved properties include hTLR9 antagonist activity. In some embodiments, the improved properties include increased hTLR9 antagonist activity.

[0142] Generally, the properties of an oligonucleotide composition as described herein can be assessed using any suitable assay.

[0143] Those skilled in the art will recognize and / or will be readily able to develop appropriate assays for particular oligonucleotide compositions.

[0144] definition

[0145] As used herein, unless otherwise indicated, the following definitions apply. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version) and Handbook of Chemistry and Physics, 75th edition. In addition, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th edition, Smith, MB and March, J., ed., John Wiley & Sons, New York: 2001.

[0146] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0147] The term "about" is intended to represent an approximation of plus or minus 10% of the nominal value to which it refers, or in one embodiment, an approximation of plus or minus 5%, or in another embodiment, an approximation of plus or minus 2%. For the field of the present disclosure, this level of approximation is appropriate unless it is specifically stated that the value requires a more stringent range.

[0148] Aliphatic: As used herein, "aliphatic" means a straight chain (i.e., unbranched) or branched substituted or unsubstituted hydrocarbon chain that is completely saturated or contains one or more unsaturated units, or a substituted or unsubstituted monocyclic, bicyclic or polycyclic hydrocarbon ring that is completely saturated or contains one or more unsaturated units (but does not include aromatic units), or a combination thereof. In some embodiments, aliphatic groups contain 1-50 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-7 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 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, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0149] Alkenyl: As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds.

[0150] Alkyl: As used herein, the term "alkyl" has its usual meaning in the art and may include saturated aliphatic groups, including straight chain alkyls, branched chain alkyls, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyls, and cycloalkyl-substituted alkyls. In some embodiments, the alkyl group has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl group has about 1-20 carbon atoms in its main chain (e.g., for a straight chain alkyl group having about 1 to 20 carbon atoms, the ... 1 -C 20 , for the branched chain is C 2-C 20 ), and alternatively has about 1-10 carbon atoms. In some embodiments, the cycloalkyl ring has about 3-10 carbon atoms in its ring structure, wherein such ring is monocyclic, bicyclic or polycyclic, and alternatively has about 5, 6 or 7 carbons in the ring structure. In some embodiments, the alkyl group can be a lower alkyl group, wherein the lower alkyl group contains 1-4 carbon atoms (e.g., C for a straight chain lower alkyl group is C 1 -C 4 ).

[0151] Alkynyl: As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds.

[0152] Antisense: As used herein, the term "antisense" refers to the characteristics of an oligonucleotide or other nucleic acid having a base sequence that is complementary or substantially complementary to a target nucleic acid that can hybridize therewith. In some embodiments, the target nucleic acid is a target gene mRNA. In some embodiments, hybridization is necessary for an activity or leads to an activity, for example, a reduction in the level, expression or activity of a target nucleic acid or its gene product. As used herein, the term "translation oligonucleotide" refers to an oligonucleotide that is complementary to a target nucleic acid. In some embodiments, an antisense oligonucleotide can guide a reduction in the level, expression or activity of a target nucleic acid or its gene product. In some embodiments, an antisense oligonucleotide can guide a reduction in the level, expression or activity of a target nucleic acid or its gene product by a mechanism involving RNase H, steric hindrance and / or RNA interference.

[0153] Approximately: As used herein, unless otherwise stated or otherwise apparent from the context, the term "approximately" or "about" with respect to a numerical value is generally deemed to include values ​​that fall within 5%, 10%, 15%, or 20% of the stated value in either direction (greater than or less than) (except where such values ​​will be less than 0% or more than 100% of the possible value). In some embodiments, the use of the term "about" with respect to dosage means ± 5 mg / kg / day.

[0154] Aryl: As used herein, the term "aryl", used alone or as part of a larger moiety such as "aralkyl", "aralkyloxy" or "aryloxyalkyl", refers to a monocyclic, bicyclic or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic. In some embodiments, aryl is a monocyclic, bicyclic or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. In some embodiments, aryl is a biaryl. The term "aryl" is used interchangeably with the term "aryl ring". In certain embodiments of the present disclosure, "aryl" refers to an aromatic ring system that may carry one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, binaphthyl, anthracenyl, and the like. 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, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0155] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide is a portion containing a stretch of consecutive amino acids or a collection of consecutive stretches of amino acids that are collectively characteristic of a protein or polypeptide. Each such continuous stretch will generally contain at least two amino acids. In general, a characteristic portion is a portion that, in addition to the sequence identity specified above, also shares at least one functional characteristic with the relevant intact protein.

[0156] Characteristic structural element: The term "characteristic structural element" or "structural element" refers to a unique structural element that is present in all members of a polypeptide, small molecule or nucleic acid family and can therefore be used by one of ordinary skill in the art to define members of the family. In some embodiments, the structural elements of a single-stranded RNAi agent include, but are not limited to, 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' cap, a modification pattern, a stereochemical pattern in the main chain, an additional chemical portion, and the like.

[0157] Comparable: The term "comparable" is used herein to describe two (or more) groups of conditions or situations that are sufficiently similar to each other to allow comparison of the results obtained or the observed phenomena. In some embodiments, multiple groups of comparable conditions or situations are characterized by a variety of substantially identical features and one or a few different features. It will be appreciated by those of ordinary skill in the art that when multiple groups of conditions are characterized by a sufficient number and type of substantially identical features, the multiple groups of conditions are comparable to each other to ensure that the following reasonable conclusions are drawn: the differences in the results obtained or the observed phenomena under different groups of conditions or situations are caused by or indicate changes in those different features.

[0158] Cycloaliphatic: The terms "cycloaliphatic," "carbocycle," "carbocyclyl," "carbocyclic radical," and "carbocyclic ring" are used interchangeably and as used herein refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems having from 3 to 30 ring members as described herein unless otherwise specified. Cycloaliphatic radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic radical has 3-6 carbons. In some embodiments, the cycloaliphatic radical is saturated and is a cycloalkyl. The term "cycloaliphatic" may also include aliphatic rings fused to one or more aromatic or non-aromatic rings such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, the alicyclic group is a bicyclic ring. In some embodiments, the alicyclic group is a tricyclic ring. In some embodiments, the alicyclic group is a polycyclic ring. In some embodiments, "alicyclic" refers to C 3 -C 6 Monocyclic hydrocarbons, or C 8 -C 10 A bicyclic or polycyclic hydrocarbon which is fully saturated or contains one or more unsaturated but non-aromatic units and which has a single point of attachment to the rest of the molecule, or C 9 -C 16 Polycyclic hydrocarbons, which are either fully saturated or contain one or more units that are unsaturated but are not aromatic, have a single point of attachment to the rest of the molecule.

[0159] Heteroaliphatic: As used herein, the term "heteroaliphatic" has its usual meaning in the art and refers to aliphatic groups described herein in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, the group selected from C, CH, CH 2 and CH 3 One or more units of are independently replaced by one or more heteroatoms (including oxidation and / or substitution forms thereof). In some embodiments, the heteroaliphatic group is a heteroalkyl group. In some embodiments, the heteroaliphatic group is a heteroalkenyl group.

[0160] Heteroalkyl: As used herein, the term "heteroalkyl" has its usual meaning in the art and refers to an alkyl group as described herein in which one or more carbon atoms are independently replaced by 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.

[0161] Heteroaryl: As used herein, the terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety (e.g., "heteroaralkyl" or "heteroaralkoxy"), refer to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, heteroaryl is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), and in some embodiments, is a group having 5, 6, 9, or 10 ring atoms. In some embodiments, heteroaryl has 6, 10, or 14 pi electrons shared in a cyclic array; and has one to five heteroatoms in addition to carbon atoms. Heteroaryl includes, but is not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl and pteridinyl. In some embodiments, heteroaryl is a heterobiaryl, such as bipyridyl, etc. As used herein, the terms "heteroaryl" and "heteroaromatic-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic or heterocyclyl rings, wherein the linking group or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolyl, tetrahydroisoquinolyl and pyrido [2,3-b] -1,4-oxazine -3 (4H) -one. Heteroaryl can be monocyclic, bicyclic or polycyclic. The term "heteroaryl" can be used interchangeably with the term "heteroaryl ring", "heteroaryl group" or "heteroaromatic", any of which includes optionally substituted rings. The term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl, wherein the alkyl and heteroaryl moieties are independently optionally substituted.

[0162] Heteroatom: As used herein, the term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any quaternized form of basic nitrogen; or a substitutable nitrogen of a heterocyclic ring (e.g., N in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (such as in N-substituted pyrrolidinyl) etc.).

[0163] Heterocycle: As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical" and "heterocyclic ring" are used interchangeably and refer to a monocyclic, bicyclic or polycyclic 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 is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably one to four heteroatoms as defined above. When used in reference to the ring atoms of the heterocycle, the term "nitrogen" includes substituted nitrogen. For 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 the pyrrolidinyl substituted by N-).Heterocycle can be connected to its side group at any heteroatom or carbon atom producing a stable structure, and any ring atom can be optionally substituted.The example of such saturated or partially unsaturated heterocyclic radical includes but is not limited to tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolane, diazepine, oxazolidinyl, thioazepine, morpholinyl and quinuclidinyl. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic group", "heterocyclic moiety" and "heterocyclic radical" are used interchangeably herein and also include groups in which the heterocyclyl ring is fused to one or more aryl, heteroaryl or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl or tetrahydroquinolinyl. The heterocyclyl group may be monocyclic, bicyclic or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group, wherein the alkyl and heterocyclyl moieties are independently optionally substituted.

[0164] Lower alkyl: The term "lower alkyl" refers to a C 1-4 Straight or branched chain alkyl. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl.

[0165] Lower haloalkyl: The term "lower haloalkyl" refers to a C 1-4 Straight chain or branched chain alkyl.

[0166] Optionally substituted: As described herein, the compounds (e.g., oligonucleotides) of the present disclosure may contain optionally substituted and / or substituted parts. In general, the term "substituted", whether or not preceded by the term "optional", refers to one or more hydrogens of a specified part being replaced by 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 substituent may be the same or different at each position. In some embodiments, the optionally substituted group is unsubstituted. The combination of substituents contemplated by the present disclosure is preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that do not substantially change when subjected to conditions that allow them to be produced, detected, and in certain embodiments, to be recovered, purified, and used for one or more purposes disclosed herein.

[0167] Suitable monovalent substituents on a substitutable atom (e.g., a suitable carbon atom) are independently halogen; -(CH 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 o ;-(CH 2 ) 0-4 CH(OR o ) 2 ; o Substituted -(CH 2 ) 0-4 Ph; by R o Substituted -(CH 2 ) 0-4 O(CH 2 ) 0-1 Ph; by R o -CH=CHPh substituted; can be R o Substituted -(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 o ;-(CH 2 ) 0-4 SC(O)R o ;-(CH 2 ) 0-4 C(O)NR o 2 ;-C(S)NR o 2 ;-C(S)SRo ;-SC(S)SR o ;-(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 NR o 2 ;-N(R o )S(O) 2 R o ;-N(OR o )R o ;-C(NH)NR o 2 ;-Si(R o ) 3 ;-OSi(R o ) 3 ;-B(R o ) 2 ;-OB(R o ) 2 ;-OB(OR o ) 2 ;-P(R o ) 2 ;-P(OR o ) 2 ;-OP(R o) 2 ;-OP(OR o ) 2 ;-P(O)(R o ) 2 ;-P(O)(OR o ) 2 ;-OP(O)(R o ) 2 ;-OP(O)(OR o ) 2 ;-OP(O)(OR o )(SR o );-SP(O)(R o ) 2 ;-SP(O)(OR o ) 2 ;-N(R o )P(O)(R o ) 2 ;-N(R o )P(O)(OR o ) 2 ;-P(R o ) 2 [B(R o ) 3 ];-P(OR o ) 2 [B(R o ) 3 ];-OP(R o ) 2 [B(R o ) 3 ];-OP(OR o ) 2 [B(R o ) 3 ];-(C 1-4 Straight chain or branched alkylene)ON(R o ) 2 ; or -(C 1-4 Straight or branched alkylene)C(O)ON(R o ) 2 , where each R o may be substituted as defined below and are independently hydrogen, C 1-20 aliphatic, C having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus 1-20 Heteroaliphatic, -CH 2 -(C 6-14 aryl), -O(CH 2 ) 0-1 (C 6-14 aryl), -CH 2-(5-14 membered heteroaryl ring), a 5-20 membered monocyclic, bicyclic or polycyclic, saturated, partially unsaturated or aromatic ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or, notwithstanding the above definition, two independent occurrences of R o and its intervening atoms together 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 substituted as defined below.

[0168] R o (or two independent occurrences of R o Suitable monovalent substituents on the ring formed by the intermediate atoms thereof are independently halogen, -(CH 2 ) 0-2 R · 、-(halogenated R · )、-(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR · 、-(CH 2 ) 0-2 CH(OR · ) 2 、-O(halogenated 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 SR · 、-(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 Straight or branched alkylene)C(O)OR · or -SSR · , where each R · is unsubstituted or, when preceded by "halo", substituted only by one or more halogens, and is independently selected from 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. o Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0169] For example, suitable divalent substituents on suitable carbon atoms are independently the following: =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-, where each independent occurrence of R * is selected from hydrogen, C which may be substituted as defined below 1-6 aliphatic, and unsubstituted 5-6 membered saturated, partially unsaturated or aromatic rings having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. Suitable divalent substituents bonded to the ortho-substitutable carbon of the "optionally substituted" group include: -O(CR * 2 ) 2–3 O-, where each independent occurrence of R * Selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, and unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0170] R * Suitable substituents on the aliphatic group are independently halogen, -R · 、-(halogenated R · ), -OH, -OR · 、-O(halogenated R ·)、-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", substituted only by one or more halogens, and is independently C 1-4 Aliphatic, -CH 2 Ph,-O(CH 2 ) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated and or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0171] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation and is not intended to include aryl or heteroaryl moieties as defined herein.

[0172] RNA interference: As used herein, the term "RNA interference" or "RNAi" refers to a post-transcriptional targeted gene silencing process involving RISC (RNA-induced silencing complex). It is reported that the process of RNAi occurs naturally when ribonuclease III (Dicer) cuts longer dsRNA into shorter fragments (called siRNA). Naturally produced siRNA (small interfering RNA) is usually about 21 to 23 nucleotides in length, has about 19 base pair duplexes and two single-stranded overhangs, and is usually RNA. These RNA fragments are then reported to guide the degradation of target nucleic acids (such as mRNA or pre-mRNA). It is reported that Dicer also involves the excision of 21 and 22 nucleotides of small sequential RNA (stRNA) involved in translation control from precursor RNA with a conserved structure. Hutvagner et al. 2001, Science, 293, 834. Those skilled in the art will appreciate that RNAi can be mediated by single-stranded or double-stranded oligonucleotides, which include sequences that are complementary or substantially complementary to the target sequence (e.g., in the target mRNA). Therefore, in some embodiments of the present disclosure, single-stranded oligonucleotides as described herein can serve as RNAi agents; In some embodiments, double-stranded oligonucleotides as described herein can serve as RNAi agents. In some embodiments, RNAi reactions involve endonuclease complexes, commonly referred to as RNA-induced silencing complexes (RISC), which guide the cutting of single-stranded mRNAs complementary to the antisense strand of siRNA. In some embodiments, RISC guides the cutting of target RNAs complementary to the oligonucleotides provided, and the oligonucleotides can be used as single-stranded RNAi agents. In some embodiments, the cutting of target RNA occurs in the middle of a region complementary to the antisense strand of a siRNA duplex or a single-stranded RNAi agent. In some embodiments, RNA interference is guided by single-stranded oligonucleotides, which serve as single-stranded RNAi agents, which can guide RNA interference in a mechanism involving RISC approaches.

[0173] RNAi agent: As used herein, the terms "RNAi agent", "iRNA agent", etc. refer to a PNPLA3 oligonucleotide that reduces the level and / or activity (e.g., translation) of a target gene product (e.g., a transcript of a target gene, such as pre-mRNA or mRNA) when administered to a system in which the target gene product is being expressed or has been expressed. In some embodiments, the RNAi agent may be or comprise a single-stranded oligonucleotide or a double-stranded oligonucleotide. In some embodiments, the RNAi agent may have a structure recognized in the art, such as siRNA (short inhibitory RNA), shRNA (short or small hairpin RNA), dsRNA (double-stranded RNA), microRNA, etc. In some embodiments, the RNAi agent may specifically bind to an RNA target (e.g., a transcript of a target gene). In some embodiments, after binding to its target, the RNAi agent is then loaded into RISC (RNA-induced silencing complex). In some embodiments, the RNAi agent, in some embodiments, directs the degradation of its target and / or inhibits the translation of its target by 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, the single-stranded RNAi agent oligonucleotide may be or comprise a sense or antisense strand sequence, such as Sioud 2005 J. Mol. Biol. 348: 1079-1090. In some embodiments, the RNAi agent is a compound capable of directing RNA interference. In some embodiments, the RNAi agent may have a structure or format such as that present in a "canonical" siRNA structure). In some embodiments, the RNAi agent may have a structure different 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, may be blunt-ended, and / or may comprise one or more modifications, mismatches, gaps, and / or nucleotide substitutions. In some embodiments, the RNAi agent contains a 3'-terminal cap as described in the present disclosure. Without wishing to be bound by any particular theory, applicants propose that, in certain embodiments, the 3'-end cap can allow two functions: (1) allowing RNA interference; and (2) increasing the duration of activity and / or biological half-life of the RNAi agent (which can be achieved, for example, by increasing binding to 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 those in serum or intestinal fluid). In some embodiments, the RNAi agents of the present disclosure target (e.g., bind to, anneal to, etc.) a target mRNA.In some embodiments, exposure of the RNAi agent to its target results in a reduction in activity, level, and / or expression, such as a "knockdown" or "knockout" of the target. In particular, in some embodiments, in the case of a disease, disorder, and / or condition characterized by overexpression and / or overactivity of the target gene, administration of the RNAi agent to a cell, tissue, or subject sufficiently knocks down the target gene to restore normal activity levels or reduce activity to a level that can alleviate, improve, mitigate, inhibit, prevent the disease, disorder, and / or condition, delay its onset, reduce its severity, and / or reduce the occurrence of one or more symptoms or features thereof. In some embodiments, the RNAi agent is double-stranded, comprising an antisense strand, which is a single-stranded RNAi agent as described herein, which in combination with the sense strand can guide RNA interference.

[0174] Single-stranded RNA interference: As used herein, the phrase "single-stranded RNAi" or "single-stranded RNA interference" and the like refer to a process or method of gene silencing directed, at least in part, by administering a single-stranded RNAi agent to a system (e.g., a cell, a tissue, an organ, a subject, etc.), wherein the RNAi will be directed by the RNAi agent and a RISC pathway is required. The term may be used herein in some cases to distinguish from "double-stranded RNAi" or "double-stranded RNA interference", wherein a double-stranded RNAi agent is administered to a system and can be further processed, such as causing one of its two strands to be loaded into RISC to, for example, inhibit translation, cleave target RNA, etc.

[0175] Single-stranded RNAi agent: As used herein, the phrase "single-stranded RNAi agent" refers to a single-stranded oligonucleotide that can direct single-stranded RNA interference (RNAi or iRNA) gene silencing through the RISC pathway. A single-stranded RNAi agent may include a polymer of one or more single-stranded nucleotides.

[0176] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which provided compounds or compositions are administered, e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes, according to the present invention. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0177] Substantially: As used herein, the term "substantially" refers to a qualitative condition that exhibits an overall or near-overall range or degree of a target feature or property. 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. In addition, one of ordinary skill in the biological field will appreciate that biological and chemical phenomena rarely, if ever, reach completeness and / or proceed to completeness or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the lack of potential completeness inherent in many biological and / or chemical phenomena.

[0178] Suffering from: An individual who is "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.

[0179] 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 members of the general public. 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.

[0180] Systemic: As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" have their art-understood meanings and refer to the administration of a compound or composition so that it enters the system of the recipient.

[0181] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces 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, mitigate, inhibit, prevent, delay the onset of, lessen the severity of, and / or reduce the occurrence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0182] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to the 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 appreciated by one of ordinary skill in the art, the effective amount of a substance may vary according to factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, the effective amount of a compound for treating a disease, disorder, and / or condition in a formulation is an amount that alleviates, improves, alleviates, inhibits, prevents a disease, disorder, and / or condition, delays its onset, reduces its severity, and / or reduces the occurrence of one or more symptoms or characteristics thereof. In some embodiments, the therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0183] Treatment: As used herein, the terms "treat", "treatment" or "treating" refer to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and / or reducing the occurrence of one or more symptoms or characteristics of a disease, disorder and / or condition. Treatment may be administered to a subject who does not show signs of a disease, disorder and / or condition. In some embodiments, treatment may be administered to a subject who only shows early signs of a disease, disorder and / or condition, for example, to achieve the purpose of reducing the risk of developing lesions associated with the disease, disorder and / or condition.

[0184] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0185] Wild-type: As used herein, the term "wild-type" has its meaning as understood in the art and refers to an entity having structure and / or activity as found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or background. One of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in a variety of different forms (e.g., alleles).

[0186] 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 (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule, and therefore include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. These terms include analogs of RNA or DNA made from modified nucleotides and / or modified polynucleotides (such as, but not limited to, methylated, protected and / or capped nucleotides or polynucleotides) as equivalents. The term encompasses polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or 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 ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise indicated, the prefix poly refers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units, and wherein the prefix oligo refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0187] 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 linkages. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but it is understood that natural and non-natural base analogs are also included. Naturally occurring sugars are pentoses (five-carbon sugars) deoxyribose (which forms DNA) or ribose (which forms RNA), but it is understood that natural and non-natural sugar analogs are also included. Nucleotides are linked by internucleotide linkages to form nucleic acids or polynucleotides. Many internucleotide linkages are known in the art (such as, but not limited to, phosphates, phosphorothioates, borophosphates, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriester, phosphorothioate, H-phosphonate, phosphoramidate, borophosphate, 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 linkages. As used herein, the term "nucleotide" also encompasses structural analogs for replacing natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs.

[0188] Modified nucleotides: The term "modified nucleotides" includes any chemical moiety that is structurally different from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, the modified nucleotides comprise modifications at the sugar, base, and / or internucleotide linkages. In some embodiments, the modified nucleotides comprise modified sugars, modified nucleobases, and / or modified internucleotide linkages. In some embodiments, the modified nucleotides are capable of having at least one function of a nucleotide, such as forming a subunit in a polymer that is capable of base pairing with a nucleic acid comprising at least a complementary base sequence.

[0189] Analog: The term "analog" means any functional analog in which a chemical moiety is structurally different from a reference chemical moiety or class of moieties but is capable of performing at least one function of such reference chemical moiety or class of moieties. As non-limiting examples, nucleotide analogs are structurally different from nucleotides but perform at least one function of nucleotides; nucleobase analogs are structurally different from nucleobases but perform at least one function of nucleobases; etc.

[0190] Nucleoside: The term "nucleoside" refers to a moiety wherein a nucleobase or modified nucleobase is covalently bonded to a sugar or modified sugar.

[0191] Modified nucleosides: The term "modified nucleosides" refers to a portion derived from or chemically similar to a natural nucleoside, but it comprises a chemical modification that distinguishes it from a natural nucleoside. Non-limiting examples of modified nucleosides include those nucleosides comprising modifications at the base and / or sugar. Non-limiting examples of modified nucleosides include those nucleosides having 2' modifications at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack a core base). In some embodiments, the modified nucleosides can have at least one function of a nucleoside, such as forming a portion in a polymer that can base pair with a nucleic acid comprising at least a complementary base sequence.

[0192] Nucleoside analogs: The term "nucleoside analogs" refers to chemical moieties that are chemically different from natural nucleosides but are capable of performing at least one function of a nucleoside. In some embodiments, nucleoside analogs comprise analogs of sugars and / or analogs of nucleobases. In some embodiments, modified nucleosides are capable of having at least one function of a nucleoside, such as forming a moiety in a polymer that is capable of base pairing with a nucleic acid comprising a complementary base sequence.

[0193] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in a closed and / or open form. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranosyl, pentopyranose, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs for replacing conventional sugar molecules, such as diols, polymers forming the backbone of nucleic acid analogs, diol nucleic acids ("GNA"), etc. As used herein, the term "sugar" also encompasses structural analogs for replacing natural or naturally occurring nucleotides, such as modified sugars and nucleotide sugars.

[0194] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar.

[0195] Nucleobase: The term "nucleobase" refers to the portion of a nucleic acid that is involved in hydrogen bonding that binds one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase is a "modified nucleobase," such as 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, the modified nucleobase simulates the spatial arrangement, electronic properties or some other physicochemical properties of the nucleobase, and retains the property of hydrogen bonding that binds one nucleic acid chain to another nucleic acid chain in a sequence-specific manner. In some embodiments, the modified nucleobase can be paired with all five naturally occurring bases (uracil, thymine, adenine, cytosine or guanine) without substantially affecting the unwinding behavior, the recognition by intracellular enzymes or the activity of oligonucleotide duplexes. As used herein, the term "nucleobase" also encompasses structural analogs used to replace natural or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs.

[0196] Modified nucleobase: The terms "modified nucleobase", "modified base", etc. refer to a chemical moiety that is chemically different from a nucleobase but is capable of performing at least one function of a nucleobase. In some embodiments, a modified nucleobase is a nucleobase comprising a modification. In some embodiments, a modified nucleobase is capable of having at least one function of a nucleobase, such as forming a moiety in a polymer that is capable of base pairing with a nucleic acid comprising at least a complementary base sequence.

[0197] 3'-Terminal Cap: The term "3'-terminal cap" refers to a non-nucleotide chemical moiety that is bound to the 3'-terminus of a PNPLA3 oligonucleotide (e.g., an RNAi agent). In some embodiments, the 3'-terminal cap replaces the 3'-terminal dinucleotide. In some embodiments, the 3'-terminal cap of a PNPLA3 oligonucleotide performs at least one of the following functions: allows oligonucleotide-directed RNA interference, protects the oligonucleotide from degradation or reduces the amount or rate of oligonucleotide degradation (e.g., by nucleases), reduces off-target effects of the sense strand, or increases the activity, duration, or efficacy of oligonucleotide-directed RNA interference. By describing the 3'-terminal cap as "non-nucleotide," it is meant that the 3'-terminal cap is not a nucleotide moiety or oligonucleotide moiety that is attached to the sugar moiety of the remainder of the PNPLA3 oligonucleotide, as if it were part of the PNPLA3 oligonucleotide chain. Certain exemplary 3'-terminal caps are described herein. One of ordinary skill in the art will appreciate that other 3'-terminal caps known in the art can be used in accordance with the present disclosure.

[0198] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group can be subsequently unmasked by removing the blocking group. In some embodiments, the blocking group is a protecting group.

[0199] Moiety: The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often identified as a chemical entity embedded in or attached to a molecule.

[0200] Solid support: The term "solid support" refers to any support that enables the synthesis of nucleic acids. In some embodiments, the term refers to glass or polymers that are insoluble in the medium used to perform the reaction steps for synthesizing nucleic acids and are 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).

[0201] Linker or connecting moiety: The terms "linker," "connecting moiety," and the like refer to any chemical moiety that connects one chemical moiety to another. In some embodiments, a linker is a moiety that connects one oligonucleotide to another oligonucleotide in a polymer. In some embodiments, a linker is a moiety that is optionally located between a terminal nucleoside and a solid support or between a terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0202] 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 encodes a protein or a portion thereof. A DNA molecule may contain an open reading frame (such as an exon sequence) encoding a protein, and may also include 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 present between exons in some but not all cases. It may be desirable that the gene be operably linked to (or it may contain) one or more promoters, enhancers, repressors and / or other regulatory sequences to regulate the activity or expression of the gene, as is well known in the art.

[0203] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes recombinant polynucleotides synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.

[0204] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, and may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0205] Oligonucleotide can be single-stranded or double-stranded.As used herein, the term "oligonucleotide chain" encompasses single-stranded oligonucleotides.Single-stranded oligonucleotides can have double-stranded regions (formed by two parts of single-stranded oligonucleotides), and double-stranded oligonucleotides comprising two oligonucleotide chains can, for example, have single-stranded regions at regions where two oligonucleotide chains are not complementary to each other.In some embodiments, oligonucleotides can guide the expression and / or level reduction of target genes or their gene products.In some embodiments, oligonucleotides can guide the expression and / or level reduction of target genes or their gene products by RNA interference.In some embodiments, oligonucleotides can guide the expression and / or level reduction of target genes or their gene products by biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNA enzyme H-mediated knocking down or spatial steric hindrance of gene expression).In some embodiments, oligonucleotides can guide the expression and / or level reduction of target genes or their gene products by RNA interference and / or RNA enzyme H-mediated knocking down. Exemplary oligonucleotides include, but are not limited to, structural genes, genes including control and termination regions, self-replicating systems (such as viral or plasmid DNA), single-stranded and double-stranded RNAi agents and other RNA interference agents (RNAi agents or iRNA agents), shRNAs, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, super microRNAs, aptamers, antisense microRNAs, microRNA antagonists, Ul adapters, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0206] Double-stranded and single-stranded oligonucleotides that effectively induce RNA interference are also referred to herein as RNAi agents or iRNA agents. In some embodiments, these oligonucleotides that induce RNA interference associate with a cytoplasmic multiprotein complex called the RNAi-induced silencing complex (RISC). In many embodiments, double-stranded RNAi agents are long enough so that they can be cut by endogenous molecules (e.g., by Dicer), producing smaller oligonucleotides that can enter the RISC mechanism and participate in the cutting and / or translation inhibition of RISC-mediated target sequences (e.g., target mRNA sequences).

[0207] The oligonucleotides disclosed herein may have various lengths. In certain embodiments, the length of the oligonucleotide may be in the range of about 2 to about 200 nucleotides. In various related embodiments, the length of the single-stranded, double-stranded, and triple-stranded oligonucleotides may be in the range of 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 some embodiments, the length of the PNPLA3 oligonucleotide is about 10 to about 40 nucleotides. In some embodiments, the length of the PNPLA3 oligonucleotide is about 9 to about 39 nucleotides. In some embodiments, the length of the oligonucleotide is at least 4 nucleotides. In some embodiments, the length of the oligonucleotide is at least 5 nucleotides. In some embodiments, the length of the oligonucleotide is at least 6 nucleotides. In some embodiments, the length of the oligonucleotide is at least 7 nucleotides. In some embodiments, the length of the oligonucleotide is at least 8 nucleotides. In some embodiments, the length of the oligonucleotide is at least 9 nucleotides. In some embodiments, the length of the oligonucleotide is at least 10 nucleotides. In some embodiments, the length of the oligonucleotide is at least 11 nucleotides. In some embodiments, the length of the oligonucleotide is at least 12 nucleotides. In some embodiments, the length of the oligonucleotide is at least 15 nucleotides. In some embodiments, the length of the oligonucleotide is at least 20 nucleotides. In some embodiments, the length of the oligonucleotide is at least 25 nucleotides. In some embodiments, the length of the oligonucleotide is at least 30 nucleotides. In some embodiments, the oligonucleotide is a duplex of a complementary chain of at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a duplex of a complementary chain of at least 21 nucleotides in length. In some embodiments, each nucleotide counted in length independently comprises an optionally substituted nucleobase selected from the group consisting of adenine, cytosine, guanosine, thymine and uracil.

[0208] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to the linkage that connects the nucleoside units of a PNPLA3 oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkage is a phosphodiester linkage (natural phosphate linkage) as found in naturally occurring DNA and RNA molecules. In some embodiments, the term "internucleotide linkage" includes modified internucleotide linkages. In some embodiments, the internucleotide linkage is 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, such an organic or inorganic moiety is selected from, but not limited to, =S, =Se, =NR', -SR', -SeR', -N(R') 2 , B(R') 3 , -S-, -Se-, and -N(R')-, wherein each R' is independently defined and described in the present disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a or modified phosphorothioate triester linkages.

[0209] One of ordinary skill in the art will appreciate that at a given pH, an internucleotide linkage may exist as an anion or a cation due to the presence of an acid or base moiety in the linkage.

[0210] In some embodiments, "all (Rp)" or "all (Sp)" is used to indicate that all chiral-bonded phosphorus atoms in an oligonucleotide have the same Rp or Sp configuration, respectively.

[0211] Oligonucleotide type: As used herein, the phrase "oligonucleotide type" is used to define an oligonucleotide having a specific base sequence, backbone linkage pattern (i.e., internucleotide linkage type pattern, such as phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linkage phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I). 1 In some embodiments, oligonucleotides of a commonly designated "type" are structurally identical to one another.

[0212] Those skilled in the art will appreciate that the synthetic methods of the present disclosure provide a degree of control during the synthesis of the PNPLA3 oligonucleotide chain, such that each nucleotide unit of the oligonucleotide chain can be designed and / or selected in advance to have a specific stereochemistry at the linkage phosphorus and / or a specific modification at the linkage phosphorus and / or a specific base and / or a specific sugar. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or selected in advance to have a specific combination of stereocenters at the linkage phosphorus. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or determined to have a specific combination of modifications at the linkage phosphorus. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or selected to have a specific combination of bases. In some embodiments, the PNPLA3 oligonucleotide chain is designed and / or selected to have a specific combination of one or more of the above structural features. In some embodiments, the present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chirality-controlled oligonucleotide compositions). In some embodiments, all such molecules are of the same type (i.e., structurally identical to one another). However, in many embodiments, the provided compositions typically comprise a plurality of different types of oligonucleotides in predetermined relative amounts.

[0213] Chirality control: As used herein, "chirality control" refers to control over the stereochemical design of a chiral linkage phosphorus in a chiral internucleotide linkage within a PNPLA3 oligonucleotide. In some embodiments, control is achieved by chiral elements that are not present in the sugar and base portions of the PNPLA3 oligonucleotide, for example, in some embodiments, control is achieved by using one or more chiral auxiliary agents during oligonucleotide preparation as disclosed herein, which are typically part of a chiral phosphoramidite used during oligonucleotide preparation. In contrast to chirality control, one of ordinary skill in the art understands that if a chiral internucleotide linkage is formed using conventional oligonucleotide synthesis that does not use a chiral auxiliary, such conventional oligonucleotide synthesis does not control the stereochemistry at the chiral internucleotide linkage. In some embodiments, the stereochemical design of each chiral linkage phosphorus in a chiral internucleotide linkage within a PNPLA3 oligonucleotide is controlled.

[0214] Chirality-controlled oligonucleotide composition: As used herein, the terms "chirality-controlled oligonucleotide composition", "chirality-controlled nucleic acid composition", etc. refer to a composition comprising a plurality of oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common backbone linkage pattern, and 3) a common backbone phosphorus modification pattern, wherein the plurality of oligonucleotides (or nucleic acids) have the same stereochemistry at one or more chiral internucleotide linkages (chirality-controlled internucleotide linkages), and the levels of the plurality of oligonucleotides (or nucleic acids) in the composition are predetermined (e.g., by chirality-controlled oligonucleotide preparation to form one or more chiral internucleotide linkages). In some embodiments, about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%) of all oligonucleotides in the chirality-controlled oligonucleotide composition. 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the plurality of oligonucleotides. In some embodiments, about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%) of all oligonucleotides in a chiral controlled oligonucleotide composition share a common base sequence. 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the plurality of oligonucleotides.In some embodiments, about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%) of all oligonucleotides in a chiral controlled oligonucleotide composition share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. , or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) are the plurality of oligonucleotides. In some embodiments, the predetermined level is about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-40% of all oligonucleotides in the composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern as all oligonucleotides in the composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern. %, 93%, 94%, 95%, 96%, 97%, 98% or 99%).In some embodiments, a plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides is present in an amount between about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%-100%, about 10 ... In some embodiments, the chiral internucleotide linkages are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% (or at least 5%) 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, not 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 a single oligonucleotide or nucleic acid type. For example, in some embodiments, the oligonucleotide composition of chirality control comprises a kind of oligonucleotide type.In some embodiments, the oligonucleotide composition of chirality control comprises more than one oligonucleotide type.In some embodiments, the oligonucleotide composition of chirality control comprises multiple oligonucleotide types.In some embodiments, the oligonucleotide composition of chirality control is a composition of the oligonucleotide of oligonucleotide type, and the composition comprises the oligonucleotide of the multiple oligonucleotide type of predetermined level.

[0215] Chirally pure: As used herein, the phrase "chirally pure" is used to describe the relative amounts of PNPLA3 oligonucleotides (eg, single-stranded RNAi agents) wherein all of the oligonucleotides exist in a single diastereomeric form with respect to the bound phosphorus.

[0216] Chirality: As used herein, the phrase "chirality" is used to describe a PNPLA3 oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the phosphorus linkage. For example, a PNPLA3 oligonucleotide in which all nucleotide units have an Rp stereochemistry at the phosphorus linkage is chirality-uniform. Similarly, a PNPLA3 oligonucleotide in which all nucleotide units have an Sp stereochemistry at the phosphorus linkage is chirality-uniform.

[0217] Predetermined: predetermined means deliberately selected, for example, in contrast to random occurrence or in the absence of control. Those of ordinary skill in the art who read this specification will appreciate that the present disclosure provides a technique that allows the selection of specific chemistry and / or stereochemical features to be incorporated into the oligonucleotide composition, and also allows the controlled preparation of oligonucleotide compositions with such chemistry and / or stereochemical features. Such compositions provided are "predetermined" as described herein. Because compositions that may contain certain oligonucleotides happen to have been produced by processes that cannot be controlled to intentionally produce specific chemistry and / or stereochemical features, they are not "predetermined" compositions. In some embodiments, a predetermined composition is a composition that can intentionally be regenerated (for example, by repeating a controlled process). In some embodiments, the predetermined level of multiple oligonucleotides in the composition means the absolute amount and / or relative amount (ratio, percentage, etc.) of multiple oligonucleotides in the control composition. In some embodiments, the predetermined level of multiple oligonucleotides in the composition is achieved by preparing the oligonucleotides controlled by chirality.

[0218] Phosphorus bond: As defined herein, the phrase "phosphorus bond" is used to indicate that the particular phosphorus atom referred to is a phosphorus atom present in an internucleotide linkage corresponding to the phosphorus atom of a phosphodiester internucleotide linkage as present in naturally occurring DNA and RNA. In some embodiments, the phosphorus bond 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 phosphorus bond atom is a phosphorus atom of Formula I. L In some embodiments, the bonded phosphorus atom is chiral.

[0219] P modification: As used herein, the term "P modification" refers to any modification at the linkage phosphorus other than a stereochemical modification. In some embodiments, the P modification comprises the addition, substitution, or removal of a pendant moiety covalently attached to the linkage phosphorus. In some embodiments, the "P modification" is -XLR 1 , where X, L and R 1 Each of is independently as defined and described in this disclosure.

[0220] For purposes of this disclosure, chemical elements are identified according to the CAS version of the Periodic Table of the Elements on the inside cover of Handbook of Chemistry and Physics, 67th edition, 1986-87.

[0221] The methods and structures described herein with respect to the compounds and compositions of the present disclosure also apply to the pharmaceutically acceptable acid or base addition salts and all stereoisomeric forms of these compounds and compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0222] Figure 1. Figure 1, including Figures 1A to 1L , cartoon diagrams showing various ssRNAi formats and hybridization formats.

[0223] Figure 2 . Figure 2 Cartoons of various antisense oligonucleotide formats are shown.

[0224] Figure 3. Figure 3A Exemplary polymer formats are shown. Oligonucleotides can be joined directly and / or through joints. As shown, a polymer can include oligonucleotide monomers of the same or different structures / types. In some embodiments, the monomer of the polymer is an ssRNAi agent. In some embodiments, the monomer of the polymer is an RNase H dependent antisense oligonucleotide (ASO). Monomers can be joined by various positions, such as 5'-end, 3'-end or intermediate positions. Figure 3B Exemplary chemistries are shown for joining monomers that can perform their functions in various ways to form multimers. DETAILED DESCRIPTION

[0225] Synthetic oligonucleotides are provided in a wide variety of available molecular tools in applications. For example, oligonucleotides can be used for treatment, diagnosis, research and novel nanomaterial applications. The use of naturally occurring nucleic acids (for example, unmodified DNA or RNA) is, for example, restricted because they are susceptible to endonucleases and exonucleases. Therefore, various synthetic counterparts have been developed to avoid these shortcomings. These include synthetic oligonucleotides containing chemical modifications (for example, base modifications, sugar modifications, main chain modifications, etc.), which especially make these molecules difficult for degradation and improve other properties of oligonucleotides. From a structural perspective, chirality can be introduced into the modification of phosphate linkages between nucleotides, and some properties of oligonucleotides may be affected by the configuration of the phosphorus atom forming the oligonucleotide main chain. For example, in vitro studies have shown that the properties of antisense oligonucleotides (such as binding affinity, sequence specificity in conjunction with complementary RNA, stability to nucleases) are especially affected by the chirality of the main chain phosphorus atom.

[0226] The present disclosure encompasses, inter alia, the recognition that structural elements of oligonucleotides, such as chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) 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], can have significant effects on properties and activities (e.g., stability, specificity, selectivity, activity in reducing target gene product (transcript and / or protein) levels, etc.). In some embodiments, oligonucleotide properties can be modulated by optimizing chemical modifications (modifications of bases, sugars, and / or internucleotide linkages), chemical modification patterns, stereochemistry, and / or stereochemical patterns.

[0227] In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides having controlled structural elements (e.g., controlled chemical modifications and / or controlled main chain stereochemistry patterns) provide unexpected properties and activities, including but not limited to those described herein. In some embodiments, the compositions provided comprising oligonucleotides having 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: guiding the reduction of expression and / or levels of a target gene or its gene product; and / or guiding RNA interference; and / or guiding RNA enzyme 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.

[0228] In some embodiments, RNA interference is reported to be a post-transcriptional targeted gene silencing technology that uses RNAi agents to target RNA (e.g., gene transcripts such as messenger RNA (mRNA)) containing sequences complementary to the RNAi agents for cleavage mediated by the RISC (RNA-induced silencing complex) pathway. In essence, it is reported that when ribonuclease III (Dicer) cuts long dsRNA (double-stranded RNA) (e.g., exogenous dsRNA introduced into mammalian cells) into shorter fragments called siRNA, a type of RNAi will occur. siRNA (small interfering RNA or short inhibitory RNA) is generally about 21 to 23 nucleotides long and contains about 19 base pair duplexes. It is reported that then the smaller RNA fragments mediate the degradation of the target mRNA. It is reported that the RNAi reaction is also characterized by an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which guides the cutting of a single-stranded mRNA complementary to the antisense strand of the siRNA. It is reported that the cutting of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex. It is reported that the use of RNAi agents to target transcripts results in a decrease in gene activity, level and / or expression, such as a "knockdown" or "knockout" of the target gene or target sequence. Artificial siRNAs can be used as both therapeutic agents and for experimental purposes.

[0229] On the one hand, RNA interference agent includes single-stranded RNA that interacts with target RNA sequence to guide target RNA cutting. Without wishing to be bound by theory, it is reported that the type III endonuclease referred to as Dicer decomposes the long double-stranded RNA introduced into plant and invertebrate cells into siRNA (Sharp et al., Genes Dev.2001, 15:485). It is reported that a ribonuclease III-like enzyme Dicer processes dsRNA into short interfering RNA of 19-23 base pairs, which has the characteristics of two base 3' overhangs (Bernstein et al., (2001) Nature 409:363). It is reported that siRNA is then incorporated into the RNA-induced silencing complex (RISC), wherein one or more helicases unwind the siRNA duplex, thereby enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). After binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15: 188). Thus, in one aspect, the present disclosure relates to single-stranded RNA that promotes RISC complex formation to achieve target gene silencing.

[0230] In some embodiments, appropriate RNAi agents can be selected by any method known in the art or any method that would occur to a person of ordinary skill in the art in light of the present disclosure. For example, the selection criteria may include one or more of the following steps: preliminary analysis of the target gene sequence and design of the RNAi agent; such design may take into account sequence similarities between species (human, cynomolgus monkey, mouse, etc.) and differences from other (non-target) genes; in vitro screening of RNAi agents (e.g., 10 nM in cells expressing the target transcript); determining the EC50 or IC50 in cells; determining the viability of cells treated with the RNAi agent, wherein in some embodiments, it is desired that the RNAi agent of the target does not inhibit the viability of these cells; testing with human PBMCs (peripheral blood mononuclear cells), for example, testing the level of TNF-α to estimate immunogenicity, wherein immunostimulatory sequences are generally less required; testing in a human whole blood assay, wherein fresh human blood is treated with the RNAi agent and cytokine / chemokine levels are determined [e.g., TNF-α (tumor necrosis factor-α) and / or MCP1 (monocyte chemoattractant protein 1)], wherein immunostimulatory sequences are generally less required; determining in vivo gene knockdown using cells or tumors in test animals; and optimizing specific modifications of the RNAi agent.

[0231] The so-called canonical siRNA structure is reported to be a double-stranded RNA molecule in which each strand is about 21 nucleotides long. The two strands are reported to be an antisense (or "leader") 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 reported to be largely complementary, typically forming two 3' overhangs of 2 nucleotides at both ends.

[0232] Although the canonical siRNA structure is reported to be double-stranded, the RNAi agent can also be single-stranded. In some embodiments, the single-stranded RNAi agent corresponds to the antisense strand of the double-stranded siRNA, and the single-stranded RNAi agent lacks the corresponding passenger strand.

[0233] However, it has been reported that not all structural elements of single-stranded RNAi agents tested are effective; introduction of some structural elements into PNPLA3 oligonucleotides has been reported to interfere with single-stranded RNA interference activity.

[0234] In some embodiments, the present disclosure provides oligonucleotides and compositions that can be used as RNAi agents. In some embodiments, the present disclosure provides oligonucleotides and compositions that can be used as single-stranded RNAi agents. The present disclosure particularly provides novel structures of single-stranded oligonucleotides that can guide RNA interference. Without wishing to be bound by any particular theory, the present disclosure points out that single-stranded RNAi agents have advantages over double-stranded RNAi agents. For example, the commodity cost of single-stranded RNAi agents is low because only one chain needs to be constructed. Additionally or alternatively, only one chain (antisense chain) is applied to target the target transcript. The source of the off-target effect guided by dsRNA is to load the sense chain into RISC and bind to and knock down unwanted targets (Jackson et al. 2003 Nat. Biotech. 21: 635-637), and single-stranded RNAi agents can cause fewer off-target effects compared to corresponding double-stranded RNAi agents. In addition, some single-stranded RNAi agents, including some disclosed herein, can target specific sequences that have not been successfully targeted by double-stranded RNAi agents before (e.g., they can significantly reduce the level of sequence and / or sequence products (transcripts and / or proteins) compared to double-stranded RNAi agents). The present disclosure provides, among other things, novel formats (modifications, stereochemistry, combinations thereof, etc.) of oligonucleotides that can direct single-stranded RNA interference.

[0235] Oligonucleotides

[0236] In some embodiments, the oligonucleotide provided can guide the reduction of the expression and / or level of the target gene or its gene product. In some embodiments, the oligonucleotide provided can guide the reduction of the target product level. In some embodiments, the oligonucleotide provided can reduce the transcript level of the target gene. In some embodiments, the oligonucleotide provided can reduce the mRNA level of the target gene. In some embodiments, the oligonucleotide provided can reduce the protein level encoded by the target gene. In some embodiments, the oligonucleotide provided can guide the reduction of the expression and / or level of the target gene or its gene product by RNA interference. In some embodiments, the oligonucleotide provided can guide the reduction of the expression and / or level of the target gene or its gene product by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the reduction of the expression and / or level of the target gene or its gene product by RNA interference and / or RNA enzyme H mediated knocking down. In some embodiments, the provided oligonucleotides can guide the reduction of expression and / or level of the target gene or its gene product by spatially blocking translation after binding to the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides include one or more structural elements described herein or known in the art according to the present disclosure, such as base sequence; modification; stereochemistry; internucleotide linkage pattern; main chain linkage pattern; main chain chiral center pattern; main chain phosphorus modification pattern; additional chemical parts, including but not limited to one or more targeting parts, lipid parts and / or carbohydrate parts, etc.; seed region; post-seed region; 5'-terminal structure; 5'-terminal region; 5' nucleotide part; 3'-terminal region; 3'-terminal dinucleotide; 3'-terminal cap; etc. In some embodiments, the seed region of the PNPLA3 oligonucleotide is or comprises the second to eighth, second to seventh, second to sixth, third to eighth, third to seventh, third to seventh, or fourth to eighth, or fourth to seventh nucleotides counting from the 5' end; and the post-seed region of the oligonucleotide is the region immediately 3' to the seed region and between the seed region and the 3' terminal region.

[0237] In some embodiments, provided compositions comprise a PNPLA3 oligonucleotide. In some embodiments, provided compositions comprise one or more lipid moieties, one or more carbohydrate moieties (unless otherwise indicated, in addition to the sugar moieties of the nucleoside units forming the oligonucleotide chain having an internucleotide linkage), and / or one or more targeting components.

[0238] In some embodiments, where the presence and / or activity of a particular allele (and / or one or more of its products (e.g., RNA and / or protein products)) (disease-associated allele) is associated (e.g., correlated) with the presence, incidence, and / or severity of one or more diseases and / or conditions, different alleles of the same gene (e.g., gene) are present but are not so associated or are less associated (e.g., exhibit less significant or statistically insignificant correlation). In some such embodiments, oligonucleotides and methods thereof as described herein can preferentially or specifically target associated alleles relative to one or more less associated / non-associated alleles.

[0239] In some embodiments, the target sequence is a sequence that binds to a PNPLA3 oligonucleotide as described herein. In many embodiments, the target sequence is identical or exactly complementary to a sequence of contiguous residues in a provided oligonucleotide or therein (e.g., the provided oligonucleotide includes a targeted binding sequence that is identical or exactly complementary to the target sequence). In some embodiments, a small amount of difference / mismatch is tolerated between a PNPLA3 oligonucleotide (a relevant portion thereof) and its target sequence. In many embodiments, the target sequence is present within a target gene. In many embodiments, the target sequence is present in a transcript (e.g., mRNA and / or pre-mRNA) produced by a target gene.

[0240] In some embodiments, the target sequence includes one or more allelic sites (i.e., the location where allelic variation occurs within the target gene). In some such embodiments, the oligonucleotides provided preferentially or specifically bind to one allele relative to one or more other alleles. In some embodiments, the targeted binding sequence is identical or precisely complementary to the target sequence of one allele. In some embodiments, the targeted binding sequence is identical to the target sequence of one allele. In some embodiments, the targeted binding sequence is precisely complementary to the target sequence of one allele. In some embodiments, the oligonucleotides provided preferentially bind to disease-associated alleles. In some embodiments, the oligonucleotides provided preferentially bind to disease-associated alleles and include a target binding sequence that is identical or precisely complementary to the target sequence of the disease-associated allele, but not to other alleles. For example, in some embodiments, the PNPLA3 oligonucleotides (or their targeted binding sequence portions) provided herein have a sequence that is identical or precisely complementary to a specific allelic form of the target sequence. In some embodiments, the target sequence is the sequence of a specific allele. In some embodiments, the PNPLA3 oligonucleotides provided herein (or targeted binding sequence portions thereof) have a sequence that is identical or exactly complementary to the allelic locus of a disease-associated allele.

[0241] As will be appreciated by those skilled in the art, various allele sites may be included in the target sequence in accordance with the present disclosure. In some embodiments, the target sequence comprises a SNP. In some embodiments, the target sequence comprises a mutation. In some embodiments, the SNP is a SNP in PNPLA3.

[0242] According to the present disclosure, various linkers, lipid moieties, carbohydrate moieties, and targeting moieties can be used, including many moieties known in the art. In some embodiments, the lipid moiety is a targeting moiety. In some embodiments, the carbohydrate moiety is a targeting moiety. In some embodiments, the targeting moiety is a lipid moiety. In some embodiments, the targeting moiety is a carbohydrate moiety. As will be readily appreciated by those skilled in the art, according to the present disclosure, various linkers, including those described in the present disclosure, can be used to connect two moieties, such as a lipid / carbohydrate / targeting component and a PNPLA3 oligonucleotide moiety. As will be readily appreciated by those skilled in the art, linkers described for connecting two moieties can also be used to connect other moieties, for example, linkers used to connect lipid and PNPLA3 oligonucleotide moieties can also be used to connect carbohydrate or targeting moieties to PNPLA3 oligonucleotide moieties, and vice versa.

[0243] In some embodiments, the present disclosure provides chirality controlled oligonucleotides and oligonucleotide compositions. For example, in some embodiments, provided compositions contain predetermined levels of one or more individual oligonucleotide types, wherein PNPLA3 oligonucleotide types are defined by: 1) base sequence; 2) backbone linkage pattern; 3) backbone chiral center pattern; and 4) backbone P modification pattern. In some embodiments, a particular oligonucleotide type can be defined by: 1A) base identity; 1B) base modification pattern; 1C) sugar modification pattern; 2) backbone linkage 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 present disclosure provides an oligonucleotide composition for controlling the chirality of oligonucleotides, wherein the composition comprises a predetermined level of a plurality of oligonucleotides, wherein the plurality of oligonucleotides share a common base sequence and comprise the same linkage phosphorus configuration at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 chiral internucleotide linkages (chirality-controlled internucleotide linkages).

[0244] In some embodiments, the oligonucleotides provided contain 2-30 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 5-30 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 10-30 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 1 chirality-controlled internucleotide linkage. In some embodiments, the oligonucleotides provided contain 2 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 3 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 4 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 5 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 6 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 7 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 8 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided contain 9 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided comprise 10 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided comprise 11 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided comprise 12 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided comprise 13 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided comprise 14 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided have 15 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided have 16 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided have 17 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided have 18 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided have 19 chirality-controlled internucleotide linkages. In some embodiments, the oligonucleotides provided have 20 chirality-controlled internucleotide linkages.

[0245] In some embodiments, the oligonucleotide provided is a monomer. In some embodiments, the oligonucleotide provided is a P-modified monomer. In some embodiments, the oligonucleotide provided is a stereo monomer. In some embodiments, the oligonucleotide provided is a stereo monomer with configuration Rp. In some embodiments, the oligonucleotide provided is a stereo monomer with configuration Sp.

[0246] In some embodiments, the oligonucleotides provided are alternators. In some embodiments, the oligonucleotides provided are P-modified alternators. In some embodiments, the oligonucleotides provided are stereo alternators.

[0247] In some embodiments, the oligonucleotides provided are block polymers. In some embodiments, the oligonucleotides provided are P-modified block polymers. In some embodiments, the oligonucleotides provided are stereoblock polymers.

[0248] In some embodiments, provided oligonucleotides are gapmers.

[0249] In some embodiments, the provided oligonucleotides are skipmers.

[0250] In some embodiments, the oligonucleotides provided are hemimers. In some embodiments, the hemimer is a PNPLA3 oligonucleotide in which the 5'-terminal or 3'-terminal region has a sequence that has a structural feature that the remaining oligonucleotides do not have. In some embodiments, the 5'-terminal or 3'-terminal region has or comprises 2 to 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 a stereochemistry of the chiral internucleotide linkage, or a combination thereof. In some embodiments, the hemimer is a PNPLA3 oligonucleotide in which each sugar moiety of the 5'-terminal region shares a common modification. In some embodiments, the hemimer is a PNPLA3 oligonucleotide in which each sugar moiety of the 3'-terminal region shares a common modification. In some embodiments, any other sugar moiety in the oligonucleotide does not share a common sugar modification of the 5' or 3'-terminal region. In some embodiments, an exemplary hemimer is a PNPLA3 oligonucleotide comprising a substituted or unsubstituted 2'-O-alkyl sugar modified nucleoside, a bicyclic sugar modified nucleoside, a β-D-ribonucleoside or a β-D-deoxyribonucleoside (e.g., a 2'-MOE modified nucleoside, and an LNA TM or ENA TMIn some embodiments, the oligonucleotide provided is a single polymer, an alternating polymer, a block polymer, a spacer polymer, a semipolymer and a jump polymer. In some embodiments, the oligonucleotide provided is a single polymer, an alternating polymer, a block polymer, a spacer polymer and a jump polymer. For example, in some embodiments, the oligonucleotide provided is an alternating polymer and a spacer polymer. In some embodiments, the nucleotide provided is a spacer polymer and a jump polymer. It will be appreciated by those skilled in the art of chemistry and synthesis that the combination of numerous other styles is available, and is only limited to the commercial availability and / or the synthetic accessibility of the components required for the oligonucleotide provided according to the disclosed method. In some embodiments, the semipolymer structure provides a favorable benefit. In some embodiments, the oligonucleotide provided is a 5'-semipolymer, which comprises a modified sugar moiety in the 5'-terminal sequence. In some embodiments, the provided oligonucleotides are 5'-hemimers that include a modified 2'-sugar moiety in the 5'-terminal sequence.

[0251] In some embodiments, the oligonucleotides provided comprise one or more optionally substituted nucleotides. In some embodiments, the oligonucleotides provided comprise one or more modified nucleotides. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted nucleosides. In some embodiments, the oligonucleotides provided comprise one or more modified nucleosides. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted LNAs.

[0252] In some embodiments, the oligonucleotides provided include one or more optionally substituted nucleobases. In some embodiments, the oligonucleotides provided include one or more optionally substituted natural nucleobases. In some embodiments, the oligonucleotides provided include one or more optionally substituted modified nucleobases. In some embodiments, the oligonucleotides provided include one or more 5-methylcytidines; 5-hydroxymethylcytidine, 5-formylcytosine or 5-carboxylcytosine. In some embodiments, the oligonucleotides provided include one or more 5-methylcytidines.

[0253] In some embodiments, each base (BA) is independently a core base of optionally substituted or protected adenine, cytosine, guanosine, thymine or uracil. As will be appreciated by those skilled in the art, various protected core bases, including those core bases well known in the art, such as those used in the preparation of oligonucleotides (for example, 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 protected core bases, each of which is incorporated herein by reference), and can be used according to the disclosure.

[0254] In some embodiments, the oligonucleotides provided comprise one or more optionally substituted sugars. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted sugars found in naturally occurring DNA and RNA. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted ribose or deoxyribose sugars. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted ribose or deoxyribose sugars, wherein one or more hydroxyl groups of the ribose or deoxyribose moiety are optionally and independently replaced by: halogen, R', -N(R') 2 , -OR' or -SR', wherein each R' is independently as defined above and described herein. In some embodiments, the provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2' position of the deoxyribose sugar is optionally and independently substituted with halogen, R', -N(R') 2 , -OR' or -SR', wherein each R' is independently as defined above and as described herein. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted deoxyriboses, wherein the 2' position of the deoxyribose is optionally and independently substituted with a halogen. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted deoxyriboses, wherein the 2' position of the deoxyribose is optionally and independently substituted with one or more -F halogens. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted deoxyriboses, wherein the 2' position of the deoxyribose is optionally and independently substituted with -OR', wherein each R' is independently as defined above and as described herein. In some embodiments, the oligonucleotides provided comprise one or more optionally substituted deoxyriboses, wherein the 2' position of the deoxyribose is optionally and independently substituted with -OR', wherein each R' is independently an optionally substituted C 1 -C 6In some embodiments, provided oligonucleotides comprise one or more optionally substituted deoxyribose sugars, wherein the 2' position of the deoxyribose sugar is optionally and independently substituted with -OR', wherein each R' is independently an optionally substituted C 1 -C 6 In some embodiments, the oligonucleotides provided include one or more optionally substituted deoxyriboses, wherein the 2' position of the deoxyribose is optionally and independently substituted with -OMe. In some embodiments, the oligonucleotides provided include one or more optionally substituted deoxyriboses, wherein the 2' position of the deoxyribose is optionally and independently substituted with -O-methoxyethyl.

[0255] In some embodiments, the oligonucleotide provided is a hybridized oligonucleotide chain. In certain embodiments, the oligonucleotide provided is a partially hybridized oligonucleotide chain. In certain embodiments, the oligonucleotide provided is a fully hybridized oligonucleotide chain. 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).

[0256] In some embodiments, any of the structures of the PNPLA3 oligonucleotides described in WO2012 / 030683 can be modified according to the methods of the present disclosure to provide compositions having controlled chirality thereof. For example, in some embodiments, the chirality controlled compositions comprise stereochemical control at any one or more chiral bonded phosphorus atoms, optionally by incorporation of 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 of WO2012 / 030683 is preselected to place a stereochemical modification at the bonded phosphorus of the nucleotide unit and / or to be P modified with chirality control at the bonded phosphorus of the nucleotide unit.

[0257] In some embodiments, provided oligonucleotides comprise nucleic acid analogs, e.g., 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.

[0258] In some embodiments, the oligonucleotide provided is characterized as being able to indirectly or directly increase or decrease the activity of a protein or inhibit or promote the expression of a protein. In some embodiments, the oligonucleotide provided is characterized in that it can be used to control cell proliferation, viral replication and / or any other cell signaling process.

[0259] In some embodiments, the 5'-end and / or 3'-end of the oligonucleotide provided is modified. In some embodiments, the 5'-end and / or 3'-end of the oligonucleotide provided is modified by a terminal cap portion. Examples of such modifications including terminal cap portions are widely described herein and in the art, such as, but not limited to, those modifications described in U.S. Patent Application Publication US2009 / 0023675A1.

[0260] In some embodiments, oligonucleotides of the type of PNPLA3 oligonucleotides characterized by 1) a common base sequence and length, 2) a common backbone linkage pattern, and 3) a common backbone chiral center pattern have the same chemical structure. For example, they have the same base sequence, the same nucleoside modification pattern, the same backbone linkage pattern (i.e., the pattern of internucleotide linkage types, such as phosphate, phosphorothioate, etc.), the same backbone chiral center pattern (i.e., the linkage phosphorus stereochemistry pattern (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., "-XLR" in Formula I). 1 " group style).

[0261] Single-stranded RNAi agents and antisense oligonucleotides

[0262] In some embodiments, the disclosure provides oligonucleotides. In some embodiments, the disclosure provides oligonucleotides that reduce the expression and / or level of a target gene or its gene product. Those of ordinary skill in the art who read the disclosure will appreciate that in some embodiments, the oligonucleotides provided can serve as RNAi agents. Alternatively or additionally, in some embodiments, the oligonucleotides provided can act through an RNase H-dependent mechanism and / or another biochemical mechanism that does not involve RNA interference.

[0263] The present disclosure defines, among other things, certain structural attributes that may be particularly desirable and / or effective in PNPLA3 oligonucleotides. The present disclosure defines, among other things, certain structural attributes that may be particularly desirable and / or effective in 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 in PNPLA3 oligonucleotides that act by 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 in single-stranded ssRNAi agents (ssRNAi or ssRNAi agents); in some such embodiments, such structural attributes may be different from those structural attributes that are particularly desirable and / or effective in the corresponding strands of double-stranded RNAi agents (dsRNAi or dsRNAi agents), as further described below. In some embodiments, the oligonucleotide provided is a single-stranded RNAi agent (e.g., it can be loaded into RISC and / or can direct or enhance RISC-mediated targeting). In some embodiments, the oligonucleotides provided are antisense oligonucleotides (eg, which can be loaded into RNase H and / or direct or enhance RNase H-mediated cleavage of a target and / or operate via a different biochemical mechanism).

[0264] In some embodiments (including in 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 those oligonucleotides that act by an RNase H-dependent mechanism. In some embodiments, PNPLA3 oligonucleotides may direct a decrease in the expression and / or level of a target gene or its gene product by sterically blocking translation after binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion (e.g., skipping). In some embodiments, PNPLA3 oligonucleotides may perform a function or a significant percentage of a function (e.g., 10%-100%, not less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) independently of RNA interference or RISC.

[0265] In some embodiments, the oligonucleotides provided are antisense oligonucleotides (ASOs) that direct cleavage of the target RNA mediated by RNase H rather than RISC (RNA interference silencing complex).

[0266] In some embodiments, the oligonucleotides provided are single-stranded RNAi (ssRNAi) agents that direct cleavage of a target mRNA mediated by RISC (RNA interference silencing complex) rather than the enzyme RNase H. In some embodiments, the PNPLA3 oligonucleotides can perform a function or a significant percentage of function (e.g., 10%-100%, not less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) independent of RNase H.

[0267] Double-stranded RNAi agents can also use RISC instead of the enzyme RNase H to guide the cutting of target mRNA. In some embodiments, the difference between single-stranded RNAi agents and double-stranded RNAi agents is that ssRNAi agents only include one oligonucleotide chain and generally do not include a double-stranded region of significant length, while dsRNAi agents include a double-stranded region of significant length (e.g., at least about 15bp or about 19bp in "canonical" siRNA). In some embodiments, dsRNAi includes two separate complementary chains (which are not covalently linked) that form a double-stranded region (e.g., in "canonical" siRNA), or a long single strand that includes 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 that forms two chains. In some embodiments, the sequence and / or structural elements (chemical modification, stereochemistry, etc.) required for the disclosure of effective single-stranded RNAi agents may be different from those required for effective double-stranded RNAi agents.

[0268] The present disclosure particularly encompasses 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 in the provided formats described herein), and vice versa. In some embodiments, the present disclosure provides designs for effective ssRNAi. In some embodiments, the present invention demonstrates that certain base sequences, when combined with structural elements (modifications, stereochemistry, one or more additional chemical moieties, etc.) according to the present disclosure, can, for example, provide oligonucleotides with unexpectedly high activity when administered as ssRNAi agents, particularly compared to oligonucleotides comprising the same sequence but double-stranded and administered as dsRNAi agents. In some embodiments, the present invention demonstrates that certain base sequences, when combined with structural elements (modifications, stereochemistry, one or more additional chemical moieties, etc.) according to the present disclosure, can provide oligonucleotides with unexpectedly high activity, for example, the ability to reduce the expression and / or level of a target gene or its gene product.

[0269] Structural and functional differences between single-stranded RNAi (ssRNAi) agents, double-stranded RNAi (dsRNAi) agents, and RNase H-dependent antisense oligonucleotides (ASOs)

[0270] In some embodiments, single-stranded RNAi (ssRNAi) agents, double-stranded RNAi (dsRNAi) agents, and RNase H-dependent antisense oligonucleotides (ASOs) all involve binding of an agent or oligonucleotide (or a portion thereof) to a complementary (or substantially complementary) target RNA (e.g., mRNA or pre-mRNA), followed by cleavage of the target RNA and / or reduction in expression and / or levels of a target gene or its gene product. In some embodiments, RNAi agents, whether double-stranded or single-stranded, use a RISC or RNA interference silencing complex including the enzyme Ago-2 (Argonaute-2). In some embodiments, RNase H-dependent antisense oligonucleotides are single-stranded and use a different enzyme, RNase H. RNase H is reported to be a cellular endonuclease that cuts the RNA strand of an RNA:DNA duplex; see those linkers in 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 that can direct RNase H-mediated knockdown include, but are not limited to, those consisting of or comprising a region of consecutive 2'-deoxynucleotide units that do not contain a 2'-modification. In some embodiments, oligonucleotides that can direct RNase H-mediated knockdown are gap-enlarged oligonucleotides or gapmers. In some embodiments, the spacer comprises an internal region comprising a plurality of nucleotides supporting RNAseH cleavage and is located between external regions having a plurality of nucleotides chemically different from the nucleosides in the internal region. In some embodiments, the spacer comprises 2'-deoxynucleotides that do not contain 2'-modifications within a certain range flanked or adjacent to one or two flanks. In some embodiments, the spacer guides the RNAseH cleavage of the corresponding RNA target. In some embodiments, the flank does not guide RNAseH cleavage or serves as a substrate for RNAseH cleavage. The flanks can have different lengths (including but not limited to 1 to 8nt) and can include various modifications or analogs (including but not limited to 2'-modifications, including but not limited to 2'-OMe and 2'-MOE).As non-limiting examples, see U.S. Pat. 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 such modifications or analogs can be associated with modified (e.g., increased, decreased, or altered) RNase H cleavage of the target.

[0271] In some embodiments, double-stranded RNAi agents, even their antisense strands, are structurally distinct from RNase H-dependent antisense oligonucleotides. In some embodiments, RNase H-dependent antisense oligonucleotides and siRNA oligonucleotides appear to have diametrically opposite characteristics with respect to both 5'-end structure and overall duplex stability.

[0272] It is reported that double-stranded RNAi agents can be naturally produced in cells by Dicer enzymes, which can cut larger RNA molecules (such as double-stranded RNA from invading viruses) into dsRNA. The canonical structure of dsRNA agents includes two RNA chains, each about 19 to 23nt long, and the two RNA chains are annealed to form a double-stranded region of about 19-21bp and two 3' dinucleotide overhangs. For double-stranded RNAi agents, it is reported that the sense strand has been unwound from the duplex before the antisense strand is incorporated into RISC. In addition to the natural separation of double-stranded RNAi agents into antisense strands and sense strands, single-stranded RNAi agents have not yet been reported to be naturally produced in human cells.

[0273] The present disclosure provides, inter alia, the following teachings: in many cases, a single-stranded RNAi agent is not merely an isolated antisense strand of a double-stranded RNAi agent, because, for example, the antisense strand of an effective dsRNAi agent may be far less effective than a dsRNAi agent, and a ssRNAi agent may be far less effective than a ssRNAi agent when formulated as a dsRNAi agent (e.g., by annealing of the sense strand). In some embodiments, double-stranded and single-stranded RNAi agents differ in many important respects. The structural parameters of a double-stranded RNAi agent are not necessarily reflected in a single-stranded RNAi agent.

[0274] In some embodiments, the present disclosure teaches that a target sequence suitable for a double-stranded RNAi agent may not be suitable for a single-stranded RNAi agent, and vice versa. For example, in at least some cases, a single-stranded form of a double-stranded RNAi agent may not be effective.

[0275] In some embodiments, double-stranded and single-stranded RNAi agents also differ in sensitivity to the incorporation of chiral controlled internucleotide linkages. For example, Matranga et al. (2005 Cell 123: 607-620) reported that the introduction of a single Sp internucleotide linkage (e.g., a single Sp PS) into the sense strand of a double-stranded RNAi agent greatly reduced RISC assembly and RNA interference activity. In contrast, in some embodiments, the data shown herein surprisingly demonstrate that the incorporation of Sp internucleotide linkages (e.g., Sp PS) can perform two functions for single-stranded RNAi agents: (a) it increases stability against nucleases; and (b) does not interfere with RNA interference activity. Many exemplary oligonucleotides can be used as effective single-stranded RNAi agents comprising one or more chiral controlled internucleotide linkages (e.g., shown herein as Sp internucleotide linkages or Sp PS (phosphorothioate)).

[0276] Alternatively or additionally, double-stranded and single-stranded RNAi agents may differ in immunogenicity. In some embodiments, it is reported that some single-stranded RNAi agents are more immunogenic than double-stranded RNAi agents. Sioud J.Mol.Biol.(2005)348,1079–1090. In some embodiments, it is reported that several double-stranded RNAi agents do not induce an immune response, while the corresponding single-stranded RNAi agents induce an immune response. In some embodiments, the present disclosure provides oligonucleotides with low immunogenicity. In some embodiments, such oligonucleotides can be used as ssRNAi agents.

[0277] The present disclosure particularly encompasses the recognition that conventional designs of certain single-stranded RNAi agents derived from double-stranded RNAi agents (including base sequences) often fail to provide effective single-stranded RNAi agents. In some embodiments, the present disclosure surprisingly demonstrates that ssRNAi agents derived from base sequences of effective RNase H-dependent ASOs can produce effective ssRNAi agents.

[0278] In some embodiments, the present disclosure provides oligonucleotides useful as effective RNase H-dependent ASOs, comprising a region of 2'-deoxynucleotides without 2'-modifications, and complementary or substantially complementary to an RNA sequence or a portion thereof. In some embodiments, the region may be, for example, a core sequence of about 10 nt, flanked on one or both sides by flanks, wherein the flanks are chemically distinct from the core and may, as non-limiting examples, comprise 2'-modifications or internucleotide linkage modifications.

[0279] Oligonucleotides

[0280] In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference and / or RNA enzyme H mediated knocking down. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by spatially blocking translation after binding to the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion.

[0281] In some embodiments, oligonucleotides are provided having structural elements described herein, or formats or portions thereof.

[0282] In some embodiments, provided oligonucleotides capable of directing a decrease in expression and / or levels of a target gene or its gene product have structural elements described herein, or formats or portions thereof.

[0283] In some embodiments, provided oligonucleotides capable of directing a decrease in the expression and / or level of a target gene or its gene product are in the format of any oligonucleotide disclosed herein, such as an oligonucleotide format disclosed in Table 1A or a figure or table or elsewhere disclosed herein.

[0284] In some embodiments, the provided oligonucleotides have any of the formats shown in FIG. 1 .

[0285] The present disclosure provides data demonstrating that various oligonucleotides in various formats are capable of directing the reduction of expression and / or levels of a target gene or its gene product targeted to any of a variety of different sequences in a variety of different genes in a variety of different species; additional data generated support the efficacy of ssRNAi agents in the disclosed formats and those not shown.

[0286] In some embodiments, provided oligonucleotides capable of directing RNase H-mediated knockdown have a structural element described herein, or a format or portion thereof.

[0287] In some embodiments, provided oligonucleotides capable of directing RNase H-mediated knockdown have the format of any oligonucleotide disclosed herein, such as an oligonucleotide format disclosed in Table 1A or a figure or table or elsewhere disclosed herein.

[0288] In some embodiments, the provided oligonucleotides have any of the formats shown in FIG. 1 .

[0289] The present disclosure provides data demonstrating that various oligonucleotides in various formats are capable of directing RNase H-mediated knockdown of any of a variety of different sequences in a variety of different genes in a variety of different species; additional data generated support the efficacy of ssRNAi agents in the disclosed formats and those not shown.

[0290] In some embodiments, provided oligonucleotides capable of directing single-stranded RNA interference have a structural element described herein, or a format or portion thereof.

[0291] In some embodiments, provided oligonucleotides capable of directing single-stranded RNA interference have the format of any oligonucleotide disclosed herein, such as an oligonucleotide format disclosed in Table 1A or a figure or table or elsewhere disclosed herein.

[0292] In some embodiments, a single-stranded RNAi agent is provided having any of the formats shown in FIG. 1 .

[0293] The present disclosure provides data demonstrating that various RNAi agents in various formats are capable of directing RNA interference against any of a variety of different sequences in any of a variety of different genes; additional data generated support the efficacy of ssRNAi agents in the disclosed formats and those not shown.

[0294] 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 an early stop 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 oligonucleotides provided in the provided composition, such as the first plurality of oligonucleotides, comprise base modifications, sugar modifications, and / or internucleotide linkage modifications. In some embodiments, the oligonucleotides provided comprise base modifications and sugar modifications. In some embodiments, the oligonucleotides provided comprise base modifications and internucleotide linkage modifications. In some embodiments, the oligonucleotides provided comprise sugar modifications and internucleotide modifications. In some embodiments, the oligonucleotides provided comprise sugar modifications and internucleotide modifications. In some embodiments, the compositions provided comprise base modifications, sugar modifications, and internucleotide linkage modifications. Exemplary chemical modifications, such as base modifications, sugar modifications, internucleotide linkage modifications, etc., are well known in the art and include, but are not limited to, those described in the present 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 1 It is an 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 with 5-20 ring atoms, wherein one or more ring atoms are optionally and independently heteroatoms. Exemplary ring structures are well known in the art, such as those structures present in BNA, LNA, etc. In some embodiments, the oligonucleotide provided comprises both one or more modified internucleotide linkages and one or more natural phosphate linkages. In some embodiments, oligonucleotides and compositions thereof comprising modified internucleotide linkages and natural phosphate linkages simultaneously provide improved properties, such as activity, etc. In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In some embodiments, the modified internucleotide linkage is a thiophosphate linkage. In some embodiments, the modified internucleotide linkage is a substituted thiophosphate linkage.

[0295] The present disclosure especially encompasses the following understanding: the stereo-random oligonucleotide products contain a plurality of different chemical entities that are different from each other in the stereochemical structure of each main chain chiral center in the oligonucleotide chain. In the case of not controlling the stereochemistry of the main chain chiral center, the stereo-random oligonucleotide products provide uncontrolled compositions, which comprise the oligonucleotide stereoisomers of undetermined levels. Even if these stereoisomers may have the same base sequence, at least due to their main chain stereochemistry difference, they are different chemical entities, and as demonstrated herein they can have different properties, such as activity etc. The present disclosure especially provides new compositions, which are or contain the specific stereoisomer of the target oligonucleotide. In some embodiments, specific stereoisomer can be defined, for example, by its base sequence, its length, its main chain bonding pattern and its main chain chiral center pattern. As understood in the art, in some embodiments, base sequence can refer to the identity and / or modification status of nucleoside residues in a PNPLA3 oligonucleotide (e.g., sugar and / or base composition relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues). In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising oligonucleotides of a single oligonucleotide type that are chemically identical at a predetermined level, e.g., they have the same base sequence, the same nucleoside modification pattern (modifications of the sugar and base moieties, if any), the same backbone chiral center pattern, and the same backbone phosphorus modification pattern. The present invention demonstrates, inter alia, that individual stereoisomers of a particular oligonucleotide can exhibit different stability and / or activity from one another. In some embodiments, the improved properties achieved by including and / or positioning specific chiral structures within a PNPLA3 oligonucleotide can be comparable to or even better than the improved properties achieved by using specific backbone linkages, residue modifications, and the like (e.g., by using certain types of modified phosphates [e.g., phosphorothioates, substituted phosphorothioates, and the like], sugar modifications [e.g., 2'-modifications, and the like], and / or base modifications [e.g., methylation, and the like]). The present disclosure recognizes, inter alia, that in some embodiments, the properties (e.g., activity, and the like) of a PNPLA3 oligonucleotide can be modulated by optimizing its backbone chiral center pattern, optionally in combination with modulation / optimization of one or more other features of the oligonucleotide (e.g., linkage pattern, nucleoside modification pattern, and the like). As illustrated by the various examples in the present disclosure, provided chirality-controlled oligonucleotide compositions can exhibit improved properties, such as improved single-stranded RNA interference activity, RNase H-mediated knockdown, improved delivery, and the like.

[0296] In some embodiments, oligonucleotide properties can be modulated by optimizing stereochemistry (pattern of backbone chiral centers) and chemical modifications (modifications of bases, sugars, and / or internucleotide linkages) or their pattern.

[0297] In some embodiments, the common main-chain chiral center pattern (e.g., the main-chain chiral center pattern in a single-stranded RNAi agent) includes the following pattern: OSOSO, OSSSO, OSSSOS, SOSO, SOSO, SOSOS, SOSOSO, SOSOSOSO, SOSSSO, SSOSSSOSS, SSSOSOSSS, SSSSOSOSSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS, or RRR, wherein S represents a thiophosphate in the Sp configuration and O represents a phosphodiester, wherein R represents a thiophosphate in the Rp configuration.

[0298] In some embodiments, the achiral center is a phosphodiester linkage. In some embodiments, the chiral center of the Sp configuration is a phosphorothioate linkage. In some embodiments, the achiral center is a phosphodiester linkage. In some embodiments, the chiral center of the Sp configuration is a phosphorothioate linkage.

[0299] In some embodiments, the oligonucleotide provided comprises any stereochemical pattern as described herein. In some embodiments, the oligonucleotide provided comprises any stereochemical pattern as described herein and can guide RNA interference. In some embodiments, the oligonucleotide provided comprises any stereochemical pattern as described herein and can guide RNA enzyme H mediation to strike low. In some embodiments, the oligonucleotide provided comprises any stereochemical pattern as described herein and can guide RNA interference and RNA enzyme H mediation to strike low. In some embodiments, the oligonucleotide provided comprises any stereochemical pattern as described herein and can guide RNA interference, wherein the stereochemical pattern is in the seed and / or seed post region. In some embodiments, the oligonucleotide provided comprises any stereochemical pattern as described herein and can guide RNA interference and RNA enzyme H mediation to strike low, wherein the stereochemical pattern is in the seed and / or seed post region.

[0300] In some embodiments, the oligonucleotides provided include any modification or modification pattern described herein. In some embodiments, the oligonucleotides provided include any modification or modification pattern described herein and can guide RNA interference. In some embodiments, the oligonucleotides provided include any modification pattern described herein and can guide RNA enzyme H mediated knockdown. In some embodiments, the oligonucleotides provided include any modification pattern described herein and can guide RNA interference and RNA enzyme H mediated knockdown. In some embodiments, the oligonucleotides provided include any modification pattern described herein and can guide RNA interference, wherein the modification pattern is in the seed and / or post-seed region. In some embodiments, the oligonucleotides provided include any modification pattern described herein and can guide RNA interference and RNA enzyme H mediated knockdown, wherein the modification pattern is in the seed and / or post-seed region. 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 modification or modification pattern of a sugar, 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 R 1 is an optionally substituted C1-6 alkyl group.

[0301] In some embodiments, the disclosure particularly demonstrates that 2'-F modification can improve single-stranded RNA interference. In some embodiments, the disclosure particularly demonstrates that Sp internucleotide linkage can improve oligonucleotide stability at 5'-end and 3'-end. In some embodiments, the disclosure demonstrates that, among others, natural phosphate linkage and / or Rp internucleotide linkage can improve removal of oligonucleotides from the system. As will be appreciated by those of ordinary skill in the art, according to the disclosure, various assays known in the art can be used to assess such properties.

[0302] In some embodiments, provided oligonucleotides capable of directing single-stranded RNA interference comprise one or more modified sugar moieties. In some embodiments, 5% or more of the sugar moieties of provided oligonucleotides are modified.

[0303] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein:

[0304] The first plurality of oligonucleotides have the same base sequence; and

[0305] The first plurality of oligonucleotides comprises one or more modified sugar moieties, or comprises one or more native phosphate linkages and one or more modified internucleotide linkages.

[0306] In some embodiments, the first plurality of oligonucleotides comprises one or more modified sugar moieties. In some embodiments, the provided oligonucleotides comprise one or more modified sugar moieties.

[0307] In some embodiments, the compositions provided alter transcript single-stranded RNA interference, resulting in suppression of unwanted targets and / or biological functions. In some embodiments, in such cases, the compositions provided may also induce cleavage of the transcript after hybridization.

[0308] In some embodiments, each oligonucleotide 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 oligonucleotide of the first plurality of oligonucleotides comprises no more than about 95% unmodified sugar moieties. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 50% unmodified sugar moieties. In some embodiments, each oligonucleotide 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.

[0309] In some embodiments, each oligonucleotide in the first plurality of oligonucleotides comprises two or more modified internucleotide linkages. In some embodiments, each oligonucleotide in the first plurality of oligonucleotides comprises three or more modified internucleotide linkages. In some embodiments, each oligonucleotide in the first plurality of oligonucleotides comprises four or more modified internucleotide linkages. In some embodiments, each oligonucleotide in the first plurality of oligonucleotides comprises five or more modified internucleotide linkages. In some embodiments, each oligonucleotide in the first plurality of oligonucleotides comprises ten or more modified internucleotide linkages.

[0310] In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 30% natural phosphate linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 20% natural phosphate linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 10% natural phosphate linkages. In some embodiments, each oligonucleotide of the first plurality of oligonucleotides comprises no more than about 5% natural phosphate linkages.

[0311] In some embodiments, the oligonucleotides provided include one or more isotopes of increased levels. In some embodiments, the oligonucleotides provided are, for example, labeled with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the oligonucleotides provided in the provided compositions, such as the first plurality of oligonucleotides, include base modifications, sugar modifications, and / or internucleotide linkage modifications, wherein the oligonucleotides include an enriched level of deuterium. In some embodiments, the oligonucleotides provided are labeled with deuterium (with -) at one or more positions. 1 H Substitution - 2 H) In some embodiments, one or more of the PNPLA3 oligonucleotides 1 H or any moiety conjugated to the oligonucleotide (e.g., targeting moiety, lipid moiety, etc.) is 2 Such oligonucleotides can be used in any of the compositions or methods described herein.

[0312] The present invention includes all pharmaceutically acceptable isotopically labeled compounds wherein 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.

[0313] Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen, such as 2 H and 3 H; carbon, such as 11 C. 13 C and 14 C; chlorine, such as 36 Cl; fluorine, such as 18 F; iodine, such as 123 I. 124 I and 125 I; Nitrogen, such as 13 N and 15 N; oxygen, such as 15 O. 17 O and 18 O; phosphorus, such as 32 P; and sulfur, such as 35 S.

[0314] Certain isotopically-labeled compounds of formula (I), for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. 3 H) and carbon-14 (i.e. 14 C) is particularly useful for this purpose due to its ease of incorporation and readily available means of detection.

[0315] Heavier isotopes such as deuterium (i.e. 2H) substitutions may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and thus may be preferred in some circumstances.

[0316] Using positron-emitting isotopes such as 11 C. 18 F. 15 O and 13 N) substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy.

[0317] 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 Preparations using an appropriate isotopically labeled reagent in place of the non-labeled reagent previously used.

[0318] The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. Unless otherwise indicated, it is contemplated that all stereoisomeric forms of the compounds of the present invention and mixtures thereof (including racemic mixtures) form part of the present invention. In addition, all geometric and positional isomers are encompassed by the present invention. For example, if the compounds of the present invention incorporate double bonds or fused rings, cis- and trans-forms and mixtures are encompassed within the scope of the present invention.

[0319] The chiral compounds of the present invention (and chiral precursors thereof) can be obtained in an enantiomerically enriched form on a resin having an asymmetric stationary phase and mobility using chromatography (usually high pressure liquid chromatography (HPLC) or supercritical fluid chromatography (SFC)), wherein the mobile phase consists of a hydrocarbon (usually heptane or hexane) containing 0% to 50% isopropanol (usually 2% to 20%) and 0% to 5% alkylamine (usually 0.1% diethylamine (DEA) or isopropylamine). The eluent is concentrated to obtain an enriched mixture.

[0320] By methods well known to those skilled in the art, such as by chromatography and / or fractional crystallization, the physicochemical differences of diastereoisomer mixtures can be separated into their single diastereomers. Enantiomers can be separated by: by reacting with an appropriate optically active compound (e.g., chiral auxiliary, such as chiral alcohol or Mosher's acyl chloride) enantiomeric mixtures converted into diastereomeric mixtures, separating the diastereomers and converting (e.g., hydrolyzing) the single diastereomers into the corresponding pure enantiomers. Chiral HPLC columns can also be used to separate enantiomers. Alternatively, a specified stereoisomer can be synthesized by using an optically active starting material, by using an asymmetric synthesis of an optically active reagent, substrate, catalyst or solvent, or by asymmetric transformation a stereoisomer is converted into another stereoisomer.

[0321] In some embodiments, control of structural elements of oligonucleotides, such as chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages) or patterns thereof, changes in stereochemistry (e.g., stereochemistry of backbone chiral internucleotide linkages) or patterns thereof, substitution of atoms with isotopes of the same element, and / or conjugation with additional chemical moieties (e.g., lipid moieties, targeting moieties, etc.), can have a significant impact on the desired biological effect. In some embodiments, the desired biological effect is enhanced by more than 2-fold.

[0322] In some embodiments, the desired biological effect is to direct the reduction of expression and / or level of a target gene or its gene product. In some embodiments, the desired biological effect is improved single-stranded RNA interference. In some embodiments, the desired biological effect is improved RNase H-mediated knockdown. In some embodiments, the desired biological effect is improved single-stranded RNA interference and / or RNase H-mediated knockdown.

[0323] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides:

[0324] 1) having a common base sequence that is complementary to a target sequence in a transcript; and

[0325] 2) Contain one or more modified sugar moieties and modified internucleotide linkages.

[0326] In some embodiments, the oligonucleotide compositions provided are characterized in that, when contacted with a transcript in a single-stranded RNA interference system, RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, wherein the reference conditions are selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0327] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the PNPLA3 oligonucleotide type is defined by:

[0328] 1) Base sequence;

[0329] 2) Main chain linkage pattern;

[0330] 3) Main chain chiral center pattern; and

[0331] 4) Main chain phosphorus modification pattern,

[0332] The composition is chirality controlled in that it is enriched in oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence,

[0333] The oligonucleotide composition is characterized in that, when it is contacted with a transcript in a single-stranded RNA interference system, RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, wherein the reference conditions are selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0334] In some embodiments, each of the consecutive nucleoside units is independently before and / or after the modified internucleotide linkage. In some embodiments, each of the consecutive nucleoside units is independently before and / or after the thiophosphate linkage. In some embodiments, each of the consecutive nucleoside units is independently before and / or after the chiral controlled internucleotide linkage. In some embodiments, each of the consecutive nucleoside units is independently before and / or after the chiral controlled thiophosphate 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.

[0335] In some embodiments, the present disclosure provides a single-stranded RNAi agent comprising a predetermined level of a first plurality of oligonucleotides, wherein:

[0336] The first plurality of oligonucleotides have the same base sequence;

[0337] The first plurality of oligonucleotides comprises a seed region having 2, 3, 4, 5, 6, 7 or more consecutive Sp modified internucleotide linkages, a post-seed region having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive Sp modified internucleotide linkages.

[0338] In some embodiments, the seed region comprises 2 or more consecutive Sp-modified internucleotide linkages.

[0339] 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.

[0340] As demonstrated by the present disclosure, in some embodiments, provided oligonucleotide compositions are characterized in that, when contacted with a transcript in a single-stranded RNA interference system, RNAi-mediated knockdown of the transcript is improved compared to the knockdown observed under reference conditions, wherein the reference conditions are selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0341] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides defined by having:

[0342] 1) Common base sequence and length;

[0343] 2) a common backbone linkage pattern; and

[0344] 3) a common backbone chiral center pattern, the composition is a substantially pure preparation of individual oligonucleotides in that the oligonucleotides in the composition at predetermined levels have a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern.

[0345] In some embodiments, the common base sequence and length can be referred to as a common base sequence. In some embodiments, oligonucleotides with a common base sequence can have the same nucleoside modification pattern, such as sugar modification, base modification, etc. In some embodiments, the nucleoside modification pattern can be represented by a combination of position and modification. In some embodiments, the backbone linkage pattern includes the position and type of each internucleotide linkage (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.). The backbone chiral center pattern of the PNPLA3 oligonucleotide can be specified by a combination of linkage phosphorus stereochemistry (Rp / Sp) from 5' to 3'. As exemplified above, the position of the achiral linkage can be obtained, for example, by the backbone linkage pattern.

[0346] As understood by those of ordinary skill in the art, the stereo-random or racemic products of oligonucleotides are prepared by diastereoselective and / or low stereoselective coupling of nucleotide monomers, usually without the use of any chiral auxiliary, chiral modification agent and / or chiral catalyst. In some embodiments, in the substantially racemic (or chirality uncontrolled) products of oligonucleotides, all or most of the coupling steps are not chirality controlled, because no coupling step is specifically performed to provide enhanced stereoselectivity. Exemplary substantially racemic products of oligonucleotides are products of thiophosphate oligonucleotides, which are prepared by sulfurizing the phosphite triesters synthesized by commonly used phosphoramidite oligonucleotides with tetraethylthiuram disulfide or (TETD) or 1,1-dioxidation 3H-1,2-phenylenedisulfide-3-ketone (BDTD) (methods well known in the art). In some embodiments, the substantially racemic products of oligonucleotides provide substantially racemic oligonucleotide compositions (or chirality uncontrolled oligonucleotide compositions).

[0347] As will be appreciated by one of ordinary skill in the art, in some embodiments, the diastereoselectivity of a coupling or linkage can be assessed by the diastereoselectivity of dimer formation under the same or comparable conditions, wherein the dimers have identical 5'- and 3'-nucleosides and internucleotide linkages.

[0348] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide composition of a first plurality of oligonucleotides, because the composition is enriched in oligonucleotides of a single oligonucleotide type relative to a substantially racemic preparation of the same oligonucleotides. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide composition of a first plurality of oligonucleotides, because the composition is enriched in oligonucleotides of a single oligonucleotide type relative to a substantially racemic preparation of the same oligonucleotides, which share:

[0349] 1) Common base sequence and length;

[0350] 2) a common backbone linkage pattern; and

[0351] 3) Common main chain chiral center pattern.

[0352] In some embodiments, the present disclosure provides a PNPLA3 oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the oligonucleotides are of a particular oligonucleotide type characterized by:

[0353] 1) Common base sequence and length;

[0354] 2) a common backbone linkage pattern; and

[0355] 3) Common main chain chiral center patterns;

[0356] The composition is chirality controlled in that it is enriched for oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides of the same base sequence and length.

[0357] In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have the same structure.

[0358] In some embodiments, oligonucleotides of the PNPLA3 oligonucleotide type have a common backbone phosphorus modification pattern and a common sugar modification pattern. In some embodiments, oligonucleotides of the PNPLA3 oligonucleotide type have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides of the PNPLA3 oligonucleotide type have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides of the PNPLA3 oligonucleotide type are identical.

[0359] In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common sugar modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides having a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern are identical.

[0360] In some embodiments, the oligonucleotides in the provided compositions have a common backbone phosphorus modification pattern. In some embodiments, the common base sequence is a base sequence of a PNPLA3 oligonucleotide type. In some embodiments, the provided compositions are chirality-controlled PNPLA3 oligonucleotide compositions in that the compositions contain a first plurality of oligonucleotides of a single oligonucleotide type at a predetermined level, wherein the PNPLA3 oligonucleotide type is defined by:

[0361] 1) Base sequence;

[0362] 2) Main chain linkage pattern;

[0363] 3) Main chain chiral center pattern; and

[0364] 4) Main chain phosphorus modification pattern.

[0365] As noted above and as understood in the art, in some embodiments, the base sequence of a PNPLA3 oligonucleotide can refer to the identity and / or modification status of the nucleoside residues in the oligonucleotide (e.g., sugar and / or base composition relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues).

[0366] In some embodiments, oligonucleotides of a particular type are identical in that they have the same base sequence (including length), the same chemical modification pattern of the sugar and base moieties, the same backbone linkage pattern (e.g., a pattern of natural phosphate linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same backbone chiral center pattern (e.g., the stereochemical pattern of the chiral internucleotide linkages (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., the modification pattern of the internucleotide phosphorus atoms, such as -S - and -LR of formula I 1 ).

[0367] The present disclosure recognizes, among other things, that combinations of oligonucleotide structural elements (eg, chemical modification patterns, backbone linkages, backbone chiral centers, and / or backbone phosphorus modifications) can provide surprisingly improved properties, such as biological activity.

[0368] In some embodiments, the chirally controlled (and / or stereochemically pure) preparations provided are RNAi agent oligonucleotides.

[0369] In some embodiments, chirality-controlled (and / or stereochemically pure) preparations are provided having oligonucleotides comprising one or more modified backbone linkages, bases, and / or sugars.

[0370] In some embodiments, the composition provided has an oligonucleotide comprising one or more residues modified at a sugar moiety. In some embodiments, the composition provided has an oligonucleotide comprising one or more residues modified (referred to herein as "2'-modification") at the 2' position of the sugar moiety. Such modified examples are described above and herein, and include but are not limited to 2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, S-cEt, etc. In some embodiments, the composition provided has an oligonucleotide comprising one or more 2'-modified residues. For example, in some embodiments, the oligonucleotide provided includes one or more residues, and the residue is a 2'-O-methoxyethyl (2'-MOE)-modified residue. In some embodiments, the composition provided has an oligonucleotide not comprising any 2'-modification. In some embodiments, the composition provided is an oligonucleotide not comprising any 2'-MOE residue. That is, in some embodiments, the oligonucleotide provided is not MOE modified. The present disclosure describes other exemplary sugar modifications.

[0371] In some embodiments, one or more is one. In some embodiments, one or more is two. In some embodiments, one or more is three. In some embodiments, one or more is four. In some embodiments, one or more is five. In some embodiments, one or more is six. In some embodiments, one or more is seven. In some embodiments, one or more is eight. In some embodiments, one or more is nine. In some embodiments, one or more is ten. In some embodiments, one or more is at least one. In some embodiments, one or more is at least two. In some embodiments, one or more is at least three. In some embodiments, one or more is at least four. In some embodiments, one or more is at least five. In some embodiments, one or more is at least six. In some embodiments, one or more is at least seven. In some embodiments, one or more is at least eight. In some embodiments, one or more is at least nine. In some embodiments, one or more is at least ten.

[0372] In some embodiments, the sugar moiety without the 2'-modification is a sugar moiety that is present in a natural DNA nucleoside.

[0373] Those of ordinary skill in the art understand that various regions of the target transcript can be targeted by the provided compositions and methods. In some embodiments, the base sequence of the provided oligonucleotides comprises an intron sequence. In some embodiments, the base sequence of the provided oligonucleotides comprises an exon sequence. In some embodiments, the base sequence of the provided oligonucleotides comprises an intron and exon sequence.

[0374] As will be appreciated by those of ordinary skill in the art, the oligonucleotides and compositions provided can, among other things, target a large number of nucleic acid polymers. For example, in some embodiments, the oligonucleotides and compositions provided can target a transcript of a nucleic acid sequence, wherein the common base sequence of the oligonucleotide (e.g., a base sequence of a PNPLA3 oligonucleotide type) includes or is a sequence complementary to the sequence of the transcript. In some embodiments, the common base sequence includes a sequence complementary to the sequence of the target. In some embodiments, the common base sequence is a sequence complementary to the sequence of the target. In some embodiments, the common base sequence includes or is a sequence that is 100% complementary to the sequence of the target. In some embodiments, the common base sequence includes a sequence that is 100% complementary to the sequence of the target. In some embodiments, the common base sequence is a sequence that is 100% complementary to the sequence of the target.

[0375] I

[0376] In some embodiments, common base sequence includes or is a sequence complementary to a characteristic sequence element. In some embodiments, common base sequence includes a sequence complementary to a characteristic sequence element. In some embodiments, common base sequence is a sequence complementary to a characteristic sequence element. In some embodiments, common base sequence includes or is a sequence 100% complementary to a characteristic sequence element. In some embodiments, common base sequence includes a sequence 100% complementary to a characteristic sequence element. In some embodiments, common base sequence is a sequence 100% complementary to a characteristic sequence element. In some embodiments herein, characteristic sequence element is a seed region, a post-seed region, or a portion of a seed region, or a portion of a post-seed region, or a 3'-terminal dinucleotide as non-limiting examples.

[0377] In some embodiments, the signature sequence element includes or is a mutation. In some embodiments, the signature sequence element includes a mutation. In some embodiments, the signature sequence element is a mutation. In some embodiments, the signature sequence element includes or is a point mutation. In some embodiments, the signature sequence element includes a point mutation. In some embodiments, the signature sequence element is a point mutation. In some embodiments, the signature sequence element includes or is a SNP. In some embodiments, the signature sequence element includes a SNP. In some embodiments, the signature sequence element is a SNP.

[0378] In some embodiments, the common base sequence matches 100% of the target sequence, but does not match 100% of a similar sequence to the target sequence.

[0379] For example, in some embodiments, the common base sequence matches a mutation in the pathogenic copy or allele of the target nucleic acid sequence, but does not match a non-pathogenic copy or allele at the mutation site; in some other embodiments, the common base sequence matches a SNP in the pathogenic allele of the target nucleic acid sequence, but does not match a non-pathogenic allele at the corresponding site.

[0380] The present disclosure recognizes, among other things, that base sequence may have an effect on oligonucleotide properties. In some embodiments, when an oligonucleotide having a base sequence is used to inhibit a target, for example, by a pathway involving RNase H, the base sequence may have an effect on the cleavage pattern of the target: for example, structurally similar (all phosphorothioate linkages, all stereo-random) oligonucleotides having different sequences may have different cleavage patterns.

[0381] In some embodiments, the common base sequence is a base sequence comprising a SNP.

[0382] As understood by those of ordinary skill in the art, the oligonucleotide compositions and methods provided have various uses known to those of ordinary skill in the art. The method for evaluating the provided compositions and their properties and uses is also well known and practiced by those of ordinary skill in the art. Exemplary properties, uses and / or methods include but are not limited to those described in WO / 2014 / 012081 and WO / 2015 / 107425.

[0383] In some embodiments, the chiral internucleotide linkage has the structure of Formula I. In some embodiments, the chiral internucleotide linkage is a phosphorothioate. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of a provided composition independently has the structure of Formula I. In some embodiments, each chiral internucleotide linkage in a single oligonucleotide of a provided composition independently is a phosphorothioate.

[0384] In some embodiments, the oligonucleotides of the present disclosure include one or more modified sugar moieties. In some embodiments, the oligonucleotides of the present disclosure include one or more modified base moieties. As known to those of ordinary skill in the art and as described in the present disclosure, various modifications can be introduced into sugar and / or moieties. For example, in some embodiments, modification is the modification described in US9006198, WO2014 / 012081 and WO / 2015 / 107425, each of which is incorporated herein by reference in its entirety.

[0385] In some embodiments, the sugar modification is a 2'-modification. Common 2'-modifications include, but are not limited to, 2'-OR 1 , where R 1 In some embodiments, the modification is 2'-OR, wherein 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 disclosure proves that the inclusion and / or position of the linkage between specific chiral pure nucleotides can provide comparable or better stability improvement with the stability improvement obtained by using modified backbone linkages, bases and / or sugars. In some embodiments, the single oligonucleotide provided of the provided composition is not modified on sugar. In some embodiments, the single oligonucleotide provided of the provided composition is not modified on the 2'-position of sugar (that is, two groups at the 2'-position are-H / -H or-H / -OH). In some embodiments, the single oligonucleotide provided of the provided composition does not have any 2'-MOE modification.

[0386] In some embodiments, the 2'-modification is -OL- or -L-, which connects 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 connects 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.

[0387] In some embodiments, the 2'-modification is -F. In some embodiments, the 2'-modification is FANA. In some embodiments, the 2'-modification is FRNA.

[0388] In some embodiments, the sugar modification is a 5'-modification, eg, R-5'-Me, S-5'-Me, etc.

[0389] In some embodiments, the sugar modification changes the size of the sugar ring. In some embodiments, the sugar modification is a sugar moiety in FHNA.

[0390] In some embodiments, the sugar modification replaces the sugar moiety with another cyclic or non-cyclic moiety. Examples of such moieties are well known in the art and include, but are not limited to, those used in morpholinos (optionally with their diamidophosphoryl linkages), diol nucleic acids, and the like.

[0391] In some embodiments, the ssRNAi agent is or comprises a PNPLA3 oligonucleotide selected from the group consisting of any ssRNAi in any format described in Figure 1 or elsewhere herein. A skilled artisan reading this specification will appreciate that the present disclosure expressly does not exclude the possibility that any oligonucleotide labeled as a ssRNAi agent described herein may also or alternatively function by another mechanism (e.g., as an antisense oligonucleotide; mediating knockdown by an RNase H mechanism; sterically hindered translation; or any other biochemical mechanism).

[0392] In some embodiments, the antisense oligonucleotide (ASO) is or comprises a PNPLA3 oligonucleotide selected from the group consisting of: Figure 2 Any oligonucleotide in any format described. Those skilled in the art who read this specification will understand that the present disclosure explicitly does not exclude the possibility that any oligonucleotide labeled as an antisense oligonucleotide (ASO) described herein may also or alternatively act via another mechanism (e.g., using RISC as ssRNAi); the present disclosure also points out that various ASOs may act via different mechanisms (using RNase H, sterically blocking translation or other post-transcriptional processes, changing the conformation of the target nucleic acid, etc.).

[0393] In some embodiments, the hybridizing 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 Figure 1L 1 ; or any oligonucleotide in any of the formats S40 to S42 of Figure 1 ; or any of the formats 30-32, 66-69 or 101-103 of Figure 1 . Those skilled in the art who read this specification will understand that the present disclosure explicitly does not exclude the possibility that any oligonucleotide labeled as a hybridization oligonucleotide described herein may also or alternatively function by another mechanism (e.g., as an antisense oligonucleotide; mediating knockdown by an RNase H mechanism; steric hindrance of translation; or any other biochemical mechanism).

[0394] Chirality-controlled oligonucleotides and chirality-controlled oligonucleotide compositions

[0395] In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference and / or RNA enzyme H mediation knocking down. In some embodiments, the oligonucleotide provided can block translation in space after annealing the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion, to guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided is chiral controlled.

[0396] The present disclosure provides chiral controlled oligonucleotides and chiral controlled oligonucleotide compositions with high crude purity and 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.

[0397] In some embodiments, the single-stranded RNAi agent is a substantially pure preparation of a PNPLA3 oligonucleotide type, as oligonucleotides in the composition that are not of that oligonucleotide type are impurities formed during the preparation of that oligonucleotide type, and in some cases, after certain purification procedures.

[0398] In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomeric pure internucleotide linkages with respect to chiral linkage phosphorus. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomeric pure internucleotide linkages with respect to chiral linkage phosphorus and one or more phosphodiester linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomeric pure internucleotide linkages with respect to chiral linkage phosphorus and one or more phosphodiester linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomeric pure internucleotide linkages with respect to chiral linkage phosphorus and one or more phosphodiester linkages. In some embodiments, the present disclosure provides oligonucleotides comprising one or more diastereomeric pure internucleotide linkages with respect to 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 diastereomeric pure internucleotide linkages with respect to chiral linkage phosphorus. Exemplary internucleotide linkages are further described below, including those with structure of formula I.

[0399] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide, wherein at least two individual internucleotide linkages within the oligonucleotide have different stereochemistry and / or different P modifications relative to each other.

[0400] Internucleotide linkage

[0401] In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference and / or RNA enzyme H mediation knocking down. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by spatially blocking translation after annealing the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the oligonucleotide provided includes any internucleotide linkage described herein or known in the art.

[0402] In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that directs RNA interference, a PNPLA3 oligonucleotide that directs RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that directs both RNA interference and RNase H-mediated knockdown can comprise any internucleotide linkage described herein or known in the art.

[0403] A non-limiting example of an internucleotide linkage or an unmodified internucleotide linkage is a phosphodiester; non-limiting examples of modified internucleotide linkages include those internucleotide linkages in which one or more oxygens of the phosphodiester have been replaced by, as non-limiting examples, sulfur (such as sulfur in phosphorothioate), H, an alkyl group, or another moiety or element that is not oxygen. A non-limiting example of an internucleotide linkage is a moiety that does not contain phosphorus but is used to link two sugars. A non-limiting example of an internucleotide linkage is a moiety that does not contain phosphorus but is used to link two sugars in the backbone of a PNPLA3 oligonucleotide. Other non-limiting examples of nucleotides, modified nucleotides, nucleotide analogs, internucleotide linkages, modified internucleotide linkages, bases, modified bases and base analogs, sugars, modified sugars and sugar analogs, and nucleosides, modified nucleosides, and nucleoside analogs are disclosed herein.

[0404] In certain embodiments, the internucleotide linkage has the structure of Formula I:

[0405]

[0406] wherein each variable is defined and described below. In some embodiments, the linkage of Formula I is chiral. In some embodiments, the disclosure provides a chiral controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I. In some embodiments, the disclosure provides a chiral controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the single internucleotide linkages of Formula I within the oligonucleotide have different P modifications relative to each other. In some embodiments, the disclosure provides a chiral controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the single internucleotide linkages of Formula I within the oligonucleotide have different -XLR relative to each other. 1 In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the individual internucleotide linkages of Formula I within the oligonucleotide have different X relative to each other. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising one or more modified internucleotide linkages of Formula I, and wherein the individual internucleotide linkages of Formula I within the oligonucleotide have different -LR relative to each other. 1In some embodiments, the chirality-controlled oligonucleotide is a PNPLA3 oligonucleotide of a specific oligonucleotide type in the provided compositions. In some embodiments, the chirality-controlled oligonucleotide is a PNPLA3 oligonucleotide in the provided compositions that has a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern. In some embodiments, the chirality-controlled oligonucleotide is a PNPLA3 oligonucleotide of a specific oligonucleotide type in the chirality-controlled compositions, and the chirality-controlled oligonucleotide is of the type. In some embodiments, the chirality-controlled oligonucleotide is a PNPLA3 oligonucleotide in the provided compositions that contains a plurality of oligonucleotides at predetermined levels, the plurality of oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern, and the chirality-controlled oligonucleotides share a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern.

[0407] In some embodiments, chirality-controlled oligonucleotides comprise different internucleotide phosphorus linkages.

[0408] In some embodiments, the phosphorothioate triester linkage comprises a chiral auxiliary, e.g., for controlling the stereoselectivity of the reaction. In some embodiments, the phosphorothioate triester linkage does not comprise a chiral auxiliary. In some embodiments, the phosphorothioate triester linkage is intentionally maintained to guide administration to a subject, and / or the phosphorothioate triester linkage is intentionally maintained during administration to a subject.

[0409] In some embodiments, the chirality-controlled oligonucleotide is attached to a solid support. In some embodiments, the chirality-controlled oligonucleotide is cleaved from a solid support.

[0410] In some embodiments, the chirality-controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive modified internucleotide linkages. In some embodiments, the chirality-controlled oligonucleotide comprises at least one phosphodiester internucleotide linkage and at least two consecutive phosphorothioate triester internucleotide linkages.

[0411] In some embodiments, the present disclosure provides compositions (e.g., chiral controlled oligonucleotide compositions) comprising or consisting of a plurality of provided oligonucleotides. In some embodiments, all such provided oligonucleotides are of the same type, i.e., all have the same base sequence, backbone linkage pattern (i.e., internucleotide linkage type pattern, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., linkage phosphorus stereochemistry pattern (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I disclosed herein). 1"group pattern". In some embodiments, all oligonucleotides of the same type are identical. However, in many embodiments, provided compositions typically contain predetermined relative amounts of multiple oligonucleotide types.

[0412] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that directs RNA interference, the PNPLA3 oligonucleotide that directs RNase H-mediated knockdown, or the PNPLA3 oligonucleotide that directs both RNA interference and RNase H-mediated knockdown can comprise any internucleotide linkage described herein or known in the art. In some embodiments, the moiety that binds ASPGR is, for example, a GalNAc moiety, any GalNAc described herein or known in the art, or a variant or modification thereof. In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that guides RNA interference, a PNPLA3 oligonucleotide that guides RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that guides both RNA interference and RNase H-mediated knockdown may comprise any internucleotide linkage described herein or known in the art in combination with any other structural element or modification described herein, including but not limited to, base sequence, sugar, base (nucleobase) or a portion thereof; stereochemistry or pattern 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 moiety; 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 element thereof, and / or any other structural element or modification described herein; and in some embodiments, the present disclosure relates to multimers of any such oligonucleotides.

[0413] In some embodiments, the chirality-controlled oligonucleotide comprises one or more modified internucleotide phosphorus linkages. In some embodiments, the chirality-controlled oligonucleotide comprises, for example, phosphorothioate or phosphorothioate triester linkages.

[0414] In some embodiments, the modified internucleotide linkage is a phosphorothioate. In some embodiments, the modified internucleotide linkage is selected from, for example, those described in US 20110294124, US20120316224, US20140194610, US20150211006, US20150197540, WO 2015107425, PCT / US2016 / 043542, and PCT / US2016 / 043598, Whittaker et al. 2008 Tetrahedron Letters 49: 6984-6987.

[0415] Non-limiting examples of internucleotide linkages also include those described 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. NY Acad. Sci. 1988, 507, 220, and Vasseur et al. J. Am. Chem. Soc. 1992, 114, 4006.

[0416] In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein one or more U is replaced by T. In some embodiments, the present disclosure provides a chirality-controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 50% identity to the sequence of any oligonucleotide disclosed herein.

[0417] In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising the sequence present in any oligonucleotide disclosed herein, wherein the oligonucleotide has a backbone linkage pattern, a backbone chiral center pattern, and / or a backbone phosphorus modification pattern as described herein.

[0418] In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein one or more T is replaced by U. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 50% identity with the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 60% identity with the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 70% identity with the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 80% identity with the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 90% identity to the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence present in any oligonucleotide disclosed herein, wherein the sequence has more than 95% identity to the sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide comprising a sequence of any oligonucleotide disclosed herein. In some embodiments, the present disclosure provides a chiral controlled oligonucleotide having a sequence of any oligonucleotide disclosed herein.

[0419] The present disclosure provides a chirality-controlled oligonucleotide comprising the sequence of any oligonucleotide disclosed herein, wherein at least one internucleotide linkage is The present disclosure provides a chirality-controlled oligonucleotide comprising the sequence of any oligonucleotide disclosed herein, wherein each internucleotide linkage is The present disclosure provides a chirality-controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein at least one internucleotide linkage is The present disclosure provides a chirality-controlled oligonucleotide having the sequence of any oligonucleotide disclosed herein, wherein each internucleotide linkage is 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.

[0420] Base (nucleobase)

[0421] In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference and / or RNA enzyme H mediation knocking down. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by spatially blocking translation after annealing the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the oligonucleotide provided includes any core base described herein or known in the art.

[0422] 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 whose corresponding amino groups are protected by acyl protecting groups; 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs (such as pseudoisocytosine and pseudouracil) and other modified nucleobases, such as 8-substituted purines, xanthines, or hypoxanthines (the latter two are natural degradation products). Exemplary modified nucleobases are disclosed 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 of uracil, thymine, adenine, cytosine or guanine, for example, with respect to hydrogen bonds and / or base pairing. In some embodiments, the nucleobase is an optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine or guanine. In some embodiments, the nucleobase is a uracil, thymine, adenine, cytosine, 5-methylcytosine or guanine.

[0423] In some embodiments, the modified base is an optionally substituted adenine, cytosine, guanine, thymine or uracil. In some embodiments, the modified nucleobase is independently adenine, cytosine, guanine, thymine or uracil, which is modified by one or more modifications, by which:

[0424] (1) the nucleobase is modified with one or more optionally substituted groups independently selected from the group consisting of acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silane, and combinations thereof;

[0425] (2) one or more atoms of the nucleobase are independently replaced by different atoms selected from carbon, nitrogen and sulfur;

[0426] (3) one or more double bonds in the nucleobase are independently hydrogenated; or

[0427] (4) One or more aryl or heteroaryl rings are independently inserted into the nucleobase.

[0428] Various additional nucleobases are described in the art.

[0429] In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference and / or RNA enzyme H mediation knocking down. In some embodiments, the oligonucleotide provided can block translation in space after annealing the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion, to guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided includes any sugar described herein or known in the art.

[0430] In some embodiments, provided oligonucleotides capable of directing single-stranded RNA interference comprise one or more modified sugar moieties in addition to native sugar moieties.

[0431] The most common naturally occurring nucleotides comprise a ribose sugar linked to the nucleobases adenosine (A), cytosine (C), guanine (G) and thymine (T) or uracil (U). Modified nucleotides are also contemplated, wherein the phosphate group or phosphorus linkage in the nucleotide may be linked to various positions of a sugar or modified sugar. As a non-limiting example, the phosphate group or phosphorus linkage may be linked to the 2", 3", 4" or 5" hydroxyl moiety of a sugar or modified sugar. In this context, nucleotides incorporating modified nucleobases as described herein are also contemplated. In some embodiments, nucleotides or modified nucleotides comprising an unprotected -OH moiety are used according to the methods disclosed herein.

[0432] In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that directs RNA interference, a PNPLA3 oligonucleotide that directs RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that directs both RNA interference and RNase H-mediated knockdown can comprise any base (nucleobase), modified base, or base analog described herein or known in the art. In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that guides RNA interference, a PNPLA3 oligonucleotide that guides RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that guides 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 element or modification described herein, including but not limited to, a base sequence or portion thereof; a sugar; an internucleotide linkage; 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 modified pattern of sugars, bases, or internucleotide linkages; a format or any structural element thereof, and / or any other structural element or modification described herein; and in some embodiments, the disclosure relates to multimers of any such oligonucleotides.

[0433] In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that directs RNA interference, a PNPLA3 oligonucleotide that directs RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that directs both RNA interference and RNase H-mediated knockdown can comprise any sugar.

[0434] Various additional sugars are described in the art.

[0435] Base sequence of PNPLA3 oligonucleotide

[0436] In some embodiments, the oligonucleotide provided can guide the expression and / or level reduction of the target gene or its gene product. In some embodiments, the oligonucleotide provided can guide the expression and / or level reduction of the target gene or its gene product by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression and / or level reduction of the target gene or its gene product by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knockdown or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression and / or level reduction of the target gene or its gene product by RNA interference and / or RNA enzyme H mediated knockdown. In some embodiments, the oligonucleotide provided can guide the expression and / or level reduction of the target gene or its gene product by spatially blocking translation after annealing the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the oligonucleotide provided may include any base sequence described herein or a portion thereof, wherein a portion is a span of at least 15 consecutive bases or a span of at least 15 consecutive bases with 1-5 mismatches.

[0437] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that guides RNA interference, the PNPLA3 oligonucleotide that guides RNA enzyme H-mediated knockdown, or the PNPLA3 oligonucleotide that guides RNA interference and RNA enzyme H-mediated knockdown simultaneously may comprise any base sequence described herein. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that guides RNA interference, the PNPLA3 oligonucleotide that guides RNA enzyme H-mediated knockdown, or the PNPLA3 oligonucleotide that guides RNA interference and RNA enzyme H-mediated knockdown simultaneously may comprise any base sequence described herein or a portion thereof. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that guides RNA interference, the PNPLA3 oligonucleotide that guides RNA enzyme H-mediated knockdown, or the PNPLA3 oligonucleotide that guides RNA interference and RNA enzyme H-mediated knockdown simultaneously may comprise any base sequence described herein or a portion thereof, wherein a portion is a span of 15 consecutive bases or a span of 15 consecutive bases with 1-5 mismatches.

[0438] The sequence of the single-stranded RNAi agent is of 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.

[0439] The base sequence of the single-stranded RNAi agent is complementary to the base sequence of the target transcript. As used herein, "target transcript sequence", "target sequence", "target gene" and the like refer to a continuous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, such as a target gene, including mRNA as a product of RNA processing of a primary transcription product.

[0440] The terms "complementary", "fully complementary" and "substantially complementary" may be used herein with respect 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 can be understood from the context in which they are used. A strand of a single-stranded RNAi agent, antisense oligonucleotide or other oligonucleotide is complementary to a strand of a target sequence when maximally aligned when each base of the single-stranded RNAi agent, antisense oligonucleotide or other oligonucleotide is capable of base pairing with sequential bases on the target strand. As a non-limiting example, if a target sequence has a base sequence of, for example, 5'-GCAUAGCGAGCGAGGGAAAAC-3', a PNPLA3 oligonucleotide having a base sequence of 5'GUUUUCCCUCGCUCGCUAUGC-3' is complementary or fully complementary to such a target sequence. Of course, it should be noted that replacing U with T or vice versa does not change the amount of complementarity.

[0441] As used herein, a polynucleotide that is "substantially complementary" to a target sequence is complementary to a large extent or for the most part, but not 100% complementary. In some embodiments, a substantially complementary sequence (e.g., a chain or antisense oligonucleotide of a single-stranded RNAi agent) has 1, 2, 3, 4 or 5 mismatches with a sequence that is 100% complementary to the target sequence. In the case of a single-stranded RNAi agent, the disclosure indicates that in many cases the 5' terminal nucleotide (N1) has a mismatch with the complementary sequence of the target sequence. Similarly, in a single-stranded RNAi agent, the 3'-terminal dinucleotide, if present, can be mismatched with the complementary sequence of the target sequence. As a non-limiting example, if the target sequence has, for example, a base sequence of 5'-GCAUAGCGAGCGAGGGAAAAC-3', a single-stranded RNAi agent with a base sequence of 5'TUUUUCCCUCGCUCGCUAUTU-3' is substantially complementary to this target sequence.

[0442] The present disclosure presents various single-stranded RNAi agents and antisense oligonucleotides and other oligonucleotides in Table 1A and elsewhere, each of which has a defined base sequence. In some embodiments, the present disclosure encompasses any oligonucleotide having the following base sequence, which is, comprises, or comprises a portion of: the base sequence of any of the various single-stranded RNAi agents, antisense oligonucleotides, and other oligonucleotides disclosed herein. In some embodiments, the present disclosure encompasses any oligonucleotide having the following base sequence, which is, comprises, or comprises a portion of: the base sequence of any of the various single-stranded RNAi agents, antisense oligonucleotides, and other oligonucleotides disclosed herein, with any chemical modification, stereochemistry, format, structural feature (e.g., if the oligonucleotide is a single-stranded RNAi agent, it is the 5'-terminal structure, 5'-terminal region, 5' nucleotide portion, seed region, post-seed region, 3'-terminal region, 3'-terminal dinucleotide, 3'-terminal cap, or any structure, pattern, or portion thereof) and / or any other modification described herein (e.g., conjugation with another portion such as a targeting portion, a carbohydrate portion, a GalNAc portion, a lipid portion, etc.; and / or multimerization).

[0443] In some embodiments, the base sequence of the PNPLA3 oligonucleotide is, comprises, or comprises a portion of: the base sequence of any oligonucleotide disclosed herein.

[0444] In some embodiments, the present disclosure discloses PNPLA3 oligonucleotides of sequences described herein. In some embodiments, the present disclosure discloses PNPLA3 oligonucleotides of sequences described herein, wherein the oligonucleotide is capable of directing a reduction in the expression and / or level of a target gene or its gene product. In some embodiments, the PNPLA3 oligonucleotide of the sequence is a single-stranded RNAi agent. In some embodiments, the PNPLA3 oligonucleotide of the sequence is an antisense oligonucleotide that directs RNAse H-mediated knockdown. In some embodiments, the PNPLA3 oligonucleotide of the sequence simultaneously directs RNA interference and RNAse H-mediated knockdown. In some embodiments, the PNPLA3 oligonucleotide of the sequence comprises any structure described herein (e.g., any 5'-terminal structure described herein, 5'-terminal region, 5' nucleotide portion, seed region, post-seed region, 3'-terminal dinucleotide, 3'-terminal cap, or any portion of any of these structures, or any chemical, stereochemical, additional chemical portion, etc.). If the oligonucleotide is a ssRNAi agent, the sequence may be preceded by a T (as a non-limiting example, 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 (as a non-limiting example, 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 In some embodiments, the length of the PNPLA3 oligonucleotide does not exceed a total of about 49, 45, 40, 30, 35, 25, 23 nucleotides. In some embodiments, the portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 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 nucleotides with 0-3 mismatches, wherein a span with 0 mismatches is complementary and a span with 1 or more mismatches is a non-limiting example of a substantially complementary sequence.In some embodiments, wherein the above sequence starts with a U at the 5'-terminus, the U may be deleted and / or replaced by another base. In some embodiments, the present disclosure encompasses any oligonucleotide having the following base sequence, the base sequence is or comprises or comprises a portion of: any oligonucleotide disclosed herein having a format disclosed herein or a portion of a format disclosed herein.

[0445] In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that guides RNA interference, the PNPLA3 oligonucleotide that guides RNA enzyme H-mediated knockdown, or the PNPLA3 oligonucleotide that guides RNA interference and RNA enzyme H-mediated knockdown simultaneously may comprise any base sequence described herein. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that guides RNA interference, the PNPLA3 oligonucleotide that guides RNA enzyme H-mediated knockdown, or the PNPLA3 oligonucleotide that guides RNA interference and RNA enzyme H-mediated knockdown simultaneously may comprise any base sequence described herein or a portion thereof. In some embodiments, the PNPLA3 oligonucleotide, the PNPLA3 oligonucleotide that guides RNA interference, the PNPLA3 oligonucleotide that guides RNA enzyme H-mediated knockdown, or the PNPLA3 oligonucleotide that guides RNA interference and RNA enzyme H-mediated knockdown simultaneously may comprise any base sequence described herein or a portion thereof, wherein a portion is a span of 15 consecutive bases or a span of 15 consecutive bases with 1-5 mismatches. In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that guides RNA interference, a PNPLA3 oligonucleotide that guides RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that guides both RNA interference and RNase H-mediated knockdown may comprise any base sequence described herein or a combination of a portion thereof with any other structural element or modification described herein, including but not limited to, a sugar; a base; an internucleotide linkage; stereochemistry or a 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 modified pattern of sugar, base, or internucleotide linkage; a format or any structural element thereof, and / or any other structural element or modification described herein; and in some embodiments, the present disclosure relates to multimers of any such oligonucleotides.

[0446] Non-limiting examples of oligonucleotides having various base sequences are disclosed in Table 1A below.

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569] In some embodiments, provided oligonucleotide compositions are single-stranded RNAi agents listed in Table 1A or described elsewhere herein. In some embodiments, exemplary properties of provided oligonucleotides are demonstrated.

[0570] In some embodiments, an oligonucleotide is provided having a structure in any of the formats shown in FIG. 1 .

[0571] The present disclosure proposes many non-limiting examples of oligonucleotides capable of mediating single-stranded RNA interference (e.g., single-stranded RNAi agents). Experimental data (not shown) show that various putative single-stranded RNAi agents are actually capable of mediating RNA interference. In some experiments, an in vitro Ago-2 assay was used, including the use of RNA test substrate WV-2372 (which targets a different gene APOC3). In the presence of oligonucleotides WV-1308 and WV-2420, there was no band representing the RNA test substrate, indicating that these oligonucleotides are single-stranded RNAi agents capable of mediating RNA interference. The remaining lanes are controls: there is no substrate for the negative control ASO WV-2134; there is no substrate for the negative control ASO WV-2134, which does not mediate RNA interference; there is no substrate for the test oligonucleotide WV-1308; there is no substrate for the test oligonucleotide WV-2420; only substrate; no substrate, WV-2134 is added; and no substrate, WV-1308 is added. In vitro Ago-2 assays (data not shown) were also performed, using APOC3 mRNA as a test substrate in a 3'RACE assay of Hep3B cells. In the presence of test oligonucleotide WV-3021, cleavage products of APOC3 mRNA were detected, corresponding to the cleavage of mRNA at the site corresponding to the incision between positions 10 and 11 of WV-3021. Artificial cleavage products were also detected. Experimental data (not shown) demonstrate that the dual-mechanism oligonucleotide WV-2111 can simultaneously mediate knockdown by RNAse H and RNA interference. In the experiment, several oligonucleotides can mediate RNA interference. The RNA test substrate is WV-2372. Experiments show that in the presence of test oligonucleotides WV-1308, WV-2114, WV-2386 and WV-2387, the RNA test substrate disappears, indicating that all of these oligonucleotides can act as single-stranded RNAi agents mediating RNA interference. The remaining lanes are controls. Therefore, experiments show that oligonucleotides WV-1308, WV-2114, WV-2386 and WV-2387 can mediate RNA interference. Therefore, experiments show that several single-stranded RNAi agents (such as WV-1308, WV-2420, WV-3021, WV-2111, WV-2114, WV-2386 and WV-2387) can mediate RNA interference. The present disclosure proposes many non-limiting examples of oligonucleotides, which have various sequences, formats, modifications, 5'-terminal regions, seed regions, post-seed regions and 3'-terminal regions, and can mediate single-stranded RNA interference (for example, single-stranded RNAi agents).

[0572] Oligonucleotide Format

[0573] In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference. In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNA enzyme H mediated knocking down or spatial steric hindrance of gene expression). In some embodiments, the oligonucleotide provided can guide the expression of the target gene or its gene product and / or the reduction of the level by RNA interference and / or RNA enzyme H mediated knocking down. In some embodiments, the oligonucleotide provided can block translation spatially after annealing the target gene mRNA, and / or by changing or interfering with mRNA splicing and / or exon inclusion or exclusion, to guide the expression of the target gene or its gene product and / or the reduction of the level. In some embodiments, the oligonucleotide provided may have any format described herein or known in the art or its part or its structural element.

[0574] In some embodiments, the PNPLA3 oligonucleotide can have any format or structural elements thereof described herein or known in the art.

[0575] In some embodiments, a PNPLA3 oligonucleotide capable of directing a decrease in the expression and / or level of a target gene or its gene product can have any format or structural elements thereof described herein or known in the art.

[0576] In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that directs RNA interference, a PNPLA3 oligonucleotide that directs RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that directs both RNA interference and RNase H-mediated knockdown can have any format or structural elements thereof described herein or known in the art.

[0577] Additional non-limiting examples of various ssRNAi formats are embodied by the various single-stranded RNAi agents described herein.

[0578] In some embodiments, the single-stranded RNAi provided comprises a 5 '-end represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any 5 '-end of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0579] In some embodiments, the single-stranded RNAi provided comprises a 5'-terminal structure or 5'-terminal region represented by any single-stranded RNAi format depicted in Figure 1 or any 5'-terminal structure or 5'-terminal region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0580] In some embodiments, the single-stranded RNAi provided comprises a 5'-nucleotide represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any 5'-nucleotide of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0581] In some embodiments, the single-stranded RNAi provided comprises a 5'-nucleoside represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any 5'-nucleoside of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0582] In some embodiments, the single-stranded RNAi provided comprises a seed region represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any of the seed regions of any single-stranded RNAi agents or single-stranded RNAi formats described herein.

[0583] In some embodiments, the single-stranded RNAi provided comprises a post-seed region represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any post-seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0584] In some embodiments, the single-stranded RNAi provided comprises a post-seed region or component thereof represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any post-seed region or component thereof of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0585] In some embodiments, the single-stranded RNAi provided comprises a 3 '-terminal dinucleotide represented by any of the single-stranded RNAi formats depicted in FIG. 1 or any 3 '-terminal dinucleotide of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0586] In some embodiments, the single-stranded RNAi provided comprises a seed region having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any single-stranded RNAi format depicted in Figure 1, or any seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0587] In some embodiments, the single-stranded RNAi provided comprises a post-seed region having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any single-stranded RNAi format depicted in Figure 1, or any post-seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0588] In some embodiments, the single-stranded RNAi provided comprises a post-seed region or a component thereof having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any single-stranded RNAi format depicted in Figure 1, or any post-seed region or a component thereof of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0589] In some embodiments, the single-stranded RNAi provided comprises a 3'-terminal dinucleotide having an internucleotide linkage pattern represented by the internucleotide linkage pattern of any single-stranded RNAi format depicted in Figure 1, or any 3'-terminal dinucleotide of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0590] In some embodiments, the single-stranded RNAi provided comprises a seed region having a chemical modification pattern represented by the chemical modification pattern of any single-stranded RNAi format depicted in Figure 1 or any seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0591] In some embodiments, the single-stranded RNAi provided comprises a post-seed region having a chemical modification pattern represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any post-seed region of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0592] In some embodiments, the single-stranded RNAi provided comprises a post-seed region or a component thereof having a chemical modification pattern represented by any of the single-stranded RNAi formats depicted in Figure 1, or any post-seed region or a component thereof of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0593] In some embodiments, the single-stranded RNAi provided comprises a 3'-terminal dinucleotide having a chemical modification pattern represented by the chemical modification pattern of any single-stranded RNAi format depicted in Figure 1 or any 3'-terminal dinucleotide of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0594] In some embodiments, the single-stranded RNAi provided comprises a chemical modification represented by any of the single-stranded RNAi formats depicted in FIG. 1 , or any chemical modification of any single-stranded RNAi agent or single-stranded RNAi format described herein.

[0595] In some embodiments, the single-stranded RNAi provided comprises a chemical modification represented by any of the chemical modifications depicted in Figure 1 or any of the single-stranded RNAi formats described herein, wherein the chemical modification is conjugation of a moiety comprising a phosphate, a linker, or a targeting moiety.

[0596] In some embodiments, the single-stranded RNAi provided comprises a chemical modification represented by any chemical modification of any single-stranded RNAi format depicted in Figure 1 or described herein, wherein the chemical modification is conjugated to a portion comprising a phosphate, a linker, or a targeting moiety, wherein the targeting moiety comprises a GalNAc moiety. In some embodiments, the GalNAc is protected or deprotected GalNAc.

[0597] In some embodiments, the PNPLA3 oligonucleotide is capable of reducing the expression, activity and / or level of a target gene and / or its gene product and has the format of any oligonucleotide described herein. In some embodiments, the PNPLA3 oligonucleotide is capable of reducing the expression, activity and / or level of a target gene and / or its gene product by an RNase H-mediated mechanism or a mechanism associated with steric hindrance of translation and has the format of any oligonucleotide described herein. In some embodiments, the PNPLA3 oligonucleotide is capable of reducing the expression, activity and / or level of a target gene and / or its gene product by an RNase H-mediated mechanism or a mechanism associated with steric hindrance of translation and has an asymmetric format. In some embodiments, the PNPLA3 oligonucleotide having an asymmetric format comprises a first wing, a core and a second wing, wherein the core comprises a region of 5 or more consecutive 2'-deoxynucleotides that can anneal with the target mRNA and form a structure recognized by RNase H, and wherein the structures of the first wing and the second wing are different. In some embodiments, the first wing and the second wing differ in their 2'-modification and / or internucleotide linkage or stereochemical pattern of internucleotide linkage.

[0598] In some embodiments, the PNPLA3 oligonucleotide is capable of reducing the expression, activity, and / or level of a target gene and / or its gene product, comprises a neutral internucleotide linkage (eg, a neutral backbone).

[0599] In some embodiments, the PNPLA3 oligonucleotide comprises a neutral backbone. In some embodiments, the PNPLA3 oligonucleotide comprises an internucleotide linkage, which is a triazole, a neutral triazole, or an alkyne. In some embodiments, a nucleic acid (including but not limited to a PNPLA3 oligonucleotide) comprises an internucleotide linkage, which comprises a triazole, a neutral triazole, or an alkyne, wherein the internucleotide linkage is stereocontrolled and is in the Rp or Sp configuration. In some embodiments, the internucleotide linkage comprising a triazole has the following formula: In some embodiments the internucleotide linkage comprising a neutral triazole has the formula:

[0600] wherein X is O or S. In some embodiments, the alkyne-containing internucleotide linkage has the formula: Wherein X is O or S.

[0601] In some embodiments, the internucleotide linkage comprises a cyclic guanidine. In some embodiments, the internucleotide linkage comprises a cyclic guanidine moiety and has the following structure: In some embodiments, the neutral internucleotide linkage or the internucleotide linkage comprising 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.

[0602] As will be appreciated by those skilled in the art, in some cases, Can be used to indicate the site of attachment (e.g. ); in some cases, Can be used to indicate stereo-irregular connectivity.

[0603] Length of PNPLA3 oligonucleotide

[0604] In some embodiments, the oligonucleotides provided are capable of directing the reduction of expression and / or levels of a target gene or its gene product. In some embodiments, the oligonucleotides provided are capable of directing the reduction of expression and / or levels of a target gene or its gene product by RNA interference. In some embodiments, the oligonucleotides provided are capable of directing the reduction of expression and / or levels of a target gene or its gene product by a biochemical mechanism that does not involve RNA interference or RISC (including but not limited to RNAse H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the oligonucleotides provided are capable of directing the reduction of expression and / or levels of a target gene or its gene product by RNA interference and / or RNAse H-mediated knockdown. In some embodiments, the oligonucleotides provided are capable of directing the reduction of expression and / or levels of a target gene or its gene product by spatially blocking translation after annealing the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the oligonucleotides provided can have any length, wherein the length of the PNPLA3 oligonucleotide enables the oligonucleotide to direct the reduction of expression and / or levels of a target gene or its gene product.

[0605] In some embodiments, the length of the PNPLA3 oligonucleotide is such that the oligonucleotide is capable of directing a decrease in the expression and / or level of a target gene or its gene product.

[0606] In some embodiments, the length of the RNAi agent enables the RNAi agent to direct RNA interference of a specific target transcript in a sequence-specific manner. In some embodiments, the RNAi agent comprises a sufficient number of nucleobases with sufficient identity to recognize the target transcript. In some embodiments, the RNAi agent also has a length suitable for mediating RNAi interference.

[0607] The target portion of the sequence will be at least long enough to serve as a substrate for the cleavage directed by the iRNA at or near the portion. For example, the length of the target sequence is typically about 9-36 nucleotides ("nt"), such as about 15-30 nucleotides in length, including all subranges therebetween. Table 1A shows examples of single-stranded RNAi agents of various lengths.

[0608] Figure 1 shows a non-limiting example of a single-stranded RNAi agent with a length of 19 to 25. Single-stranded RNAi agents with any of these lengths were constructed and found to be able to knock down the target gene. Therefore, the single-stranded RNAi agent provided can have any of a plurality of different lengths.

[0609] Non-limiting examples of formats for ssRNAi agents that are 19 bases in length include: Formats 20-21 of FIG. 1 .

[0610] Non-limiting examples of formats for ssRNAi agents that are 20 bases in length include: Format 19 of FIG. 1 .

[0611] 5'-end of a PNPLA3 oligonucleotide comprising a single-stranded RNAi agent

[0612] In some embodiments, the structure of the 5'-end of the oligonucleotide enables the PNPLA3 oligonucleotide to direct the reduction of expression and / or levels of a target gene or its gene product.

[0613] In some embodiments, the structure of the 5 '-end of the RNAi agent enables the RNAi agent to direct RNA interference of a specific target transcript in a sequence-specific manner.

[0614] In some embodiments, the oligonucleotides provided 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 oligonucleotides provided that can guide RNA enzyme 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 oligonucleotides provided that can guide 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 oligonucleotides provided that can guide RNA interference and RNA enzyme 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.

[0615] The present disclosure particularly recognizes that the 5'-terminal structure of the oligonucleotide, optionally in combination with additional features according to the present disclosure, can provide unexpected advantages. In some embodiments, the present disclosure provides a 5'-terminal group (corresponding to the ribose present in natural RNA (or the deoxyribose present in natural DNA) 5'-HO-CH 2 -), which can surprisingly improve one or more properties and / or activities of the oligonucleotide (e.g., stability, activity, manufacturing cost, etc.).

[0616] In some embodiments, the 5'-OH groups of the provided oligonucleotides are unmodified, i.e., they exist as free -OH. In some embodiments, the 5'-terminal group is 5'-HO-CH 2 The present disclosure demonstrates, inter alia, that the provided oligonucleotides having a free 5'-OH group can achieve comparable properties and / or activities (e.g., stability, RNAi activity when used as ss-RNAi agents, etc.) as otherwise identical oligonucleotides containing a 5'-phosphate (or derivative thereof) group, despite literature reports that certain activities (e.g., RNAi activity) require the presence of a 5'-phosphate group.

[0617] 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 bioisostere 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'-terminal group does not contain a phosphorus atom or a carboxyl group. In some embodiments, the 5'-terminal group is 5'-HO-CH 2Among other things, the present disclosure demonstrates that provided oligonucleotides without a 5'-phosphate or a derivative or bioisostere thereof can surprisingly achieve comparable activity to oligonucleotides with a 5'-phosphate but otherwise identical, such as activity in knocking down mRNA levels of a target gene via an RNAi pathway.

[0618] In some embodiments, the 5'-nucleoside unit of the provided oligonucleotide (which includes the sugar and core base moieties, but does not include the internucleotide linkage between the 5'-nucleoside unit and the second nucleoside unit from the 5'-end) does not include a phosphate group or a derivative or bioisostere thereof. In some embodiments, the 5'-nucleoside unit does not include a phosphorus atom. In some embodiments, the 5'-nucleoside does not include an acidic group. In some embodiments, the 5'-nucleoside unit does not include a -COOH group or a salt form thereof.

[0619] In some embodiments, the 5'-terminal group is or comprises a phosphate group or a derivative or a bioisostere thereof. In some embodiments, the 5'-nucleoside unit comprises a 5'-group which is a phosphate group or a derivative or a bioisostere thereof. As will be appreciated by those of ordinary skill in the art, many such groups are known in the art and can be used in accordance with the present disclosure.

[0620] In some embodiments, the 5'-terminal group is -CH 2 -OP(O)(OH)-(OH) or a salt thereof.

[0621] In some embodiments, a 5'-nucleoside unit is provided having In some embodiments, the 5'-nucleoside unit provided has In some embodiments, X is O. In some embodiments, X is S. In some embodiments, R E Yes -(R)-CH(CH 3 )-OP(O)(OH)-SH or a salt thereof. In some embodiments, R E Yes -(R)-CH(CH 3 )-OP(O)(OH)-OH or a salt thereof. In some embodiments, R E Yes -(S)-CH(CH 3 )-OP(O)(OH)-SH or a salt thereof. In some embodiments, R E Yes -(S)-CH(CH 3 )-OP(O)(OH)-OH or a salt thereof. In some embodiments, the 5'-nucleoside unit provided has In some embodiments, the 5'-nucleoside unit provided has structure or a salt form thereof.

[0622] As will be readily appreciated by those of ordinary skill in the art, provided compounds, such as oligonucleotides or partial structures thereof, such as the 5'-terminal structure of the oligonucleotide, internucleotide linkages, etc., may exist partially (sometimes primarily) as one or more salt forms thereof at certain pHs (e.g., physiological pH) due to, for example, one or more acidic and / or basic moieties therein. In some embodiments, provided 5'-nucleoside units may exist partially (sometimes primarily) as one or more salt forms thereof. For example, depending on the pH, Can be used as Unless expressly stated otherwise, when a compound or structure is provided, all salt forms are included.

[0623] In some embodiments, R E Yes-LP(O)(XR) 2 or a salt thereof. In some embodiments, R E Yes-LP(O)(XR) 2 or a salt thereof, wherein each X is independently -O-, -S- or a covalent bond. E Yes - LP(O)(OR) 2 or a salt thereof. In some embodiments, R E is -LP(O)(OR)(SR) or a salt thereof. E is -LP(O)(OR)(R) or a salt thereof. In some embodiments, L is a covalent bond or a divalent, optionally substituted, linear or branched C 1-6 aliphatic, wherein one or more methylene units are optionally and independently substituted by -O-, -S-, or -N(R')-. E Yes -LR 5s In some embodiments, R E In some embodiments, R E yes In some embodiments, R E Yes-C(R) 2 -. In some embodiments, X is -O-. In some embodiments, X is -S-. In some embodiments, X is -N(R)-. In some embodiments, L comprises an optionally substituted, divalent or multivalent In some embodiments, L comprises an optionally substituted In some embodiments, L comprises In some embodiments, R is independently -H or selected from C 1-10 Alkyl, C 1-10 Allyl and C 6-14 In some embodiments, R is -H. In some embodiments, R E is optionally substituted In some embodiments, R E yes

[0624] Many phosphate derivatives and / or bioisosteres and 5'-nucleoside units are described in the literature and can be used according to the present disclosure, for example, some such structures are described in, for example, US2016-0194349; US2016-0186175; US20130323836, etc. In some embodiments, the 5'-terminal group R E Or 5'-nucleoside units are described in, for example, 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, such as T-VP, T-PO, etc.

[0625] Bridged morpholino and cyclohexenyl nucleotides and nucleosides are described, for example, in U.S. Patent Application Publication No. 2016-0186175, which can be used in accordance with the present disclosure.

[0626] Exemplary embodiments of variables are described extensively throughout this disclosure. For structures having two or more variables, unless otherwise indicated, each variable can independently be any embodiment described herein.

[0627] In some embodiments, a PNPLA3 oligonucleotide capable of directing a decrease in the expression and / or level of a target gene or its gene product can comprise any 5 '-end described herein or known in the art.

[0628] In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that directs RNA interference, a PNPLA3 oligonucleotide that directs RNase H-mediated knockdown can comprise any 5 '-end described herein or known in the art.

[0629] In some embodiments, a PNPLA3 oligonucleotide, a PNPLA3 oligonucleotide that directs RNase H-mediated knockdown, or a PNPLA3 oligonucleotide that directs both RNA interference and RNase H-mediated knockdown can comprise any 5 '-end described herein or known in the art.

[0630] 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 wherein 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 of FIG. 1 .

[0631] In some embodiments, the 5'-end of a provided single-stranded RNAi agent does not comprise a phosphorus-containing moiety (e.g., the 5'-end comprises phosphorus). Non-limiting examples of ssRNAi formats in which the 5'-end does not comprise a phosphorus-containing moiety include formats 16-19, 22, 32-79, 83-91, 96, and 103 of FIG. 1 .

[0632] In some embodiments, the 5'-end of the single-stranded RNAi agent provided has a portion comprising a phosphate (such as a phosphodiester, a phosphorothioate, a phosphorodithioate, an H-phosphonate), or other portions similar or identical to internucleotide linkages comprising a phosphate. In some embodiments, the 5'-end of the single-stranded RNAi agent provided has a portion comprising a phosphate but not a phosphodiester; in some embodiments, such portions are referred to as phosphate mimetics, modified phosphates, or phosphate analogs.

[0633] In some embodiments, the 5'-end of a provided single-stranded RNAi agent does not have a phosphate-containing moiety.

[0634] In some embodiments of the single-stranded RNAi agent, the 5' terminal structure has a structure selected from the group consisting of 5'-(R)-MeOH T, 5'-(R)-Me PO T, 5'-(R)-Me PS T, 5'-(R)-Me PH T, 5'-(S)-Me OH T, 5'-(S)-Me POT, 5'-(S)-Me PS T, and 5'-(S)-PH T.

[0635] In some embodiments of the single-stranded RNAi agents provided, they comprise a phosphorus-containing moiety at the 5'-terminal structure, the 5'-terminal structure being represented by a structure selected from the group consisting of Formula IV-a (Mod022, also known as C3 PO and n-propyl), IV-b (Mod022*), IV-c (POMod023*), IV-d (PSMod023*), and IV-e (PHMod023*):

[0636]

[0637] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, the 5'-terminal structure being represented by a structure selected from the group consisting of: Structure of Formula IV-f (also known as n-propyl C3 PO or Mod022):

[0638]

[0639] n-Propyl(Mod022)

[0640] Where 5' indicates the point of attachment to the 5' carbon of the sugar.

[0641] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, the 5'-terminal structure being represented by a structure selected from the group consisting of: (also referred to as C3 PS or Mod022*) Structure:

[0642]

[0643] n-Propyl (Mod022*)

[0644] wherein 5' indicates the point of attachment to the 5' carbon of the sugar (eg N1).

[0645] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, the 5'-terminal structure being represented by a structure selected from the group consisting of: Structure of Formula IV-g (also known as dimethyl C3 or C3 dimethyl PS or Mod023*):

[0646]

[0647] Dimethyl C3 (Mod023*)

[0648] wherein 5' indicates the point of attachment to the 5' carbon of the sugar (eg N1).

[0649] In some embodiments, the single-stranded RNAi agent comprises a 5'-terminal structure selected from any one of: PO (phosphodiester), Formula IV-h; PH (H-phosphonate), Formula IV-i; and PS (phosphorothioate), Formula IV-j:

[0650]

[0651] In some embodiments of the provided single-stranded RNAi agents, they comprise a phosphorus-containing moiety at the 5'-terminal structure, the 5'-terminal structure being represented by a structure selected from any one of the following:

[0652]

[0653] wherein 5' indicates the point of attachment to the 5' carbon of the sugar (eg N1).

[0654] In some embodiments, P in any of Formulas IV-a to IV-j is stereo-random or stereo-constrained as in the Sp or Rp configuration.

[0655] In some embodiments, the 5'-terminal structure is selected from any one of the following: a phosphate, a phosphate analog, a 5'-monophosphate ((HO)2(O)PO-5'), a 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'), a 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), a 5'-monothioate (phosphorothioate; (HO)2( (HO)(HS)(S)PO-5'), 5'-monodithioate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothioate ((HO)2(O)PS-5'); any additional combination of oxygen / sulfur substituted mono-, di- and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0656] In some embodiments, 5'-ends comprising phosphorus-containing moieties can have particular advantages, as single-stranded RNAi agents comprising them may be more active in RNA interference.

[0657] In some embodiments, the 5' terminal structure has the following structure: a 5'-nucleotide or a modified 5'-nucleotide, a 5'-nucleotide analog, a 5'-nucleoside or a modified 5'-nucleoside or a 5'-nucleoside analog.

[0658] In some embodiments, the 5' terminal structure has the structure of any of the following: 5'-guanosine cap, 5'-adenosine cap, 5'-monothioate, 5'-monodithioate, 5'-phosphorothioate, 5'-phosphoramidate, 5'-alkylphosphonate and 5'-alkyletherphosphonate; 5'-monophosphate, 5'-diphosphate and 5'-triphosphate; 5'-triphosphate; monophosphate, diphosphate or triphosphate, wherein at least one oxygen atom in the monophosphate, diphosphate or triphosphate is replaced by a sulfur atom; 5'-α-thiotriphosphate and 5'-γ-thiotriphosphate; alkylphosphonate; alkylphosphonate having the formula: RP(OH)(O)--O-5' or (OH) 2 (O)P-5'-CH 2 --, where R is C 1 -C 3 Alkyl; alkyl ether phosphonate; or an alkyl ether phosphonate having the formula: RP(OH)(O)--O-5', wherein R is an alkyl ether.

[0659] Various 5'-nucleosides are described in, for example, US patent application Ser. No. 14 / 959,714, published as US2016-0194349 Al; US patent application Ser. No. 14 / 983,907, published as US2016-0186175 Al; or US patent application Ser. No. 13 / 696,796, published as US20130323836.

[0660] In some embodiments, the 5'-terminal structure is a vinylphosphonate.

[0661] In certain embodiments, oligomeric compounds are provided, wherein the 5'-terminal compound has the formula VIII-c, wherein G is F, OCH 3 or O(CH 2 ) 2 --OCH 3 .

[0662] In some embodiments, the 5' terminal structure has a structure selected from the group consisting of 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)-MePS T, and 5'-(S)-PH T.

[0663] In some embodiments, the 5' terminal structure has a structure of 5'-(R)-MeOHT.

[0664] In some embodiments, the 5' terminal structure has a structure of 5'-(R)-Me PO T.

[0665] In some embodiments, the 5' terminal structure has a structure of 5'-(R)-MePST.

[0666] In some embodiments, the 5' ter...

Claims

1. A composition comprising oligonucleotides of a specific oligonucleotide type, characterized in that: a) common base sequence; b) common backbone linkage patterns; c) a common backbone chiral center pattern; wherein the oligonucleotide comprises at least one internucleotide linkage comprising a linkage phosphorus in an Sp configuration; wherein the composition is chirality controlled in that the composition is enriched in oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence; wherein the oligonucleotide targets PNPLA3; wherein the common base sequence comprises 15 or more consecutive bases of TACUGUAGAAAGGCAUGAATU (SEQ ID NO: 153), wherein one or more U may be optionally and independently replaced by T; and The length of the common base sequence is 15 to 49 nucleotides in total.

2. The composition of claim 1, wherein the plurality of oligonucleotides: independently share the same linkage phosphorus stereochemistry at five or more chirality-controlled internucleotide linkages; and wherein at least 10% of all oligonucleotides in the composition that share the common base sequence are the plurality of oligonucleotides.

3. The composition of claim 2, wherein the plurality of oligonucleotides independently share the same linkage phosphorus stereochemistry at five or more chiral internucleotide linkages, and 50%-100% of all oligonucleotides in the composition that share the common base sequence are the plurality of oligonucleotides.

4. The composition of claim 2, wherein the plurality of oligonucleotides independently share the same linkage phosphorus stereochemistry at each phosphorothioate internucleotide linkage.

5. The composition of claim 2, wherein the plurality of oligonucleotides have the same structure.

6. The composition of claim 4, wherein the composition is a liquid composition.

7. The composition of claim 2, wherein each of the plurality of oligonucleotides independently comprises a targeting moiety.

8. The composition of claim 7, wherein the targeting moiety is 9. The composition of claim 7 or 8, wherein the targeting moiety is linked to the one or more oligonucleotides via a linker.

10. The composition of claim 9, wherein the linker has the following structure:

11. The composition of claim 7, wherein the targeting moiety is selected from:

12. Use of a therapeutically effective amount of a composition according to any one of claims 1 to 11 in the preparation of a medicament for treating fatty liver in humans, wherein the therapeutically effective amount of the composition is administered to a human in need of such treatment.

13. The use according to claim 12, wherein the fatty liver is non-alcoholic fatty liver disease.

14. The use according to claim 12, wherein the fatty liver is non-alcoholic steatohepatitis.

15. Use of a therapeutically effective amount of a composition according to any one of claims 1 to 11 in the preparation of a medicament for treating non-alcoholic steatohepatitis with liver fibrosis or non-alcoholic steatohepatitis with liver cirrhosis in humans, wherein the therapeutically effective amount of the composition is administered to a human in need of such treatment.

16. Use of a therapeutically effective amount of a composition according to any one of claims 1 to 11 for the preparation of a medicament for treating non-alcoholic steatohepatitis with cirrhosis and hepatocellular carcinoma in humans, wherein the therapeutically effective amount of the composition is administered to a human in need of such treatment.

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