Oligonucleotide compositions and methods of use thereof

Abstract

To provide designed oligonucleotides, compositions and methods thereof.SOLUTION: In one embodiment, provided oligonucleotide compositions improve single stranded RNA interference and / or RNaseH-mediated knockdown. In particular, it encompasses the recognition that structural elements of an oligonucleotide can significantly affect the properties and activities (e.g., RNA interference (RNAi) activity, stability, delivery, etc.) of the oligonucleotide, such as base sequence, chemical modifications (e.g., sugar modifications, base modifications, and / or internucleotide linkage modifications) 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. The oligonucleotide compositions can also be used, for example, to effect RNA interference and / or RNaseH-mediated knockdown.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 514,769, filed June 2, 2017, U.S. Provisional Patent Application No. 62 / 514,771, filed June 2, 2017, U.S. Provisional Patent Application No. 62 / 656,949, filed April 12, 2018, U.S. Provisional Patent Application No. 62 / 670,686, filed May 11, 2018, and U.S. Provisional Patent Application No. 62 / 670,709, filed May 11, 2018, each of which is incorporated by reference in its entirety. [Background technology]

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

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

[0004] In some embodiments, the present disclosure demonstrates that compositions comprising oligonucleotides (and particularly single-stranded oligonucleotides) with controlled structural elements can provide unexpected properties and / or activities.

[0005] In some embodiments, the present disclosure includes the recognition that stereochemistry (particularly the stereochemistry of backbone chiral centers) can unexpectedly improve the properties of oligonucleotides. In contrast to the often-held observation that some structural elements that increase stability can also reduce activity (e.g., RNA interference), the present disclosure shows that controlling stereochemistry can surprisingly increase stability without significantly reducing activity.

[0006] In some embodiments, the present disclosure provides techniques (e.g., compounds, methods, etc.) for improving the stability of oligonucleotides while maintaining or increasing activity, including compositions of oligonucleotides with improved stability.

[0007] In some embodiments, the present disclosure provides oligonucleotides having certain 5' end structures.

[0008] Among other things, the present disclosure demonstrates that such oligonucleotides can have desirable properties.

[0009] In some embodiments, the present disclosure provides 5' end structures that, when used in accordance with the present disclosure, can produce oligonucleotides with enhanced biological activity (eg, RNAi activity).

[0010] While the literature often reports that the presence of a 5' phosphate (or modified phosphate) moiety is required for RNAi activity, in some embodiments, the present disclosure surprisingly demonstrates that oligonucleotides having an unmodified 5' end (i.e., having a 5'-OH) can achieve RNAi activity comparable to identical oligonucleotides that contain a 5' phosphate (or modified phosphate) moiety. Thus, among other things, the present disclosure provides, in some embodiments, oligonucleotides whose sequences are directed to an RNAi target site, which oligonucleotides may contain one or more other structural features (described herein and / or otherwise known in the art) that are useful (or not deleterious) to RNAi activity, and which oligonucleotides contain a 5'-OH moiety.

[0011] In some embodiments, the present disclosure recognizes that various additional chemical moieties (e.g., lipid moieties and / or carbohydrate moieties) can be incorporated into an oligonucleotide to improve one or more oligonucleotide properties (e.g., knockdown of a target gene or its gene product). In some embodiments, the additional chemical moieties are optional. In some embodiments, an oligonucleotide can include more than one additional chemical moiety. In some embodiments, an oligonucleotide can include two or more additional chemical moieties, where the additional chemical moieties are the same or different, or of the same category (e.g., targeting moiety, carbohydrate moiety, moiety that binds to ASPGR, lipid moiety, etc.), or of different categories. In some embodiments, certain additional chemical moieties facilitate delivery of the oligonucleotide to desired cells, tissues, and / or organs. In some embodiments, certain additional chemical moieties facilitate internalization of the oligonucleotide and / or increase the stability of the oligonucleotide.

[0012] In some embodiments, the present disclosure provides techniques for incorporating various additional chemical moieties into oligonucleotides, e.g., reagents and methods for introducing additional chemical moieties via a nucleobase (e.g., by covalent attachment (optionally via a linker) to a site on the nucleobase).

[0013] In some embodiments, the present disclosure demonstrates that surprisingly high target specificity can be achieved using oligonucleotides having structures that include one or more of the features described herein.

[0014] In some embodiments, the present disclosure provides techniques (e.g., oligonucleotide compositions and methods) for achieving allele-specific suppression, whereby transcripts derived from one allele of a particular target gene are selectively knocked down relative to at least one other allele of the same gene.

[0015] Among other things, the present disclosure provides structural elements, techniques, and / or features that can be incorporated into an oligonucleotide to confer or adjust one or more properties thereof (e.g., compared to an otherwise identical oligonucleotide lacking the related technique or feature). In some embodiments, the present disclosure describes that one or more of the provided techniques and / or features can be usefully incorporated into oligonucleotide(s) of various sequences.

[0016] In some embodiments, the present disclosure demonstrates that certain provided structural elements, techniques, and / or features are particularly useful for oligonucleotides (e.g., RNAi agents) that participate in and / or induce the RNAi mechanism. However, the teachings of the present disclosure are not limited to oligonucleotides that participate in or function via any particular mechanism. In some embodiments, the present disclosure relates to any oligonucleotide useful for any purpose, such oligonucleotides that function via any mechanism and include any sequence, structure, or format (or portion thereof) described herein. In some embodiments, the present disclosure provides oligonucleotides useful for any purpose, such as oligonucleotides that function via any mechanism and include any sequence, structure, or format (or portion thereof) described herein, including, but not limited to, any 5'-terminal structure, 5'-terminal region, first region (including, but not limited to, a seed region), second region (including, but not limited to, a post-seed region), and 3'-terminal region (which may be a 3'-terminal dinucleotide and / or a 3'-terminal cap), optional additional chemical moieties (including, but not limited to, a targeting moiety, a carbohydrate moiety, an APGR-binding moiety, and a lipid moiety), stereochemistry or stereochemistry pattern, modification or modification pattern, internucleotide linkage or internucleotide linkage pattern, sugar(s) modification or sugar modification pattern, base(s) modification or base modification pattern. In some embodiments, provided oligonucleotides may participate in (e.g., induce) the RNAi mechanism. In some embodiments, provided oligonucleotides may participate in the RNase H (ribonuclease H) mechanism. In some embodiments, provided oligonucleotides can act as translation inhibitors (eg, sterically block translation).

[0017] In some embodiments, the provided oligonucleotides may be involved in exon skipping. In some embodiments, the provided oligonucleotides may be aptamers. In some embodiments, the provided oligonucleotides may bind to and inhibit the function of proteins, small molecules, nucleic acids, or cells. In some embodiments, the provided oligonucleotides may be involved in triple helix formation with double-stranded nucleic acids in cells. In some embodiments, the provided oligonucleotides may bind to genomic (e.g., chromosomal) nucleic acids. In some embodiments, the provided oligonucleotides may bind to genomic (e.g., chromosomal) nucleic acids and thereby inhibit or reduce expression of the nucleic acid (e.g., by inhibiting or reducing transcription, transcription enhancement, modification, etc.). In some embodiments, the provided oligonucleotides may bind to DNA quadruplexes. In some embodiments, the provided oligonucleotides may be immunomodulatory. In some embodiments, the provided oligonucleotides may be immunostimulatory. In some embodiments, the provided oligonucleotides may be immunostimulatory and may contain CpG sequences. In some embodiments, the provided oligonucleotides may be immunostimulatory and may contain CpG sequences and may be useful as adjuvants. In some embodiments, provided oligonucleotides may be immunostimulatory, may contain CpG sequences, may be useful as adjuvants in the treatment of disease (e.g., infectious disease or cancer). In some embodiments, provided oligonucleotides may be therapeutic. In some embodiments, provided oligonucleotides may be non-therapeutic. In some embodiments, provided oligonucleotides may be therapeutic or non-therapeutic. In some embodiments, provided oligonucleotides are useful in therapeutic, diagnostic, research, and / or nanomaterial applications. In some embodiments, provided oligonucleotides may be useful for experimental purposes. In some embodiments, provided oligonucleotides may be useful for experimental purposes (e.g., as probes) in microarrays, etc.In some embodiments, provided oligonucleotides may participate in multiple biological mechanisms; in certain such embodiments, for example, provided oligonucleotides may participate in both the RNAi mechanism and the RNase H mechanism.

[0018] In some embodiments, provided oligonucleotides are directed to a target (e.g., a target sequence, a target RNA, a target mRNA, a target pre-mRNA, a target gene, etc.). A target gene is a gene for which the expression and / or activity of one or more gene products (e.g., RNA and / or protein products) is intended to be modified. In many embodiments, the target gene is intended to be inhibited. Thus, when the oligonucleotides described herein act on a particular target gene, the presence of the oligonucleotide modifies the presence and / or activity of one or more gene products of that gene compared to its absence.

[0019] In some embodiments, a target is a particular allele whose expression and / or activity of one or more products (e.g., RNA and / or protein products) is intended to be modified. In many embodiments, the target allele is one whose presence and / or expression is associated with (e.g., correlated with) the presence, onset, and / or severity of one or more diseases and / or conditions. Alternatively, or in addition, in some embodiments, the target allele is one whose modification of the level and / or activity of one or more gene products is correlated with amelioration of one or more aspects of the disease and / or condition (e.g., delayed onset, reduced severity, responsiveness to other treatments, etc.).

[0020] In some embodiments, different alleles of the same gene exist where the presence and / or activity of a particular allele (a disease-associated allele) is associated (e.g., correlated) with the presence, onset, and / or severity of one or more disorders, diseases, and / or conditions, and such different alleles are not so associated, or are associated to a lesser extent (e.g., exhibit a less significant or statistically insignificant correlation). In some such embodiments, the oligonucleotides and methods described herein may preferentially or specifically target the associated allele relative to one or more less / unassociated allele(s), thereby mediating allele-specific suppression.

[0021] In some embodiments, the target sequence is a sequence to which an oligonucleotide described herein binds. In many embodiments, the target sequence is identical to or an exact complement of the sequence of a provided oligonucleotide or the sequence of consecutive residues present therein (e.g., a provided oligonucleotide comprises a target binding sequence that is identical to or an exact complement of the target sequence). In some embodiments, the target binding sequence is an exact complement of a target sequence in a transcription product (e.g., pre-mRNA, mRNA, etc.). The target binding sequence / target sequence can be of various lengths for the provided oligonucleotides to have desired activities and / or properties. In some embodiments, the target binding sequence / target sequence comprises 5-50 bases (e.g., 10-40, 15-30, 15-25, 16-25, 17-25, 18-25, 19-25, 20-25, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 or more). In some embodiments, a small number of differences / mismatches between (relevant portions of) the oligonucleotide and its target sequence are tolerated, including, but not limited to, the target sequence and / or the 5' and / or 3' terminal regions of the oligonucleotide sequence. In many embodiments, the target sequence is present within a target gene. In many embodiments, the target sequence is present within a transcription product (e.g., mRNA and / or pre-mRNA) produced from the target gene.

[0022] In some embodiments, the target sequence comprises one or more allelic sites (i.e., positions at which allelic variation occurs within the target gene). In some embodiments, the allelic site is a mutation. In some embodiments, the allelic site is a SNP. In some such embodiments, provided oligonucleotides preferentially or specifically bind to one allele compared to one or more other alleles. In some embodiments, provided oligonucleotides preferentially bind to disease-associated alleles. For example, in some embodiments, the oligonucleotides provided herein (or target binding sequence portions thereof) have a sequence that is completely or at least partially identical to the target sequence of a particular allelic version, or a sequence that is the exact complement of the target sequence of a particular allelic version.

[0023] In some embodiments, the oligonucleotides provided herein (or target binding sequence portions thereof) have a sequence identical to or an exact complement of a target sequence comprising an allelic site or an allelic site of a disease-associated allele. In some embodiments, the oligonucleotides provided herein have a target binding sequence that is an exact complement of a target sequence comprising an allelic site of a transcript of an allele (in many embodiments, a disease-associated allele), where the allelic site is a mutation. In some embodiments, the oligonucleotides provided herein have a target binding sequence that is an exact complement of a target sequence comprising an allelic site of a transcript of an allele (in many embodiments, a disease-associated allele), where the allelic site is a SNP. In some embodiments, the sequence is any sequence disclosed herein.

[0024] Unless otherwise specified, all sequences (including but not limited to base sequences and chemical, modification, and / or stereochemical patterns) are presented in 5' to 3' order.

[0025] In some embodiments, the present disclosure provides compositions and methods relating to oligonucleotides that are specific for a target and have any format, structural element, or base sequence of any of the oligonucleotides disclosed herein.

[0026] In some embodiments, the disclosure provides compositions and methods related to target-specific oligonucleotides having or comprising the base sequence of any of the oligonucleotides disclosed herein, or a region of at least 15 consecutive nucleotides of the base sequence of any of the oligonucleotides disclosed herein, wherein the first nucleotide of the base sequence or the first nucleotide of the at least 15 consecutive nucleotides can optionally be replaced by a T or a DNA T. In some embodiments, the oligonucleotide is capable of inducing ssRNAi.

[0027] In some embodiments, the present disclosure provides compositions and methods for RNA interference induced by single-stranded RNAi agents. In some embodiments, the oligonucleotides of such compositions can have the format, structural elements, or base sequences of the oligonucleotides disclosed herein.

[0028] In some embodiments, the present disclosure provides compositions and methods for RNase H-mediated knockdown of target gene RNA induced by oligonucleotides (eg, antisense oligonucleotides).

[0029] The provided oligonucleotides and oligonucleotide compositions can have any format, structural element, or base sequence of any of the oligonucleotides disclosed herein. In some embodiments, the structural element is a 5'-end structure, a 5'-end region, a 5'-nucleotide, a seed region, a post-seed region, a 3'-end region, a 3'-end dinucleotide, a 3'-end cap, or any portion of any of these structures, GC content, a long GC stretch, and / or any modification, chemistry, stereochemistry, modification pattern, chemical pattern, or stereochemical pattern, or chemical moiety (e.g., including but not limited to, a targeting moiety, a lipid moiety, a GalNAc moiety, a carbohydrate moiety, etc.), any component, or any combination of any of the above.

[0030] In some embodiments, the present disclosure provides compositions and methods of use of oligonucleotides.

[0031] In some embodiments, the present disclosure provides compositions and methods of use of oligonucleotides capable of inducing both RNA interference and RNase H-mediated knockdown of target gene RNA. In some embodiments, the oligonucleotides of such compositions can have the format, structural elements, or base sequence of the oligonucleotides disclosed herein.

[0032] In some embodiments, an oligonucleotide that induces a particular event or activity is involved in the particular event or activity, e.g., is involved in reducing the expression, level, or activity of a target gene or its gene product. In some embodiments, an oligonucleotide is considered to "induce" a particular event or activity when the presence of the oligonucleotide in a system in which the event or activity may occur correlates with a detectable increase in the occurrence, frequency, intensity, and / or level of the event or activity.

[0033] In some embodiments, the provided oligonucleotides comprise any one or more structural elements of the oligonucleotides described herein, including, for example, a base sequence (or at least a 15-contiguous base portion thereof), a pattern of internucleotide linkages (or at least a 5-contiguous internucleotide linkage portion thereof), a stereochemical pattern of internucleotide linkages (or at least a 5-contiguous internucleotide linkage portion thereof), a 5'-terminal structure, a 5'-terminal region, a first region, a second region, and a 3'-terminal region (which may be a 3'-terminal dinucleotide and / or a 3'-terminal cap), and optional additional chemical moieties; in some embodiments, at least one structural element comprises a chiral center that is chirally controlled. In some embodiments, the total number of nucleotides contained in the 3'-terminal dinucleotide may be two. In some embodiments, the oligonucleotides further comprise a chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, a lipid moiety, and any other chemical moiety described herein or known in the art. In some embodiments, the moiety that binds to APGR is a GalNAc moiety or a variant, derivative, or modified version thereof described herein and / or known in the art. In some embodiments, the oligonucleotide is a single-stranded RNAi agent. In some embodiments, the first region is a seed region. In some embodiments, the second region is a post-seed region.

[0034] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of the single-stranded RNAi agent described herein, for example, a 5'-end structure, a 5'-end region, a seed region, a post-seed region (the region between the seed region and the 3'-end region), and a 3'-end region (which may be a 3'-end dinucleotide and / or a 3'-end cap), as well as optional additional chemical moieties; in some embodiments, at least one structural element comprises a chiral center that is chirally controlled. In some embodiments, the total number of nucleotides contained in the 3'-end dinucleotide may be two. In some embodiments, the oligonucleotide further comprises a chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety. In some embodiments, the moiety that binds to APGR is any GalNAc or variant, derivative, or modification thereof described herein or known in the art.

[0035] In some embodiments, the provided oligonucleotides comprise any one or more structural elements of the oligonucleotides described herein (e.g., a 5'-terminal structure, a 5'-terminal region, a first region, a second region, a 3'-terminal region, and optional additional chemical moieties), wherein at least one structural element comprises a chiral center that is chiral-controlled. In some embodiments, the oligonucleotide comprises a section in which the total number of nucleotides without a 2'-modification is at least 5. In some embodiments, the oligonucleotide further comprises an additional chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety. In some embodiments, the provided oligonucleotides are capable of inducing RNA interference. In some embodiments, the provided oligonucleotides are capable of inducing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides are capable of inducing both RNA interference and RNase H-mediated knockdown. In some embodiments, the first region is a seed region. In some embodiments, the second region is a post-seed region.

[0036] In some embodiments, provided oligonucleotides comprise any one or more structural elements of a single-stranded RNAi agent (e.g., a 5'-end structure, a 5'-end region, a seed region, a post-seed region, and a 3'-end region, and optional additional chemical moieties), wherein at least one structural element comprises a chiral center that is chiral-controlled, and in some embodiments, the oligonucleotide also has the ability to induce RNase H-mediated knockdown of a target gene RNA. In some embodiments, the oligonucleotide comprises a section in which the total number of 2'-deoxynucleotides is at least 5. In some embodiments, the oligonucleotide further comprises a chemical moiety selected from, by way of non-limiting example, a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety, and any other additional chemical moiety described herein.

[0037] In some embodiments, the present disclosure demonstrates that oligonucleotide properties can be modulated through chemical modification. In some embodiments, the present disclosure provides an 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. In some embodiments, the present disclosure provides an oligonucleotide composition capable of inducing single-stranded RNA interference, the oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, and / or one or more sugar, and / or one or more base modifications. In some embodiments, the oligonucleotide or oligonucleotide composition is also capable of inducing RNase H-mediated knockdown of target gene RNA. In some embodiments, the present disclosure demonstrates that oligonucleotide properties (e.g., activity, toxicity, etc.) can be modulated through chemical modification of the sugar, nucleobase, and / or internucleotide linkage. In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more modified internucleotide linkages (or "unnatural internucleotide linkages") (such as the natural internucleotide phosphate linkage found in natural DNA and RNA (-OP(O)(OH)O- (which at physiological pH is in the salt form (-OP(O)(O -Provided are oligonucleotide compositions comprising a plurality of oligonucleotides comprising a linkage available in place of) (which may be present as )O-), one or more modified sugar moieties, and / or one or more natural phosphate linkages. In some embodiments, the provided oligonucleotides can comprise two or more types of modified internucleotide linkages. In some embodiments, the provided oligonucleotides comprise a non-negatively charged internucleotide linkage. In some embodiments, the non-negatively charged internucleotide linkage is a neutral internucleotide linkage. In some embodiments, the neutral internucleotide linkage comprises a triazole moiety, an alkyne moiety, or a cyclic guanidine moiety. Such moieties are optionally substituted. In some embodiments, the provided oligonucleotides comprise a neutral internucleotide linkage and another internucleotide linkage that is not a neutral backbone. In some embodiments, the provided oligonucleotides comprise a neutral internucleotide linkage and a phosphorothioate internucleotide linkage. In some embodiments, the provided oligonucleotide compositions comprising a plurality of oligonucleotides are chiral-controlled, wherein the level of the plurality of oligonucleotides in the composition is controlled or predetermined, and the plurality of oligonucleotides share a common stereochemical configuration for one or more chiral internucleotide linkages. For example, in some embodiments, a plurality of oligonucleotides share a common stereochemical configuration for 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, a plurality of oligonucleotides share a common stereochemical configuration for each chiral internucleotide linkage. In some embodiments, a chiral internucleotide linkage is referred to as a chiral-controlled internucleotide linkage when a controlled level of oligonucleotides in a composition share a common stereochemical configuration (independently Rp or Sp).In some embodiments, the modified internucleotide linkages are such that at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.)), a majority (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, etc., in some embodiments, at least 30%, in some embodiments, at least 40%, in some embodiments, at least 50%, in some embodiments, at least 60%, in some embodiments, at least 70%, in some embodiments, at least 80%, in some embodiments, at least 90%, in some embodiments, at least 99%, etc.) of the modified internucleotide linkages are in the anionic form (e.g., —OP(O)(O). - )-O- (anionic form of the natural phosphate bond), -OP(O)(S -In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that it exists in a neutral or cationic form (e.g., as compared to a nucleotide such as )-O- (the anionic form of a phosphorothioate linkage)). In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that it exists predominantly in the neutral form at a certain pH. In some embodiments, the modified internucleotide linkage is a cationic internucleotide linkage in that it exists predominantly in the cationic form at a certain pH. In some embodiments, the pH is human physiological pH (about 7.4). In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that at least 90% of the internucleotide linkage exists in its neutral form when the pH of the aqueous solution is 7.4. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage in that at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the internucleotide linkage exists in its neutral form in an aqueous solution of the oligonucleotide. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, the percentage is at least 99%. In some embodiments, a non-negatively charged internucleotide linkage (e.g., a neutral internucleotide linkage), when in its neutral form, has no moieties with a pKa of less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, or less than 14. In some embodiments, the pKa of an internucleotide linkage in the present disclosure can be represented by the pKa of a CH3-internucleotide linkage -CH3 (i.e., two -CH3 groups replace two nucleoside units linked by the internucleotide linkage).Without wishing to be bound by any particular theory, it is believed that, at least in some cases, neutral internucleotide linkages in oligonucleotides can improve properties and / or activity compared to comparable nucleic acids that do not contain neutral internucleotide linkages, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and / or improved nuclear uptake.

[0038] In some embodiments, the non-negatively charged internucleotide linkage has a structure such as, for example, Formula In-1, Formula In-2, Formula In-3, Formula II, Formula II-a-1, Formula II-a-2, Formula II-b-1, Formula II-b-2, Formula II-c-1, Formula II-c-2, Formula II-d-1, Formula II-d-2, etc. In some embodiments, the non-negatively charged internucleotide linkage comprises a triazole moiety or an alkyne moiety. In some embodiments, the non-negatively charged internucleotide linkage comprises a cyclic guanidine moiety. In some embodiments, the modified internucleotide linkage comprising a cyclic guanidine moiety is [ka] In some embodiments, the neutral internucleotide linkage comprising a cyclic guanidine moiety is chiral controlled. In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage.

[0039] In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate internucleotide linkage is an internucleotide linkage chiral controlled to the Sp configuration.

[0040] In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate is an internucleotide linkage chiral controlled to the Rp configuration.

[0041] In some embodiments, the present disclosure provides a neutral internucleotide linkage comprising an optionally substituted triazolyl group, a neutral internucleotide linkage comprising an optionally substituted alkynyl group, and a Tmg group. [ka] and at least one phosphorothioate.

[0042] In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage selected from a neutral internucleotide linkage comprising an optionally substituted triazolyl group, a neutral internucleotide linkage comprising an optionally substituted alkynyl group, and a neutral internucleotide linkage comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is an internucleotide linkage chirally controlled to the Sp configuration.

[0043] In some embodiments, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleotide linkage selected from a neutral internucleotide linkage comprising an optionally substituted triazolyl group, a neutral internucleotide linkage comprising an optionally substituted alkynyl group, and a neutral internucleotide linkage comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is an internucleotide linkage chirally controlled to the Rp configuration.

[0044] Various types of internucleotide linkages have different properties. Without wishing to be bound by any theory, the present disclosure specifies that natural phosphate linkages (phosphodiester internucleotide linkages) are anionic and may be unstable when used in vivo without other chemical modifications. Phosphorothioate internucleotide linkages are anionic, generally more stable in vivo than natural phosphate linkages, and generally more hydrophobic, while neutral internucleotide linkages containing cyclic guanidine moieties (such as those exemplified in the present disclosure) are neutral at physiological pH and may be more stable in vivo than natural phosphate linkages and more hydrophobic.

[0045] In some embodiments, the chiral-controlled neutral internucleotide linkage is neutral at physiological pH, chiral-controlled, stable in vivo, hydrophobic, and capable of increasing endosomal escape.

[0046] In some embodiments, provided oligonucleotides comprise one or more regions (e.g., a block region, a wing region, a core region, a 5' terminal region, a 3' terminal region, an intermediate region, a seed region, a post-seed region, etc.). In some embodiments, a region (e.g., a block region, a wing region, a core region, a 5' terminal region, a 3' terminal region, an intermediate region, etc.) comprises a non-negatively charged internucleotide linkage (e.g., those of Formula In-1, Formula In-2, Formula In-3, Formula II, Formula II-a-1, Formula II-a-2, Formula II-b-1, Formula II-b-2, Formula II-c-1, Formula II-c-2, Formula II-d-1, Formula II-d-2, etc.). In some embodiments, a region comprises a neutral internucleotide linkage. In some embodiments, a region comprises an internucleotide linkage comprising a triazole moiety or an alkyne moiety. In some embodiments, a region comprises an internucleotide linkage comprising a cyclic guanidine moiety. In some embodiments, a region comprises an internucleotide linkage comprising a cyclic guanidine moiety. In some embodiments, a region comprises [ka] In some embodiments, such internucleotide linkages are chiral controlled.

[0047] In some embodiments, the disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkage, sugar, and / or base modifications.

[0048] In some embodiments, the present disclosure provides oligonucleotide compositions capable of inducing single-stranded RNA interference, the oligonucleotide compositions comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, and / or one or more sugar and / or one or more base modifications. In some embodiments, the oligonucleotides or oligonucleotide compositions are also capable of inducing RNase H-mediated knockdown of target gene RNA.

[0049] In some embodiments, the nucleotide is a natural nucleotide. In some embodiments, the nucleotide is a modified nucleotide. In some embodiments, the nucleotide is a nucleotide analog. In some embodiments, the base is a modified base. In some embodiments, the base is a protected nucleobase (such as a protected nucleobase used in oligonucleotide synthesis). In some embodiments, the base is a base analog. In some embodiments, the sugar is a modified sugar. In some embodiments, the sugar is a sugar analog. In some embodiments, the internucleotide linkage is a modified internucleotide linkage. In some embodiments, the nucleotide comprises a base, a sugar, and an internucleotide linkage, each of which is independently and optionally naturally occurring or non-naturally occurring. In some embodiments, the nucleoside comprises a base and a sugar, each of which is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2'-deoxy) nucleotides and RNA (2'-OH) nucleotides, as well as those containing one or more modifications in the base, sugar, and / or internucleotide linkage. Non-limiting examples of sugars include ribose and deoxyribose, and ribose and deoxyribose with 2'-modifications, including, but not limited to, 2'-F, LNA, 2'-OMe, and 2'-MOE modifications. In some embodiments, the internucleotide linkage can have the structure of Formula I, as described herein. In some embodiments, the internucleotide linkage does not contain phosphorus, but is a moiety that serves to link two natural or unnatural sugars.

[0050] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions that have improved properties and / or activity when compared to a reference condition, such as the absence of the composition or the presence of a reference composition (e.g., a stereoirregular composition of oligonucleotides having the same base sequence and chemical modifications).

[0051] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions that more strongly induce reduction in expression, activity, and / or levels of a gene or its gene product, single-stranded RNA interference, and / or RNase H-mediated knockdown when compared to a reference condition, such as the absence of the composition or the presence of a reference composition (e.g., a stereoirregular composition of oligonucleotides having the same base sequence and chemical modifications).

[0052] In some embodiments, the oligonucleotide composition comprising a plurality of oligonucleotides is stereoirregular in that the plurality of oligonucleotides do not share a common stereochemistry at any chiral internucleotide linkage. In some embodiments, the oligonucleotide composition comprising a plurality of oligonucleotides is chiral controlled in that the plurality of oligonucleotides share a common stereochemistry at one or more chiral internucleotide linkages. In some embodiments, the oligonucleotide composition comprising a chiral controlled first plurality of oligonucleotides has reduced susceptibility to endonucleases and exonucleases compared to the oligonucleotide composition comprising a stereoirregular first plurality of oligonucleotides.

[0053] In some embodiments, the oligonucleotide composition is capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the oligonucleotide composition is capable of inducing single-stranded RNA interference. In some embodiments, the oligonucleotide composition is capable of inducing RNase H-mediated knockdown. In some embodiments, the oligonucleotide composition is capable of inducing RNase H-mediated knockdown of a target gene RNA and RNA interference. In some embodiments, the oligonucleotide composition is capable of inducing RNase H-mediated knockdown of a first RNA target and RNA interference of a second RNA target, wherein the first RNA target and the second RNA target are the same or different.

[0054] In some embodiments, the composition comprises multimers of two or more of any of the first plurality of oligonucleotides and / or the second plurality of oligonucleotides, wherein the first plurality of oligonucleotides and the second plurality of oligonucleotides are capable of independently inducing knockdown of the same or different targets via RNA interference and / or RNase H-mediated knockdown independently.

[0055] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) consensus sequence, 2) common patterns of skeletal bonds; 3) a stereochemistry independently common to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, or at least 50 chiral internucleotide linkages ("chiral controlled internucleotide linkages"); Share The composition is chiral controlled in that the level of the first plurality of oligonucleotides in the composition is predetermined.

[0056] In some embodiments, an oligonucleotide composition comprising a plurality of oligonucleotides (e.g., a first plurality of oligonucleotides) is chiral-controlled in that the plurality of oligonucleotides independently share a common stereochemistry at one or more chiral internucleotide bonds. In some embodiments, the plurality of oligonucleotides share a common stereochemistry at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral internucleotide bonds, and each of these stereochemistry is independently Rp or Sp. In some embodiments, the plurality of oligonucleotides share a common stereochemistry at each chiral internucleotide bond. In some embodiments, a chiral internucleotide linkage is referred to as a chiral controlled internucleotide linkage when a given level of oligonucleotides in a composition share a common stereochemical configuration (independently Rp or Sp).

[0057] In some embodiments, a given level of oligonucleotides in a provided composition (e.g., the first plurality of oligonucleotides in a particular example composition) comprises 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-controlled internucleotide linkages.

[0058] In some embodiments, at least 5 internucleotide linkages are chiral controlled. In some embodiments, at least 10 internucleotide linkages are chiral controlled. In some embodiments, at least 15 internucleotide linkages are chiral controlled. In some embodiments, each chiral internucleotide linkage is chiral controlled.

[0059] In some embodiments, 1% to 100% of the chiral internucleotide linkages are chiral controlled. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the chiral internucleotide linkages are chiral controlled.

[0060] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) consensus sequence, 2) common patterns of skeletal bonds, and 3) Common patterns of skeletal chiral centers Share The composition is a substantially pure preparation of a single oligonucleotide in that a given level of oligonucleotides in the composition have a common base sequence and length, a common pattern of backbone linkages, and a common pattern of backbone chiral centers.

[0061] In some embodiments, the common pattern of backbone chiral centers comprises at least one internucleotide linkage that includes a chiral center that is chiral controlled.

[0062] In some embodiments, the predetermined level of oligonucleotides is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in the provided composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides containing or sharing a common base sequence in a provided composition.In some embodiments, all oligonucleotides in a provided composition that contain or share a common base sequence are at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in a provided composition that contain or share a common base sequence, base modification, sugar modification, and / or modified internucleotide linkage.In some embodiments, all of the oligonucleotides in a provided composition that contain or share a common base sequence, base modification, sugar modification, and / or modified internucleotide linkage are at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in a provided composition that contain or share a common base sequence, base modification pattern, sugar modification pattern, and / or modified internucleotide linkage pattern.In some embodiments, all of the oligonucleotides in a provided composition that contain or share a common base sequence, base modification pattern, sugar modification pattern, and / or modified internucleotide linkage pattern are at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all of the oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in a provided composition that share a common base sequence, common pattern of base modifications, common pattern of sugar modifications, and / or common pattern of modified internucleotide linkages.In some embodiments, all oligonucleotides in a provided composition that share a common base sequence, common pattern of base modifications, common pattern of sugar modifications, and / or common pattern of modified internucleotide linkages represent at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of all oligonucleotides in the composition.

[0063] In some embodiments, the predetermined level is between 1 and 100%. In some embodiments, the predetermined level is at least 1%. In some embodiments, the predetermined level is at least 5%. In some embodiments, the predetermined level is at least 10%. In some embodiments, the predetermined level is at least 20%. In some embodiments, the predetermined level is at least 30%. In some embodiments, the predetermined level is at least 40%. In some embodiments, the predetermined level is at least 50%. In some embodiments, the predetermined level is at least 60%. In some embodiments, the predetermined level is at least 10%. In some embodiments, the predetermined level is at least 70%. In some embodiments, the predetermined level is at least 80%. In some embodiments, the predetermined level is at least 90%. In some embodiments, the predetermined level is at least 5*(½ g ), where g is the number of chiral controlled internucleotide linkages. In some embodiments, the predetermined level is at least 10*(½ g ), where g is the number of chiral controlled internucleotide linkages. In some embodiments, the predetermined level is at least 100*(½ g) where g is the number of chiral controlled internucleotide linkages. In some embodiments, the predetermined level is at least (0.80) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.80) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.80) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.85) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.90) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.95) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.96) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.97) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.98) g where g is the number of chiral internucleotide linkages. In some embodiments, the predetermined level is at least (0.99) gwhere g is the number of chiral-controlled internucleotide linkages. In some embodiments, to determine the level of an oligonucleotide having g chiral-controlled internucleotide linkages in a composition, the product of the diastereopurities of each of the g chiral-controlled internucleotide linkages is used as a level: (diastereopurity of chiral-controlled internucleotide linkage 1) * (diastereopurity of chiral-controlled internucleotide linkage 2) * ... * (diastereopurity of chiral-controlled internucleotide linkage g), where the diastereopurity of each chiral-controlled internucleotide linkage is independently represented by the diastereopurity of a dimer comprising the same internucleotide linkage and the nucleoside adjacent to that internucleotide linkage, prepared under a method equivalent to that of the oligonucleotide (e.g., an equivalent or preferably identical oligonucleotide preparation cycle, including equivalent or preferably identical reagents and reaction conditions).

[0064] In some embodiments, the level and / or diastereopurity of an oligonucleotide can be determined by analytical methods (eg, chromatographic methods, spectroscopic methods, spectroscopic methods, or any combination thereof).

[0065] Among other things, the present disclosure includes the recognition that stereoirregular oligonucleotide preparations comprise multiple distinct chemical entities that differ from one another (e.g., in the stereochemical configuration (or stereochemistry) of individual backbone chiral centers within the oligonucleotide chain). Without control of the stereochemistry of the backbone chiral centers, stereoirregular oligonucleotide preparations result in uncontrolled compositions containing indeterminate levels of oligonucleotide stereoisomers. While such stereoisomers may have the same base sequence and / or chemical modifications, they are distinct chemical entities at least by virtue of their different backbone stereochemistry, and such stereoisomers may have different properties (e.g., nuclease susceptibility, activity, distribution, etc.), as demonstrated herein. In some embodiments, a particular stereoisomer can be defined, for example, by its base sequence, its length, its backbone bonding pattern, and its backbone chiral center pattern. In some embodiments, the present disclosure demonstrates that the improved properties and activity achieved by controlling the stereochemistry within an oligonucleotide can be comparable to or even better than those achieved by the use of chemical modifications.

[0066] In some embodiments, the provided oligonucleotide (e.g., an oligonucleotide capable of inducing a decrease in the expression and / or level of a target gene or its gene product, an oligonucleotide capable of inducing single-stranded RNA interference (e.g., a single-stranded RNAi agent, ssRNA, or ssRNAi), or an oligonucleotide capable of inducing single-stranded RNA interference and RNase H-mediated knockdown, etc.) 5'-PX0-N1-PX1-N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8-N9-PX9-N10-PX 10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22)sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz -(N26-PX26-N27-PX27) yz -(CAP) zz -3', wherein each variable is independently as described in this disclosure.

[0067] In some embodiments, PX0 is a 5'-terminal structure. In some embodiments, PX1-PX26 are each independently an internucleotide bond. In some embodiments, PX27 is an internucleotide bond or OH. In some embodiments, N1-N27 independently represent a nucleoside. In some embodiments, N1-PX1 through N27-PX27 independently represent a nucleotide. Any nucleoside can be the same or different from any adjacent nucleoside. Any nucleotide can be the same or different from any adjacent nucleotide. In some embodiments, when any of mz-yz is >1, each of N18-N27 bases can be the same or different, and / or each of N18-N27 nucleosides can be the same or different and can independently include the same or different modifications (e.g., 2'-modifications). In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10. In some embodiments, -(N18-PX18) mz If mz is >1 in -, then each N18 can be the same or different, and / or each PX18 can be the same or different. -(N19-PX19) nz If nz is >1, then each N19 may be the same or different and / or each PX19 may be the same or different. -(N20-PX20) pz If pz is >1 in -, then each N20 may be the same or different, and / or each PX20 may be the same or different. -(N21-PX21) rzIf rz is >1 in -, then each N21 may be the same or different, and / or each PX21 may be the same or different. -(N22-PX22) sz If sz is >1, then each N22 may be the same or different and / or each PX22 may be the same or different. -(N23-PX23) tz If tz is >1, then each N23 may be the same or different and / or each PX23 may be the same or different. -(N24-PX24) vz If vz is >1, then each N24 may be the same or different, and / or each PX24 may be the same or different. -(N25-PX25) wz If wz is >1, then each N25 may be the same or different and / or each PX26 may be the same or different. -(N26-PX26-N27-PX27) yz - if yz > 1, then N26 may each be the same or different, N27 may each be the same or different, N26 and N27 may each be the same or different, and / or PX26 may each be the same or different, and / or PX27 may each be the same or different, and / or PX26 and PX27 may each be the same or different, etc.

[0068] In some embodiments, N or PX may each independently and optionally further comprise one or more additional chemical moieties (e.g., a targeting moiety, a carbohydrate moiety, a GalNAc moiety, a lipid moiety, etc.).

[0069] In some embodiments, PX1 to PX27 each independently represent an internucleotide bond, and PX1 to PX27 may be the same or different. In some embodiments, PX1 to PX27 each independently represent an internucleotide bond, and the internucleotide bond is a phosphorodiester, phosphorothioate, phosphorothioate in the Sp configuration, phosphorothioate in the Rp configuration, an internucleotide bond, an internucleotide bond in the Sp configuration, or an internucleotide bond in the Rp configuration, and PX1 to PX27 may be the same or different.

[0070] In some embodiments, the 3' terminal region is -(N26-PX26-N27-PX27) yz -(CAP) zz (yz=1 and zz=0, or yz=0 and zz=1, or yz=1 and zz=1), -(N26-PX26-N27-PX27) yz (yz=1), -(CAP) zz (zz=1), or -(N26-PX26-N27-PX27) yz -(CAP) zz (yz=1 and zz=1).

[0071] In some embodiments, PX27 represents an internucleotide bond or -OH. In some embodiments, zz=0 and yz=1, and PX27 is -OH. In some embodiments, zz=1 and yz=1, and PX27 is an internucleotide bond.

[0072] In some embodiments, PX27 represents an internucleotide linkage, and the internucleotide linkage is a phosphorodiester, a phosphorothioate, a phosphorothioate in the Sp configuration, a phosphorothioate in the Rp configuration, an internucleotide linkage, an internucleotide linkage in the Sp configuration, or an internucleotide linkage in the Rp configuration. In some embodiments, PX27 is -OH.

[0073] In some embodiments, when zz=1 (e.g., when CAP is present), PX27 represents an internucleotide linkage that is a phosphorodiester, a phosphorothioate, a phosphorothioate in the Sp configuration, a phosphorothioate in the Rp configuration, an internucleotide linkage, an internucleotide linkage in the Sp configuration, or an internucleotide linkage in the Rp configuration.

[0074] In some embodiments, the oligonucleotide comprises, in 5' to 3' order, a 5' terminal region, a seed region, a post-seed region, and a 3' terminal region, and optionally further comprises an additional chemical moiety.

[0075] In some embodiments, the 5' terminal region is the entire portion of the oligonucleotide located 5' to the seed region. In some embodiments, the 3' terminal region is the entire portion of the oligonucleotide located 3' to the post-seed region.

[0076] In some embodiments, the 5' end region is designated by any of PX0-, PX0-N1-, PX0-N1-PX1-, PX0-N1-PX1-N2-, PX0-N1-PX1-N2-PX2-, PX0-N1-PX1-N2-PX2-N3-, or PX0-N1-PX1-N2-PX2-N3-PX3-.

[0077] In some embodiments, the 5' terminal region is designated by either PX0-, PX0-N1-, or PX0-N1-PX1-.

[0078] In some embodiments, the 5' end structure is designated by either PX0-, PX0-N1-, or PX0-N1-PX1-.

[0079] In some embodiments, the 5' end structure is designated by PX0-.

[0080] In some embodiments, the 5' terminal structure is a 5' terminal group.

[0081] In some embodiments, the 5' terminal structure comprises a 5' terminal group.

[0082] In some embodiments, -N1-PX1- represents the 5' nucleotide moiety. In some embodiments, -N1- represents the 5' nucleoside.

[0083] In some embodiments, the 5' terminal nucleoside is -N1-.

[0084] In some embodiments, the 5' terminal nucleotide is -N1-PX1-.

[0085] In some embodiments, provided oligonucleotides may comprise a 5' terminal region, 5' terminal structure, 5' terminal group, 5' terminal nucleoside, or 5' terminal nucleotide described herein or known in the art.

[0086] In some embodiments, -PX0-, -PX0-N1-, and -PX0-N1-PX1- are represented by the structures described herein of a 5' terminal structure, a 5' terminal region, a 5' nucleotide, a modified 5' nucleotide, a 5' nucleotide analog, or a 5' nucleoside, a modified 5' nucleoside, or a 5' nucleoside analog.

[0087] In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents a seed region. In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents a seed region. In some embodiments, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents a seed region. In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents a seed region. In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents a seed region. In some embodiments, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents a seed region. In some embodiments, -N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents a seed region. In some embodiments, -N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents a seed region.

[0088] In some embodiments, -N7-PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region (eg, the region between the seed region and the 3'-end region).

[0089] In some embodiments, -N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz-(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0090] In some embodiments, -N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0091] In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- indicates a seed region, and -N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0092] In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6- indicates a seed region, and -N7-PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0093] In some embodiments, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- indicates a seed region, and -N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0094] In some embodiments, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- indicates a seed region, and -N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20)pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0095] In some embodiments, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- indicates a seed region, and -N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0096] In some embodiments, -N3-PX3-N4-PX4-N5-PX5-N6-PX6- indicates a seed region, and -N7-PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0097] In some embodiments, -N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- indicates a seed region, and -N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region.

[0098] In some embodiments, 5'-PX0-N1-PX1- indicates a 5' end region, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7- indicates a seed region, and -PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18). mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz - indicates the post-seed region, -(N26-PX26-N27-PX27) yz -(CAP) zz -3' indicates the 3' terminal region.

[0099] In some embodiments, 5'-PX0-N1-PX1- indicates a 5' end region, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6- indicates a seed region, and -N7-PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18). mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz indicates the post-seed region, -(N26-PX26-N27-PX27) yz -(CAP) zz -3' indicates the 3' terminal region.

[0100] In some embodiments, 5'-PX0-N1-PX1-N2-PX2- indicates a 5' end region, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- indicates a seed region, and -N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz indicates the post-seed region, -(N26-PX26-N27-PX27) yz -(CAP) zz -3' indicates the 3' terminal region.

[0101] In some embodiments, 5'-PX0-N1-PX1-N2-PX2- indicates a 5' end region, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- indicates a seed region, and -N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz indicates the post-seed region, -(N26-PX26-N27-PX27) yz -(CAP) zz -3' indicates the 3' terminal region.

[0102] In some embodiments, 5'-PX0-N1-PX1- indicates the 5' end region, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8- indicates the seed region, and -PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz indicates the post-seed region, -(N26-PX26-N27-PX27) yz -(CAP) zz -3' indicates the 3' terminal region.

[0103] In some embodiments, 5'-PX0-N1-PX1-N2-PX2- indicates a 5' end region, -N3-PX3-N4-PX4-N5-PX5-N6-PX6- indicates a seed region, and -N7-PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18). mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz indicates the post-seed region, -(N26-PX26-N27-PX27) yz -(CAP) zz -3' indicates the 3' terminal region.

[0104] In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz are each independently 0-10.

[0105] In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 49 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 45 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 40 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 35 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 30 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 25 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 23 nucleotides or less. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0-10, and the total length of the oligonucleotide is about 21 nucleotides or less.

[0106] In some embodiments, -(N26-PX26-N27-PX27) yz - indicates the 3' terminal dinucleotide, yz is 1. When yz=0, the 3'-terminal dinucleotide is absent. When yz=1, the 3'-terminal dinucleotide is present.

[0107] In some embodiments, -(CAP) zz - indicates an optional 3' end cap, zz is either 0 or 1. zzWhen zz=0, CAP does not exist. When zz=1, CAP exists.

[0108] In some embodiments, if yz=1, then zz =0. In some embodiments, yz If =0, zz = 1. In some embodiments, yz = 1 and zz = 1, indicating that the molecule contains both a 3' terminal dinucleotide and a CAP.

[0109] In some embodiments, the 5'-end structure, 5'-end region, 5'-nucleotide portion, seed region, post-seed region, 3'-end, dinucleotide, and / or 3'-end cap independently have any structure described herein or known in the art. In some embodiments, any of the 5'-end structures described herein or known in the art, and / or any of the 5'-nucleotide portion structures described herein or known in the art, and / or any of the seed region structures described herein or known in the art, and / or any of the post-seed region structures described herein or known in the art, and / or any of the 3'-end dinucleotide structures described herein or known in the art, and / or any of the 3'-end cap structures described herein or known in the art can be combined.

[0110] In some embodiments, provided oligonucleotides comprise one or more blocks. In some embodiments, provided oligonucleotides comprise one or more blocks, where a block comprises one or more consecutive nucleosides, nucleotides, sugars, bases, and / or internucleotide linkages. In some embodiments, a block comprises the entire seed region or a portion thereof. In some embodiments, a block comprises the entire post-seed region or a portion thereof.

[0111] In some embodiments, the provided oligonucleotide is a blockmer.

[0112] In some embodiments, the provided oligonucleotide is an altmer.

[0113] In some embodiments, the provided oligonucleotides are altmers, comprising alternating blocks. In some embodiments, blockmirs or altmers can be defined by chemical modifications (including presence or absence), such as base modifications, sugar modifications, internucleotide linkage modifications, stereochemistry, etc., or patterns thereof.

[0114] In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference. In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC (including, but not limited to, RNase H-mediated knockdown of gene expression or steric hindrance). In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. In some embodiments, provided oligonucleotides comprise one or more blocks comprising two or more different internucleotide linkages. In some embodiments, provided oligonucleotides comprise one or more blocks comprising a combined total of two or more modified internucleotide linkages and natural phosphate linkages. In some embodiments, provided oligonucleotides comprise one or more blocks comprising two or more different modified internucleotide linkages. In some embodiments, provided oligonucleotides comprise blocks comprising two or more different internucleotide linkages in alternating order. In some embodiments, provided oligonucleotides comprise alternating blocks comprising two or more modified internucleotide linkages and natural phosphate linkages. In some embodiments, provided oligonucleotides comprise alternating blocks comprising two or more different modified internucleotide linkages. In some embodiments, the blocks comprising modified internucleotide linkages have a pattern of backbone chiral centers as described herein. In some embodiments, each block comprising modified internucleotide linkages has the same pattern of backbone chiral centers. In some embodiments, the blocks comprising modified internucleotide linkages have different patterns of backbone chiral centers. In some embodiments, the blocks comprising modified internucleotide linkages have different lengths and / or modifications.In some embodiments, the blocks comprising modified internucleotide linkages have the same length and / or modification. In some embodiments, the blocks comprising modified internucleotide linkages have the same length. In some embodiments, the blocks comprising modified internucleotide linkages have the same internucleotide linkages.

[0115] In some embodiments, the provided oligonucleotides are capable of inducing single-stranded RNA interference and comprise a first block (seed region block) in the seed region and a second block (post-seed region block) in the post-seed region, each of which independently comprises one or more modified internucleotide linkages. In some embodiments, the seed region block and the post-seed region block each independently comprise two, three, four, five, six, seven, or eight or more modified internucleotide linkages. In some embodiments, the seed region block comprises four or more modified internucleotide linkages. In some embodiments, the seed region block comprises five or more modified internucleotide linkages. In some embodiments, the seed region block comprises six or more modified internucleotide linkages. In some embodiments, the seed region block comprises seven modified internucleotide linkages. In some embodiments, the post-seed region block comprises four or more modified internucleotide linkages. In some embodiments, the post-seed region block comprises five or more modified internucleotide linkages. In some embodiments, the post-seed region block comprises six or more modified internucleotide linkages. In some embodiments, the post-seed region block comprises seven or more modified internucleotide linkages. In some embodiments, the seed region block and the post-seed region block each independently comprise at least four modified internucleotide linkages. In some embodiments, the seed region block and the post-seed region block each independently comprise at least five modified internucleotide linkages. In some embodiments, the seed region block and the post-seed region block each independently comprise at least six modified internucleotide linkages. In some embodiments, the seed region block and the post-seed region block each independently comprise at least seven modified internucleotide linkages. In some embodiments, the modified internucleotide linkages within a block are contiguous. In some embodiments, the linkages of the seed region blocks are each independently phosphorothioate linkages. In some embodiments, the linkages of the seed region blocks are each independently chiral controlled. In some embodiments, the linkages of the seed region blocks are each Sp.In some embodiments, each of the linkages in the post-seed region blocks is independently a modified internucleotide linkage. In some embodiments, each of the linkages in the post-seed region blocks is independently a phosphorothioate linkage. In some embodiments, each of the linkages in the post-seed region blocks is independently chiral controlled. In some embodiments, each of the linkages in the post-seed region blocks is Sp.

[0116] In some embodiments, the provided oligonucleotides comprise one or more sugar modifications. In some embodiments, the sugar modifications are at the 2' position. In some embodiments, the sugar modifications are 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.

[0117] In some embodiments, the provided oligonucleotides are capable of inducing single-stranded RNA interference and comprise a first block (seed region block) in the seed region and a second block (post-seed region block) in the post-seed region, each of which independently comprises one or more 2'-Fs. In some embodiments, the seed region block and the post-seed region block each independently comprise two, three, four, five, six, seven, or eight or more 2'-Fs. In some embodiments, the seed region block and the post-seed region block each independently comprise two, three, four, five, six, seven, or eight or more consecutive 2'-Fs. In some embodiments, the seed region block comprises four or more 2'-Fs. In some embodiments, the seed region block comprises five or more 2'-Fs. In some embodiments, the seed region block comprises six or more 2'-Fs. In some embodiments, the seed region block comprises seven 2'-Fs. In some embodiments, the post-seed region block comprises four or more 2'-Fs. In some embodiments, the post-seed region block comprises five or more 2'-Fs. In some embodiments, the post-seed region block comprises six or more 2'-Fs. In some embodiments, the post-seed region block comprises seven or more 2'-Fs. In some embodiments, the seed region block and the post-seed region block each independently comprise at least four 2'-Fs. In some embodiments, the seed region block and the post-seed region block each independently comprise at least five 2'-Fs. In some embodiments, the seed region block and the post-seed region block each independently comprise at least six 2'-Fs. In some embodiments, the seed region block and the post-seed region block each independently comprise at least seven 2'-Fs. In some embodiments, the 2'-Fs within a block are contiguous.

[0118] In some embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more 2'-F. In some embodiments, the oligonucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more consecutive sugar moieties that comprise 2'-F.

[0119] In some embodiments, the oligonucleotide contains only two 2'-Fs. In some embodiments, the oligonucleotide contains only two 2'-Fs, and these two nucleotides are at positions 2 and 14. Non-limiting examples of such oligonucleotides include oligonucleotides of Format 70 (FIG. 1F), as well as oligonucleotides of WV-7540 and WV-7543.

[0120] In some embodiments, the oligonucleotide contains only two 2'-Fs, these two nucleotides are at the second and fourteenth positions, and the first nucleotide (the 5' terminal nucleotide or -N1-PX1-) is 2'-deoxy. Non-limiting examples of such oligonucleotides include Format 70 (Figure 1F), as well as WV-7540 and WV-7543.

[0121] In some embodiments, the oligonucleotide contains only two 2'-Fs, these two nucleotides are at the second and fourteenth positions, and the first nucleotide (the 5' terminal nucleotide or -N1-PX1-) is a 2'-deoxy T. Non-limiting examples of such oligonucleotides include Format 70 (Figure 1F), as well as WV-7540 and WV-7543.

[0122] In some embodiments, the oligonucleotide contains only two 2'-Fs, these two nucleotides being at the second and fourteenth positions, the first nucleotide (the 5' terminal nucleotide or -N1-PX1-) being 2'-deoxy, and the 5' terminal structure (PX0) being -OH. Non-limiting examples of such oligonucleotides include oligonucleotides of Format 70 (FIG. 1F), as well as oligonucleotides of WV-7540 and WV-7543.

[0123] In some embodiments, the oligonucleotide contains only two 2'-Fs, these two nucleotides are located at the second and fourteenth positions, the first nucleotide (the 5' terminal nucleotide or -N1-PX1-) is a 2'-deoxy T, and the 5' terminal structure (PX0) is -OH. Non-limiting examples of such oligonucleotides include Format 70 (Figure 1F), as well as WV-7540 and WV-7543.

[0124] In some embodiments herein, when referring to an oligonucleotide, "first" (e.g., the first nucleotide) refers to the 5' end of the oligonucleotide, and "last" or "terminal" (e.g., the last or terminal nucleotide) refers to the 3' end.

[0125] In some embodiments, provided oligonucleotides contain alternating sugars with a particular modification and sugars with no modification or different modifications, hi some embodiments, sugars with a particular modification are found in one or more blocks.

[0126] In some embodiments, provided oligonucleotides comprise one or more blocks that alternate between sugars with a particular 2'-modification and sugars that, independently, have no modification or a different modification. In some embodiments, provided oligonucleotides comprise one or more blocks that alternate between sugars with a 2'-F modification and sugars that, independently, have no modification or a different modification. In some embodiments, provided oligonucleotides comprise one or more blocks that alternate between sugars with a 2'-OMe modification and sugars that, independently, have no modification or a different modification. In some embodiments, provided oligonucleotides comprise one or more blocks that alternate between sugars with a 2'-OMe modification and sugars that, independently, have no modification or a different modification.

[0127] In some embodiments, the sugar blocks are ff, fffm, fffmm, fffmmm, fffmmmm, fffmmmmm, fffmmmmmm, fffmmmmmmf, fffmmmmmmff, fffmmmmmmffm, fffmmmmmmffmm, fffmmmmmmffmmf, fffmmmmmmffmmfm, fffmmmmmmffmmfmf, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfmf, fffmmmmmmffmmfmfmfm, fffmmmmmm mffmmfmfmfmm, ffmmffmm, ffmmmmmmffmmfmfmfmmm, fmfmfmfmfmfmfm, fmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmf mfm, fmfmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf mfmm, fmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmm, fmfm fmfmfmfmmm, fmmffmm, fmmmmmmffmmfmfmfmmm, mff, mffm, mffmf, mffmff, mffmffm, mffmmffmm, mfmfm, mfmfmfmfmfffmfmfmfmmm, mfmfmfmfmfmfmfm, mf mfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmfmfmmm, mfmfmfmfmfmfmfmfmm, mf mfmfmfmfmfmfmm, mfmfmfmfmfmfmm, mfmfmfmfmfmfmmm, mfmfmfmfmfmmm, mfm fmfmfmfmmmfm, mfmfmfmfmmmm, mfmfmfmfmmm, mfmfmfmfmmmfmfm, mfmfmfmfmmmfmmm, mfmfmfmmmmmmfm, mfmfmfmmm, mfmfmfmmmfmfmfm, mfmfmfmmmf mfmmm, mfmfmfmmmfmmmfm, mfmfmfmmmmmfmfm, mfmfmmm, mfmfmmmfmfmfmfm, mfmfmmmfmfmfmmm, mfmfmmmfmfmmmfm, mfmfmmmfmmmfmfm, mfmfmmmmmfmfmfm,mfmmm, mfmmmfmfmfmfmfm, mfmmmfmfmfmfmmm, mfmmmfmfmfmmmfm, mfmmmfmfmmmfmfm, mfm mmfmmmfmfmfm, mfmmmfmmmfmfmfm, mfmmmmmfmfmfmfm, mmffm, mmffmm, mmffmm, mmffmmf, m mffmmff, mmffmmffm, mmffmmffmm, mmffmmfmfmfmmm, mmm, mmmffmmfmfmfmmm, mmmfmfmfmfmfmfm, mmmfmfmfmfmfmmm, mmmfmfmfmfmmmfm, mmmfmfmfmmmfmfm, mmmfmfmmmfmfmfm, mmm fmmmfmfmfmfm, mmmmffmmfmfmfmmm, mmm, mmmm, mmmmm, mmmmmffmmfmfmfmmm, mmmmmmfmfmfmfmfm, mmmmmm, mmmmmmffmmfmfmfmfm, mmmmmm, mmmmmmffmmfmfmfmm, mfmf, mfmf, mfmfmf, fmfmfm, fmfmfmf, fmfmfmf, dfdf, dfdfdf, dfdfdfdf, fdfdfdfd, fdfdfmfmf, dfmfmf, mfdfmf, or dfmfdf, wherein m indicates 2'-OMe, f indicates 2'-F, and d indicates no substitution at the 2' position. In some embodiments, the seed region and / or the post-seed region may comprise blocks of sugar modifications.

[0128] In some embodiments, the blocks are stereochemical blocks. In some embodiments, the blocks are Rp blocks, in that each internucleotide bond of the block is Rp. In some embodiments, the seed region block is an Rp block. In some embodiments, the post-seed region block is an Rp block. In some embodiments, the blocks are Sp blocks, in that each internucleotide bond of the block is Sp. In some embodiments, the seed region block is an Sp block. In some embodiments, the post-seed region block is an Sp block. In some embodiments, the provided oligonucleotides include both an Rp block and an Sp block. In some embodiments, the provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, the provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, the provided oligonucleotides include one or more PO blocks, and each internucleotide bond of the blocks is a natural phosphate linkage.

[0129] In some embodiments, the seed region block is an Sp block, and each sugar moiety comprises a 2'-F modification. In some embodiments, the seed region block is an Sp block, and 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 an Sp block, and each internucleotide linkage is a phosphorothioate linkage, and each sugar moiety comprises a 2'-F modification. In some embodiments, the seed region block comprises four or more nucleoside units. In some embodiments, a nucleoside unit is a single nucleoside. In some embodiments, the seed region block comprises five or more nucleoside units. In some embodiments, the seed region block comprises six or more nucleoside units. In some embodiments, the seed region block comprises seven or more nucleoside units. In some embodiments, the post-seed region block is an Sp block, and each sugar moiety comprises a 2'-F modification. In some embodiments, the post-seed region block is an 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 an 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 four or more nucleoside units. In some embodiments, the post-seed region block comprises five or more nucleoside units. In some embodiments, the post-seed region block comprises six or more nucleoside units. In some embodiments, the post-seed region block comprises seven or more nucleoside units. In some embodiments, the seed region and / or the post-seed region may comprise blocks. In some embodiments, the seed region and / or the post-seed region comprise stereochemical blocks.

[0130] In some embodiments, in a region, block, or oligonucleotide, a certain type of nucleoside is followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, an A is followed by an Sp internucleotide linkage. In some embodiments, an A is followed by an Rp internucleotide linkage. In some embodiments, an A is followed by a natural phosphate linkage (PO). In some embodiments, a U is followed by an Sp internucleotide linkage. In some embodiments, a U is followed by an Rp internucleotide linkage. In some embodiments, a U is followed by a natural phosphate linkage (PO). In some embodiments, a C is followed by an Sp internucleotide linkage. In some embodiments, a C is followed by an Rp internucleotide linkage. In some embodiments, a C is followed by a natural phosphate linkage (PO). In some embodiments, a G is followed by an Sp internucleotide linkage. In some embodiments, a G is followed by an Rp internucleotide linkage. In some embodiments, a G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by an Sp internucleotide linkage. In some embodiments, C and U are followed by an Rp internucleotide linkage. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by an Sp internucleotide linkage. In some embodiments, A and G are followed by an Rp internucleotide linkage. In some embodiments, A and G are followed by a natural phosphate linkage (PO).

[0131] In some embodiments, provided oligonucleotides contain alternating blocks, the blocks comprising modified and unmodified sugar moieties. In some embodiments, the modified sugar moieties comprise 2'-F modifications. In some embodiments, provided oligonucleotides contain alternating 2'-OMe modified and unmodified sugar moieties.

[0132] In some embodiments, provided oligonucleotides contain one or more 2'-F modified sugar moieties, and the 3' internucleotide linkage of the sugar moiety is a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is phosphorothioate. In some embodiments, the modified internucleotide linkage is chiral controlled and Rp. In some embodiments, the modified internucleotide linkage is chiral controlled and Sp. In some embodiments, provided oligonucleotides contain one or more 2'-OR 1 It contains a modified sugar moiety, the 3' internucleotide linkage of which is a natural phosphate linkage.

[0133] In some embodiments, provided oligonucleotides have a pattern of backbone chiral centers that is or includes (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m (unless otherwise specified, modification patterns and stereochemical patterns are written in a 5' to 3' direction as commonly used in the art). In some embodiments, provided patterns of backbone chiral centers include (Sp)m(Rp)n or are (Sp)m(Rp)n. In some embodiments, provided patterns of backbone chiral centers include (Rp)n(Sp)m or are (Rp)n(Sp)m. In some embodiments, provided patterns of backbone chiral centers include (Np)t(Rp)n(Sp)m or are (Np)t(Rp)n(Sp)m. In some embodiments, a provided pattern of backbone chiral centers comprises or is (Np)tRp(Sp)m. In some embodiments, a provided pattern of backbone chiral centers comprises or is (Sp)tRp(Sp)m. In some embodiments, a provided pattern of backbone chiral centers comprises repeating units of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. In some embodiments, the repeating unit is (Sp)m(Rp)n. In some embodiments, the repeating unit is SpRp. In some embodiments, the repeating unit is SpSpRp. In some embodiments, the repeating unit is SpRpRp. In some embodiments, the repeating unit is RpRpSp. In some embodiments, the repeating unit is (Rp)n(Sp)m. In some embodiments, the repeating unit is (Np)t(Rp)n(Sp)m. In some embodiments, the repeating unit is (Sp)t(Rp)n(Sp)m.

[0134] In some embodiments, t, n, and m are each independently 1 to 20. In some embodiments, n is 1. In some embodiments, m is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15. In some embodiments, t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m and t are each independently at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15. In some embodiments, m and t are each independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, at least one of m and t is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15. In some embodiments, m is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15. In some embodiments, t is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15.

[0135] In some embodiments, the region comprises backbone chiral centers in a pattern or repeating pattern of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m (the region comprises first and second internucleotide linkages having a pattern or repeating pattern of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, respectively). The structure begins with the internucleotide bond of and ends with the last internucleotide bond. Depending on whether or not there is a repeat, they are called "(Sp)m(Rp)n (repeated) region", "(Rp)n(Sp)m (repeated) region", "(Np)t(Rp)n(Sp)m (repeated) region", or "(Sp)t(Rp)n(Sp)m (repeated) region". For example, the (Sp)t(Rp)n(Sp)m region (WV-2555:mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA *In SmU, ((Sp)7(Rp)1(Sp)3)) does not contain a 2'-OR sugar modification. In some embodiments, each sugar moiety in the region is -CH2- at the 2' position. In some embodiments, each sugar moiety in the region is an unmodified, naturally occurring 2'-deoxyribose moiety in DNA. In some embodiments, a region containing backbone chiral centers in a pattern or repeating pattern that includes (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m is adjacent to a 5'-terminal region that structurally terminates with a nucleoside moiety (the nucleoside The moiety is linked at its 3' end to the first internucleotide linkage of the region, and the region comprises (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, or a pattern or repeating pattern of backbone chiral centers that is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. For example, WV-2555:mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA *In some embodiments, the region containing backbone chiral centers of a pattern or repeating pattern that includes (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, is adjacent to the 3'-terminal region, which structurally begins with a nucleoside moiety (the nucleoside The moiety is linked at its 5' end to the last internucleotide bond of the region, and the region comprises (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, or a pattern or repeating pattern of backbone chiral centers that is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m. For example, WV-2555:mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA *In some embodiments, a region containing backbone chiral centers of a pattern or repeating pattern that includes (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m, is adjacent to the 5'-terminal region and the 3'-terminal region. In some embodiments, the adjacent 5'-terminal region and / or the adjacent 3'-terminal region comprises a modified internucleotide linkage. In some embodiments, the adjacent 5'-terminal region and / or the adjacent 3'-terminal region comprises a modified internucleotide linkage comprising Sp-bound phosphorus. In some embodiments, the adjacent 5'-terminal region and / or the adjacent 3'-terminal region comprises an Sp phosphorothioate linkage. In some embodiments, the flanking 5'-terminal region and / or the flanking 3'-terminal region comprises one or more natural phosphate linkages. In some embodiments, the flanking 5'-terminal region and / or the flanking 3'-terminal region comprises one or more consecutive natural phosphate linkages. In some embodiments, the flanking 5'-end comprises a single modified internucleotide linkage that is the 5'-terminal internucleotide linkage and one or more consecutive natural phosphate linkages (e.g., WV-2555:mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * In some embodiments, the adjacent 3' end comprises only one modified internucleotide linkage that is the 3' terminal internucleotide linkage, and one or more consecutive natural phosphate linkages (e.g., in WV-2555:mA * SmGmCmUmU * SC * ST * ST * SG * ST * SC* SC * RA * SG * SC * SmUmUmUmA * In some embodiments, the proximal 5'-terminal region and / or the proximal 3'-terminal region comprise 2'-modified sugar units. In some embodiments, each sugar unit in the 5'-terminal region and / or the 3'-terminal region is independently modified ... is independently modified. In some embodiments, each sugar unit in the 5'-terminal region is independently modified. In some embodiments, each sugar * SmGmCmUmU * SC * ST * ST * SG * ST * SC * SC * RA * SG * SC * SmUmUmUmA * In some embodiments, the sugar units in the 5'-terminal region and / or the 3'-terminal region each contain the same 2'-modification. In some embodiments, the 2'-modification is 2'-OR, where R is an optionally substituted C 1-6 In some embodiments, the 2'-modification is aliphatic. In some embodiments, the 2'-modification is 2'-OMe. In some embodiments, the 2'-modification is 2'-MOE. In some embodiments, the 2'-modification is an LNA modification (including some type of C2-C4 bridge).

[0136] In some embodiments, a provided pattern of backbone chiral centers comprises (Rp / Sp)-(all Rp or all Sp)-(Rp / Sp). In some embodiments, a provided pattern of backbone chiral centers comprises (Rp)-(all Sp)-(Rp). In some embodiments, a provided pattern of backbone chiral centers comprises (Sp)-(all Sp)-(Sp). In some embodiments, a provided pattern of backbone chiral centers comprises (Sp)-(all Rp)-(Sp). In some embodiments, a provided pattern of backbone chiral centers comprises (Rp / Sp)-(repeating (Sp)m(Rp)n)-(Rp / Sp). In some embodiments, a provided pattern of backbone chiral centers comprises (Rp / Sp)-(repeating SpSpRp)-(Rp / Sp).

[0137] In some embodiments, the pattern of backbone chiral centers provided is (Rp / Sp)-(all Rp or all Sp)-(Rp / Sp). In some embodiments, the pattern of backbone chiral centers provided is (Sp)-(all Sp)-(Sp). In some embodiments, each chiral internucleotide linkage is Sp. In some embodiments, the pattern of backbone chiral centers provided is (Rp)-(all Sp)-(Rp). In some embodiments, the pattern of backbone chiral centers provided is (Sp)-(all Rp)-(Sp). In some embodiments, the pattern of backbone chiral centers provided is (Rp / Sp)-(repeating (Sp)m(Rp)n)-(Rp / Sp). In some embodiments, the pattern of backbone chiral centers provided is (Rp / Sp)-(repeating SpSpRp)-(Rp / Sp).

[0138] In some embodiments, the seed region and / or the post-seed region, or any portion thereof, may include a pattern of backbone chiral centers.

[0139] In some embodiments, provided oligonucleotides contain alternating internucleotide linkages of a particular type with different internucleotide linkage types, including, but not limited to, phosphodiester, phosphorothioate, stereoirregular phosphorothioate, stereocontrolled phosphorothioate (Rp or Sp), phosphodithioate, or any other type of internucleotide linkage described herein or known in the art.

[0140] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) have a common base sequence; 2) contain one or more modified sugar moieties and modified internucleotide linkages.

[0141] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single-stranded RNA interference, the first plurality of oligonucleotides comprising: 1) have a common base sequence complementary to the target sequence in the transcription product; 2) contain one or more modified sugar moieties and modified internucleotide linkages.

[0142] In some embodiments, the reference condition is the absence of the composition. In some embodiments, the reference condition is the presence of the reference composition. Examples of reference compositions comprising a reference plurality of oligonucleotides are described in detail in the present disclosure. In some embodiments, the reference plurality of oligonucleotides has different structural elements (e.g., chemical modifications, stereochemistry) compared to the first plurality of oligonucleotides in the provided composition. In some embodiments, the provided oligonucleotide compositions comprising the first plurality of oligonucleotides are chiral-controlled in that the first plurality of oligonucleotides comprises one or more chiral-controlled internucleotide linkages. In some embodiments, the provided oligonucleotide compositions comprising the first plurality of oligonucleotides are chiral-controlled in that the first plurality of oligonucleotides comprises 1 to 20 chiral-controlled internucleotide linkages. In some embodiments, the first plurality of oligonucleotides comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 chiral-controlled internucleotide linkages. In some embodiments, the reference composition is a stereoirregular preparation of oligonucleotides with the same chemical modification. In some embodiments, the reference composition is a mixture of stereoisomers, while the provided composition is a single-stranded RNAi agent of one stereoisomer. In some embodiments, the reference oligonucleotides have the same base sequence as the first plurality of oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same chemical modification as the first plurality of oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same sugar modification as the first plurality of oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same base modification as the first plurality of oligonucleotides in the provided composition. In some embodiments, the reference oligonucleotides have the same internucleotide linkage modification as the first plurality of oligonucleotides in the provided composition.In some embodiments, the reference plurality of oligonucleotides has the same base sequence and the same chemical modifications as the first plurality of oligonucleotides in the provided compositions, hi some embodiments, the reference plurality of oligonucleotides has the same stereochemistry as the first plurality of oligonucleotides in the provided compositions, but different chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.).

[0143] In some embodiments, the disclosure provides a composition comprising an oligonucleotide, wherein the oligonucleotide is complementary or substantially complementary to a target RNA sequence and has a length of from about 15 to about 49 total nucleotides, and the oligonucleotide comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0144] In some embodiments, the disclosure provides oligonucleotide compositions comprising a single-stranded RNAi agent, wherein the single-stranded RNAi agent is complementary or substantially complementary to a target RNA sequence, has a length of from about 15 to about 49 total nucleotides, and is capable of inducing target-specific RNA interference, and the single-stranded RNAi agent comprises at least one unnatural base, sugar, and / or internucleotide linkage.

[0145] 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 total nucleotides.

[0146] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) have a common base sequence that is complementary or substantially complementary to a target sequence in the transcription product; 2) comprises one or more modified sugar moieties and modified internucleotide linkages; The oligonucleotide composition is characterized in that, when contacted with a transcript, it results in improved knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0147] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single-stranded RNA interference, the first plurality of oligonucleotides comprising: 1) have a common base sequence complementary to the target sequence in the transcription product; 2) comprises one or more modified sugar moieties and modified internucleotide linkages; The oligonucleotide composition is characterized in that when it is contacted with a transcript in an RNA interference system, it improves RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0148] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides comprising: 1) base sequence, 2) skeletal bond pattern; 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification It is a specific oligonucleotide type defined by:

[0149] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single-stranded RNA interference, the first plurality of oligonucleotides comprising: 1) base sequence, 2) skeletal bond pattern; 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification It is a specific oligonucleotide type defined by:

[0150] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides of an oligonucleotide type, the oligonucleotide type comprising: 1) base sequence, 2) skeletal bond pattern; 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification is defined by the composition is chiral-controlled in that it is enriched for a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence; The oligonucleotide composition is characterized in that, when contacted with a transcript, it results in improved knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0151] In some embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of inducing single-stranded RNA interference, the first plurality of oligonucleotides being of an oligonucleotide type, the oligonucleotide type being: 1) base sequence, 2) skeletal bond pattern; 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification is defined by the composition is chiral-controlled in that it is enriched for a particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence; The oligonucleotide composition is characterized in that when it is contacted with a transcript in an RNA interference system, it improves RNAi-mediated knockdown of the transcript compared to that observed under reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0152] In some embodiments, provided oligonucleotides have any of the formats shown in FIG. 1 or have any structural elements shown in any of the formats shown in FIG.

[0153] In some embodiments, a provided single stranded RNAi agent has any of the formats shown in FIG. 1 or has any structural element shown in any of the formats shown in FIG.

[0154] In particular, the present disclosure presents data showing that various oligonucleotides of the disclosed format have the ability to induce a decrease in the expression and / or level of a target gene or its gene product when targeted to any of several different sequences in any of several different genes. In some embodiments, the present disclosure presents data showing that various RNAi agents of the disclosed format have the ability to induce RNA interference to any of many different sequences in any of many different genes.

[0155] In some embodiments, the oligonucleotides are of Format 1. In some embodiments, the oligonucleotides are of Format 2. In some embodiments, the oligonucleotides are of Format 3. In some embodiments, the oligonucleotides are of Format 4. In some embodiments, the oligonucleotides are of Format 5. In some embodiments, the oligonucleotides are of Format 6. In some embodiments, the oligonucleotides are of Format 7. In some embodiments, the oligonucleotides are of Format 8. In some embodiments, the oligonucleotides are of Format 9. In some embodiments, the oligonucleotides are of Format 10. In some embodiments, the oligonucleotides are of Format 11. In some embodiments, the oligonucleotides are of Format 12. In some embodiments, the oligonucleotides are of Format 13. In some embodiments, the oligonucleotides are of Format 14. In some embodiments, the oligonucleotides are of Format 15. In some embodiments, the oligonucleotides are of Format 16. In some embodiments, the oligonucleotides are of Format 17. In some embodiments, the oligonucleotides are of Format 18. In some embodiments, the oligonucleotides are of Format 19. In some embodiments, the oligonucleotides are of Format 20. In some embodiments, the oligonucleotides are of Format 21. In some embodiments, the oligonucleotides are of Format 22. In some embodiments, the oligonucleotides are of Format 23. In some embodiments, the oligonucleotides are of Format 24. In some embodiments, the oligonucleotides are of Format 25. In some embodiments, the oligonucleotides are of Format 26. In some embodiments, the oligonucleotides are of Format 27. In some embodiments, the oligonucleotides are of Format 28. In some embodiments, the oligonucleotides are of Format 29. In some embodiments, the oligonucleotides are of Format 30. In some embodiments, the oligonucleotides are of Format 31.In some embodiments, the oligonucleotides are of Format 32. In some embodiments, the oligonucleotides are of Format 33. In some embodiments, the oligonucleotides are of Format 34. In some embodiments, the oligonucleotides are of Format 35. In some embodiments, the oligonucleotides are of Format 36. In some embodiments, the oligonucleotides are of Format 37. In some embodiments, the oligonucleotides are of Format 38. In some embodiments, the oligonucleotides are of Format 39. In some embodiments, the oligonucleotides are of Format 40. In some embodiments, the oligonucleotides are of Format 41. In some embodiments, the oligonucleotides are of Format 42. In some embodiments, the oligonucleotides are of Format 43. In some embodiments, the oligonucleotides are of Format 44. In some embodiments, the oligonucleotides are of Format 45. In some embodiments, the oligonucleotides are of Format 46. In some embodiments, the oligonucleotides are of Format 47. In some embodiments, the oligonucleotides are of Format 48. In some embodiments, the oligonucleotides are of Format 49. In some embodiments, the oligonucleotides are of Format 50. In some embodiments, the oligonucleotides are of Format 51. In some embodiments, the oligonucleotides are of Format 52. In some embodiments, the oligonucleotides are of Format 53. In some embodiments, the oligonucleotides are of Format 54. In some embodiments, the oligonucleotides are of Format 55. In some embodiments, the oligonucleotides are of Format 56. In some embodiments, the oligonucleotides are of Format 57. In some embodiments, the oligonucleotides are of Format 58. In some embodiments, the oligonucleotides are of Format 59. In some embodiments, the oligonucleotides are of Format 60. In some embodiments, the oligonucleotides are of Format 61. In some embodiments, the oligonucleotides are of Format 62.In some embodiments, the oligonucleotides are of Format 63. In some embodiments, the oligonucleotides are of Format 64. In some embodiments, the oligonucleotides are of Format 65. In some embodiments, the oligonucleotides are of Format 66. In some embodiments, the oligonucleotides are of Format 67. In some embodiments, the oligonucleotides are of Format 68. In some embodiments, the oligonucleotides are of Format 69. In some embodiments, the oligonucleotides are of Format 70. In some embodiments, the oligonucleotides are of Format 71. In some embodiments, the oligonucleotides are of Format 72. In some embodiments, the oligonucleotides are of Format 73. In some embodiments, the oligonucleotides are of Format 74. In some embodiments, the oligonucleotides are of Format 75. In some embodiments, the oligonucleotides are of Format 76. In some embodiments, the oligonucleotides are of Format 77. In some embodiments, the oligonucleotides are of Format 78. In some embodiments, the oligonucleotides are of Format 79. In some embodiments, the oligonucleotides are of Format 80. In some embodiments, the oligonucleotides are of Format 81. In some embodiments, the oligonucleotides are of Format 82. In some embodiments, the oligonucleotide is of Format 83. In some embodiments, the oligonucleotide is of Format 84. In some embodiments, the oligonucleotide is of Format 85. In some embodiments, the oligonucleotide is of Format 86. In some embodiments, the oligonucleotide is of Format 87. In some embodiments, the oligonucleotide is of Format 88. In some embodiments, the oligonucleotide is of Format 89. In some embodiments, the oligonucleotide is of Format 90. In some embodiments, the oligonucleotide is of Format 91. In some embodiments, the oligonucleotide is of Format 92. In some embodiments, the oligonucleotide is of Format 93.In some embodiments, the oligonucleotide is of Format 94. In some embodiments, the oligonucleotide is of Format 95. In some embodiments, the oligonucleotide is of Format 96. In some embodiments, the oligonucleotide is of Format 97. In some embodiments, the oligonucleotide is of Format 98. In some embodiments, the oligonucleotide is of Format 99. In some embodiments, the oligonucleotide is of Format 100. In some embodiments, the oligonucleotide is of Format 101. In some embodiments, the oligonucleotide is of Format 102. In some embodiments, the oligonucleotide is of Format 103. In some embodiments, the oligonucleotide is of Format 104. In some embodiments, the oligonucleotide is of Format 105. In some embodiments, the oligonucleotide is of Format 106. In some embodiments, the oligonucleotide is of Format 107. Various formats of stereocontrolled (chiral controlled) oligonucleotides are shown in Tables 71A-71C, by way of non-limiting example. In some embodiments, the oligonucleotide is of Format S1. In some embodiments, the oligonucleotide is of Format S2. In some embodiments, the oligonucleotide is of Format S3. In some embodiments, the oligonucleotide is of format S4. In some embodiments, the oligonucleotide is of format S5. In some embodiments, the oligonucleotide is of format S6. In some embodiments, the oligonucleotide is of format S7. In some embodiments, the oligonucleotide is of format S8. In some embodiments, the oligonucleotide is of format S9. In some embodiments, the oligonucleotide is of format S10. In some embodiments, the oligonucleotide is of format S11. In some embodiments, the oligonucleotide is of format S12. In some embodiments, the oligonucleotide is of format S13. In some embodiments, the oligonucleotide is of format S14.In some embodiments, the oligonucleotide is of format S15. In some embodiments, the oligonucleotide is of format S16. In some embodiments, the oligonucleotide is of format S17. In some embodiments, the oligonucleotide is of format S18. In some embodiments, the oligonucleotide is of format S19. In some embodiments, the oligonucleotide is of format S20. In some embodiments, the oligonucleotide is of format S21. In some embodiments, the oligonucleotide is of format S22. In some embodiments, the oligonucleotide is of format S23. In some embodiments, the oligonucleotide is of format S24. In some embodiments, the oligonucleotide is of format S25. In some embodiments, the oligonucleotide is of format S26. In some embodiments, the oligonucleotide is of format S27. In some embodiments, the oligonucleotide is of format S28. In some embodiments, the oligonucleotide is of format S29. In some embodiments, the oligonucleotide is of format S30. In some embodiments, the oligonucleotide is of format S31. In some embodiments, the oligonucleotide is of format S32. In some embodiments, the oligonucleotide is of format S33. In some embodiments, the oligonucleotide is of format S34. In some embodiments, the oligonucleotide is of format S35. In some embodiments, the oligonucleotide is of format S36. In some embodiments, the oligonucleotide is of format S37. In some embodiments, the oligonucleotide is of format S38. In some embodiments, the oligonucleotide is of format S39. In some embodiments, the oligonucleotide is of format S40. In some embodiments, the oligonucleotide is of format S41. In some embodiments, the oligonucleotide is of format S42. In some embodiments, the oligonucleotide is of format S43. In some embodiments, the oligonucleotide is of format S44.

[0156] In some embodiments, oligonucleotides having any of the structures described and / or shown herein are capable of inducing RNA interference. In some embodiments, oligonucleotides having any of the structures described and / or shown herein are capable of inducing RNase H-mediated knockdown. In some embodiments, oligonucleotides having any of the structures described and / or shown herein are capable of inducing RNA interference and / or RNase H-mediated knockdown. In some embodiments, the oligonucleotides comprise any structural element of any of the oligonucleotides described herein or any format described herein or shown in FIG. 1. In some embodiments, the oligonucleotides comprise any structural element of any of the oligonucleotides described herein or any format described herein or shown in FIG. 1 and are capable of inducing RNA interference. In some embodiments, the oligonucleotides comprise any structural element of any of the oligonucleotides described herein or any format described herein or shown in FIG. 1 and are capable of inducing RNase H-mediated knockdown. In some embodiments, the oligonucleotide comprises any structural element of any of the oligonucleotides described herein, or any format described herein or shown in FIG. 1, and is capable of inducing RNA interference and / or RNase H-mediated knockdown.

[0157] In some embodiments, the RNAi agent comprises any one or more of a 5'-end structure, a 5'-end region, a seed region, a post-seed region, and a 3'-end region, and 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 and the 3'-end region described herein or known in the art. In some embodiments, the 3'-end region can be any 3'-end region described herein or known in the art. In some embodiments, any optional additional chemical moiety can be any optional additional chemical moiety described herein or known in the art. Any individual 5'-end structure, 5'-end region, seed region, post-seed region, 3'-end region, and optional additional chemical moiety described herein or known in the art can be independently combined with any other 5'-end structure, 5'-end region, seed region, post-seed region, 3'-end region, and optional additional chemical moiety described herein or known in the art. In some embodiments, by way of non-limiting example, the region of the single-stranded RNAi agent is a 5'-end structure, a 5'-end region, a seed region, a post-seed region, a portion of the seed region, a portion of the post-seed region, or a 3'-end dinucleotide.

[0158] In some embodiments, the base sequence of the provided oligonucleotide consists of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the provided oligonucleotide comprises the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the provided oligonucleotide comprises a sequence comprising a sequence of 15 consecutive bases of the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the provided oligonucleotide comprises a sequence comprising a sequence of 20 consecutive bases with up to 5 mismatches of the base sequence of any oligonucleotide disclosed herein.

[0159] In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, provided oligonucleotides are capable of inducing single-stranded RNAi interference. In some embodiments, provided oligonucleotides are capable of inducing RNase H-mediated knockdown. In some embodiments, provided oligonucleotides are capable of inducing single-stranded RNA interference and RNase H-mediated knockdown. In some embodiments, the oligonucleotides comprise a sequence that targets any transcript or gene targeted by the oligonucleotides disclosed herein.

[0160] In some embodiments, provided oligonucleotides target ACVR2B or MSTN-R. In some embodiments, provided oligonucleotides target APOB. In some embodiments, provided oligonucleotides target APOC3. In some embodiments, provided oligonucleotides target FXI (factor XI). In some embodiments, provided oligonucleotides target KRT14. In some embodiments, provided oligonucleotides target MSTN. In some embodiments, provided oligonucleotides target PCSK9.

[0161] In some embodiments, provided oligonucleotides target PNPLA3.

[0162] In some embodiments, provided oligonucleotides can be used to reduce or inhibit the activity, level, and / or expression of a gene or its gene product. In some embodiments, provided oligonucleotides can be used to reduce or inhibit the activity, level, and / or expression of a gene or its gene product, where aberrant or excessive activity, level, and / or expression of a gene or its gene product, deleterious mutations in the gene or its gene product, or abnormal tissue or intercellular or subcellular distribution of the gene or its gene product is associated with, causes, and / or is associated with a disorder. In some embodiments, provided oligonucleotides can be used to treat and / or manufacture a medicament for treating a disorder that is associated with, causes, and / or is associated with aberrant or excessive activity, level, and / or expression of a gene or its gene product, or abnormal distribution of a gene or its gene product.

[0163] In some embodiments, the provided oligonucleotides can be used to treat disorders associated with a particular gene or gene product, or to manufacture a medicament for treating such disorders. In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein that are capable of targeting ACVR2B and are useful for treating ACVR2B-associated disorders and / or for the manufacture of a therapeutic for an ACVR2B-associated disorder. In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein that are capable of targeting APOB and are useful for treating APOB-associated disorders and / or for the manufacture of a therapeutic for an APOB-associated disorder.

[0164] In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein that are capable of targeting APOC3 and are useful in the treatment of APOC3-associated disorders and / or the manufacture of a treatment for an APOC3-associated disorder.

[0165] In some embodiments, the present disclosure relates to methods of using oligonucleotides disclosed herein that are capable of targeting FXI (factor XI) and are useful for treating FXI (factor XI)-related disorders and / or in the manufacture of a therapy for a FXI (factor XI)-related disorder. In some embodiments, the present disclosure relates to methods of using oligonucleotides disclosed herein that are capable of targeting KRT or KRT14 and are useful for treating KRT-related or KRT14-related disorders and / or in the manufacture of a therapy for a KRT-related or KRT14-related disorder. In some embodiments, the present disclosure relates to methods of using oligonucleotides disclosed herein that are capable of targeting myostatin (MSTN) and are useful for treating myostatin (MSTN)-related disorders and / or in the manufacture of a therapy for a myostatin (MSTN)-related disorder. In some embodiments, the present disclosure relates to methods of using oligonucleotides disclosed herein that are capable of targeting PCSK9 and are useful for treating PCSK9-related disorders and / or in the manufacture of a therapy for a PCSK9-related disorder.

[0166] In some embodiments, the present disclosure relates to methods of using the oligonucleotides disclosed herein that have the ability to target PNPLA3 and are useful in the treatment of PNPLA3-associated disorders and / or the manufacture of a treatment for a PNPLA3-associated disorder.

[0167] In some embodiments, the oligonucleotide capable of targeting a gene comprises a base sequence that is a part of the base sequence of target gene, or comprises a base sequence that is complementary or substantially complementary to a part of the base sequence of target gene.In some embodiments, the length of the part is at least 15 bases.In some embodiments, the base sequence of single-stranded RNAi agent can comprise or consist of the base sequence that has a specific number of maximum mismatches with a specific base sequence.

[0168] In some embodiments, a mismatch is a difference in base sequence or length when two sequences are maximally aligned and compared. As a non-limiting example, a difference between a base at a particular position in one sequence and a base at a corresponding position in another sequence is counted as a mismatch. Thus, for example, a particular base (e.g., A) at a position in one sequence and a different base (e.g., G, C, or U) at the corresponding position in the other sequence is counted as a mismatch. For example, a base (e.g., A) at a position in one sequence and no base at the corresponding position in the other sequence (e.g., an abasic nucleotide containing a phosphate-sugar backbone but no base) or a skipped position is still counted as a mismatch. A single-stranded nick in either sequence (or in the sense or antisense strand) may not be counted as a mismatch, e.g., if one sequence contains the sequence 5'-AG-3' and the other sequence contains the sequence 5'-AG-3' with a single-stranded nick between the A and G, it would not be counted as a mismatch. Base modifications generally are not considered mismatches, e.g., if one sequence contains a C and the other sequence contains a modified C (e.g., 5mC) at the same position, it may not be counted as a mismatch. In some embodiments, for purposes of counting mismatches, a substitution of T for U, or vice versa, is not considered a mismatch.

[0169] In some embodiments, an oligonucleotide is complementary to a target sequence (e.g., RNA such as mRNA), or completely complementary or 100% complementary, meaning that the base sequence of the oligonucleotide has no mismatches with a sequence that is completely complementary to the target sequence (e.g., base-pairs via Watson-Crick base pairing). Without wishing to be bound by any particular theory, the present disclosure specifies that for single-stranded RNAi agents, it is not necessary for the 5'-terminal nucleotide portion or the 3'-terminal dinucleotide to base-pair with the target. These may be mismatches. Furthermore, an antisense oligonucleotide or single-stranded RNAi agent may have a small number of internal mismatches but still induce a reduction in the expression and / or level of a target gene or its gene product, and / or NaseH-mediated knockdown and / or RNA interference. A first base sequence of an oligonucleotide (e.g., an antisense oligonucleotide or a single-stranded RNAi agent) is substantially complementary to a target sequence if the first base sequence has a small number of mismatches with a reference base sequence that is 100% complementary to the target sequence. In some embodiments, an oligonucleotide (e.g., an antisense oligonucleotide or a single-stranded RNAi agent) can 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), where guanine, cytosine, adenine, thymine, and uracil can be optionally and independently modified while maintaining their paired hydrogen-bonding pattern found in their unmodified form. In some embodiments, a sequence that is complementary to another sequence comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 bases.

[0170] In some embodiments, the oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common pattern of backbone linkages. In some embodiments, the common pattern of backbone linkages comprises at least 10 modified internucleotide linkages.

[0171] In some embodiments, a consensus pattern of backbone linkages comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 modified internucleotide linkages. In some embodiments, a consensus pattern of backbone linkages comprises at least 15 modified internucleotide linkages. In some embodiments, a consensus pattern of backbone linkages comprises at least 19 modified internucleotide linkages. In some embodiments, a consensus pattern of backbone linkages comprises 10 or fewer, 11 or fewer, 12 or fewer, 13 or fewer, 14 or fewer, 15 or fewer, 16 or fewer, 17 or fewer, 18 or fewer, 19 or fewer, or 20 or fewer modified internucleotide linkages. In some embodiments, a consensus pattern of backbone linkages comprises 19 or fewer modified internucleotide linkages. In some embodiments, a consensus pattern of backbone linkages comprises 15 or fewer modified internucleotide linkages. In some embodiments, a consensus pattern of backbone linkages comprises 11 to 21 modified internucleotide linkages. In some embodiments, the common pattern of backbone linkages does not include a phosphodiester. In some embodiments, the common pattern of backbone linkages includes one phosphodiester. In some embodiments, the common pattern of backbone linkages includes two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or nineteen phosphodiesters. In some embodiments, the common pattern of backbone linkages includes at least two to nineteen phosphodiesters. In some embodiments, the phosphodiesters are optionally contiguous or non-contiguous. In some embodiments, the modified internucleotide linkages are optionally contiguous or non-contiguous.

[0172] In some embodiments, the common pattern of backbone linkages comprises at least 10 phosphorothioate linkages. In some embodiments, the common pattern of backbone linkages comprises at least 11 phosphorothioate linkages. In some embodiments, the common pattern of backbone linkages comprises at least 12-19 phosphorothioate linkages. In some embodiments, the common pattern of backbone linkages comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 phosphorothioate linkages. In some embodiments, the common pattern of backbone linkages does not comprise a phosphodiester. In some embodiments, the common pattern of backbone linkages comprises 1-6 phosphodiester and 13-19 phosphorothioate linkages. In some embodiments, the phosphodiesters are optionally contiguous or non-contiguous. In some embodiments, the phosphorothioate linkages are optionally contiguous or non-contiguous.

[0173] In some embodiments, the oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers includes at least one internucleotide linkage in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers includes at least one internucleotide linkage that is a phosphorothioate in the Sp configuration.

[0174] In some embodiments, the common pattern of backbone chiral centers includes at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers includes at least 6-19 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers includes at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 internucleotide linkages in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers includes 8 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers includes 1-7 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers includes 8 or fewer non-chiral internucleotide linkages (for example, by way of non-limiting example, phosphodiester). In some embodiments, the common pattern of backbone chiral centers includes no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, or no more than 7 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers includes at least 10 internucleotide linkages in the Sp configuration, and the common pattern includes no more than 8 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers includes at least 11 internucleotide linkages in the Sp configuration, and the common pattern includes no more than 7 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers includes at least 12 internucleotide linkages in the Sp configuration, and the common pattern includes no more than 6 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers includes at least 13 internucleotide linkages in the Sp configuration, and the common pattern includes no more than 6 non-chiral internucleotide linkages.In some embodiments, the common pattern of backbone chiral centers includes at least 14 internucleotide linkages in the Sp configuration, and the common pattern includes no more than 5 non-chiral internucleotide linkages. In some embodiments, the common pattern of backbone chiral centers includes at least 15 internucleotide linkages in the Sp configuration, and the common pattern includes no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration are optionally contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration are optionally contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages are optionally contiguous or non-contiguous.

[0175] In some embodiments, the oligonucleotides in the provided compositions have a common pattern of backbone phosphorus modifications. In some embodiments, the provided compositions are chiral controlled oligonucleotide compositions in that the compositions contain predetermined levels of oligonucleotides of individual oligonucleotide types, where an oligonucleotide type is defined by: 1) base sequence, 2) skeletal bond pattern; 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification.

[0176] As discussed above and understood in the art, in some embodiments, the base sequence of an oligonucleotide can refer to the identity and / or modification status of the nucleoside residues in the oligonucleotide (e.g., the status of the sugar and / or base components compared to standard natural nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization properties of such residues (i.e., the ability to hybridize with a particular complementary residue).

[0177] In some embodiments, a particular oligonucleotide type may be defined by: 1A) base identity, 1B) Base modification patterns, 1C) Glycosylation pattern, 2) skeletal bond pattern; 3) the pattern of backbone chiral centers, and 4) Pattern of backbone phosphorus modification. Thus, in some embodiments, particular oligonucleotide types may share the same bases but differ in their patterns of base modifications and / or sugar modifications, hi some embodiments, particular oligonucleotide types may share the same bases and patterns of base modifications (including, for example, the absence of base modifications), but may differ in their patterns of sugar modifications.

[0178] In some embodiments, a particular oligonucleotide type has the same base sequence (including length), the same pattern of chemical modifications to the sugar and base moieties, the same backbone linkage pattern (e.g., patterns of natural phosphate linkages, non-negatively charged linkages, phosphorothioate linkages, phosphorothioate triester linkages, and combinations thereof), the same pattern of backbone chiral centers (e.g., the stereochemistry (Rp / Sp) pattern of chiral internucleotide linkages), and the same pattern of backbone phosphorus modifications (e.g., the modification pattern on the internucleotide phosphorus atom (-S - etc.), and -LR of formula I 1 ) are chemically identical.

[0179] In some embodiments, the present disclosure provides chiral controlled oligonucleotide compositions of oligonucleotides comprising oligonucleotides containing multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotide linkages, particularly oligonucleotides containing multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral internucleotide linkages. In some embodiments, in stereoirregular or racemic preparations of oligonucleotides, at least one chiral internucleotide linkage is formed with a diastereoselectivity of less than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, in stereoselective or chiral controlled preparations of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, in stereoselective or chiral controlled preparations of oligonucleotides, each chiral internucleotide linkage is formed with a diastereoselectivity of greater than 95:5. In some embodiments, in stereoselective or chiral controlled preparations of oligonucleotides, each chiral internucleotide bond is formed with a diastereoselectivity of greater than 96:4. In some embodiments, in stereoselective or chiral controlled preparations of oligonucleotides, each chiral internucleotide bond is formed with a diastereoselectivity of greater than 97:3. In some embodiments, in stereoselective or chiral controlled preparations of oligonucleotides, each chiral internucleotide bond is formed with a diastereoselectivity of greater than 98:2. In some embodiments, in stereoselective or chiral controlled preparations of oligonucleotides, each chiral internucleotide bond is formed with a diastereoselectivity of greater than 99:1.In some embodiments, the diastereoselectivity of a chiral internucleotide linkage in an oligonucleotide can be measured via a model reaction, such as dimer formation under essentially the same or equivalent conditions, where the dimer has the same internucleotide linkage as the chiral internucleotide linkage, the 5' nucleoside of the dimer is the same as the nucleoside on the 5' end of the chiral internucleotide linkage, and the 3' nucleoside of the dimer is the same as the nucleoside on the 3' end of the chiral internucleotide linkage. In some embodiments, the diastereoselectivity of the phosphorus at the linkage in the chiral-controlled internucleotide linkage is 90% to 100% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%). In some embodiments, the diastereopurity of the linked phosphorus in the chiral controlled internucleotide linkage is 95% to 100% (e.g., 95%, 96%, 97%, 98%, 99%, or 99.5%). In some embodiments, the diastereopurity of the linked phosphorus in the chiral controlled internucleotide linkage is 97% to 100% (e.g., 97%, 98%, 99%, or 99.5%). In some embodiments, the chiral controlled internucleotide linkage has a diastereopurity of at least 97%. In some embodiments, the chiral controlled internucleotide linkage has a diastereopurity of at least 98%. In some embodiments, the chiral controlled internucleotide linkage has a diastereopurity of at least 99%. In some embodiments, the diastereopurity of a non-chiral controlled (racemic / stereoirregular) internucleotide linkage is less than 90%.

[0180] Among other things, the present disclosure provides oligonucleotide compositions and techniques for optimizing properties.

[0181] Among other things, the present disclosure provides oligonucleotide compositions and techniques for optimizing properties (e.g., improving single-stranded RNA interference, RNase H-mediated knockdown, etc.). In some embodiments, the present disclosure provides methods for attenuating immune responses associated with administration of oligonucleotides and compositions thereof (i.e., administering an oligonucleotide composition such that an undesirable immune response to the oligonucleotide in the composition is reduced, e.g., compared to that observed with a reference nucleotide composition of comparable or identical nucleotide sequence). In some embodiments, the present disclosure provides methods for increasing binding of oligonucleotides and compositions thereof to a particular protein. In some embodiments, the present disclosure provides methods for increasing binding of oligonucleotides and compositions thereof to a particular protein. In some embodiments, the present disclosure provides methods for enhancing delivery of oligonucleotides and compositions thereof. Among other things, the present disclosure includes the recognition that optimal delivery of an oligonucleotide to its target requires, in some embodiments, a balance between binding of the oligonucleotide to a particular protein so that the oligonucleotide can be transported to a desired location, and proper release of the oligonucleotide from the particular protein so that the oligonucleotide can perform its desired function (e.g., hybridization to its target, cleavage of its target, inhibition of translation, modulation of transcript processing, etc.) As exemplified herein, the present disclosure recognizes that improved oligonucleotide properties can be achieved through, among other things, chemical modification and / or stereochemistry.

[0182] In some embodiments, the disclosure provides a method for treating or preventing a disease, the method comprising administering to a subject an oligonucleotide composition described herein.

[0183] In some embodiments, the disease is one that can be cured, ameliorated, or a new beneficial function introduced by knocking down a target nucleic acid via single-stranded RNA interference after administration of a provided composition.

[0184] In some embodiments, the disease is cancer.

[0185] In some embodiments, the consensus sequence comprises a sequence selected from Table 1 A. In some embodiments, the consensus sequence is a sequence selected from Table 1 A. In some embodiments, the pattern of backbone chiral centers is selected from those set forth in Table 1A.

[0186] In some embodiments, the present disclosure provides methods comprising administering a composition comprising a first plurality of oligonucleotides, wherein the composition exhibits improved delivery compared to a reference composition comprising a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides in the reference composition has a similar common base sequence, but the plurality of oligonucleotides comprises: the stereochemical structure of each oligonucleotide within the plurality of oligonucleotides of this reference is different from one another; and / or At least some of the oligonucleotides in the reference plurality have a structure that is different from the structure exhibited by the composition plurality. In this respect, they are structurally different from the first plurality of oligonucleotides.

[0187] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition capable of inducing a decrease in expression and / or levels of a target gene or its gene product, the method comprising administering a first plurality of oligonucleotides having a common nucleotide sequence, the improvement including: administration of the oligonucleotides comprising the first plurality of oligonucleotides is characterized by improved delivery compared to a reference oligonucleotide composition of the same common nucleotide sequence; Includes:

[0188] In some embodiments, the present disclosure provides a method of administering an oligonucleotide composition capable of inducing single-stranded RNA interference, the method comprising administering a first plurality of oligonucleotides having a common nucleotide sequence, the improvement including: administration of the oligonucleotides comprising the first plurality of oligonucleotides is characterized by improved delivery compared to a reference oligonucleotide composition of the same common nucleotide sequence; Includes:

[0189] In some embodiments, the present disclosure provides an oligonucleotide single-stranded RNAi agent selected from any of the Tables (including but not limited to, Table 1A) or elsewhere disclosed herein. In some embodiments, the present disclosure provides an oligonucleotide single-stranded RNAi agent selected from any of the Tables (including but not limited to, Table 1A) or elsewhere disclosed herein, wherein the oligonucleotide is conjugated to a lipid moiety.

[0190] In some embodiments, the oligonucleotides are 25 bases or less in length. In some embodiments, the oligonucleotides are 30 bases or less in length. In some embodiments, the oligonucleotides are 35 bases or less in length. In some embodiments, the oligonucleotides are 40 bases or less in length. In some embodiments, the oligonucleotides are 45 bases or less in length. In some embodiments, the oligonucleotides are 50 bases or less in length. In some embodiments, the oligonucleotides are 55 bases or less in length. In some embodiments, the oligonucleotides are 60 bases or less in length.

[0191] In some embodiments, provided oligonucleotides comprise a lipid moiety. In some embodiments, the lipid moiety is incorporated by complexation with a lipid. In some embodiments, the lipid moiety is a fatty acid. In some embodiments, the oligonucleotide is complexed to a fatty acid. In some embodiments, provided single-stranded RNAi agents further comprise a lipid. In some embodiments, provided single-stranded RNAi agents comprise a lipid moiety complexed to the 9th or 11th nucleotide (counting from the 5' end). In some embodiments, the oligonucleotide is complexed to a fatty acid at the base. In some embodiments, provided single-stranded RNAi agents comprise a lipid moiety. In some embodiments, provided single-stranded RNAi agents comprise a lipid moiety complexed to the base of the 9th or 11th nucleotide (counting from the 5' end).

[0192] In some embodiments, the disclosure provides compounds, for example, compounds having the formula OI: [ka] or a salt thereof, wherein each variable moiety is independently as described in this disclosure.

[0193] In some embodiments, the present disclosure provides: A c -[-L M -(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b , or (A c ) a -L M -(R D ) b or a salt thereof, wherein each variable is independently as described in this disclosure.

[0194] In some embodiments, A c are each independently an oligonucleotide portion of an oligonucleotide of formula OI or a salt thereof (e.g., [H] a -A c or [H] b -A c is an oligonucleotide of formula OI or a salt thereof). In some embodiments, the present disclosure provides c -[-L M -(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b , or (A c ) a -L M -(R D In some embodiments, the present disclosure provides an oligonucleotide or salt thereof having the structure: c -[-L M -(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b , or (A c ) a -L M -(R D ) b In some embodiments, the present disclosure provides an oligonucleotide composition comprising an oligonucleotide having the structure: c -[-L M-(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b , or (A c ) a -L M -(R D ) b In some embodiments, the present disclosure provides an oligonucleotide composition comprising a predetermined level (as described in this disclosure) of an oligonucleotide having the structure: c -[-L M -(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b , or (A c ) a -L M -(R D ) b or a salt thereof. In some embodiments, a plurality (e.g., a first plurality) of oligonucleotides, or oligonucleotides of a single oligonucleotide type, have the structure: c -[-L M -(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b, or (A c ) a -L M -(R D ) b or a salt thereof. In some embodiments, the oligonucleotide in the provided compositions (e.g., provided chiral controlled oligonucleotide compositions) has the structure: c -[-L M -(R D ) a ] b , [(A c ) a -L M ] b -R D , (A c ) a -L M -(A c ) b , or (A c ) a -L M -(R D ) b In some embodiments, the structure is: c -[-L M -(R D ) a ] b or a salt thereof. In some embodiments, the structure is [(A c ) a -L M ] b -R D or a salt thereof. In some embodiments, the structure is c ) a -L M -(A c ) b or a salt thereof. In some embodiments, the structure is A c -[-L M -(R D ) a ] b or a salt thereof.

[0195] In some embodiments, A care each independently 5'-PX0-N1-PX1-N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8-N9-PX9-N10-PX10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz -(N26-PX26-N27-PX27) yz -(CAP) zz The oligonucleotide portion of an oligonucleotide having the structure -3' or a salt thereof.

[0196] In some embodiments, the complex comprises A c -[-L LD -(R LD ) a ] b wherein each variable is independently as described in this disclosure.

[0197] In some embodiments, the present disclosure provides: A c -[-L LD -(R LD ) a ] b , or [(A c ) a -L LD ] b -R LD and providing an oligonucleotide composition comprising a plurality of oligonucleotides having the structure: During the ceremony: A c are each independently an oligonucleotide moiety (e.g., [H] a -A c or [H] b -Ac is an oligonucleotide), a is 1 to 1000; b is 1 to 1000; Each L LD is independently a linker moiety, Each R LD is independently a lipid moiety or a targeting moiety.

[0198] In some embodiments, the present disclosure provides: A c -[-L LD -(R LD ) a ] b , or [(A c ) a -L LD ] b -R LD and providing an oligonucleotide composition comprising a plurality of oligonucleotides having the structure: During the ceremony: A c are each independently an oligonucleotide moiety (e.g., [H] a -A c or [H] b -A c is an oligonucleotide), a is 1 to 1000; b is 1 to 1000; Each L LD are independently a covalent bond or an optionally substituted C-C 80 wherein one or more methylene units are optionally, and independently, T LDor by an optionally substituted group selected from C-C alkylene, C-C alkenylene, -C≡C-, C-C heteroaliphatic moiety, -C(R')-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, and -C(O)O-; Each R LD are independently hydrogen or optionally substituted C-C 80 wherein one or more methylene units are optionally, and independently, C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, C1-C6 heteroaliphatic moiety, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N( -R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)-, -S(O)N(R')-, -N(R')S(O)-, -SC(O)-, -C(O)S-, -OC(O)-, and -C(O)O-; T LD is of formula I': [ka] having the structure W is O, S or Se; Each of X, Y and Z independently represents -O-, -S-, -N(-LR 1 )-, or L, L is a covalent bond or an optionally substituted linear or branched C-C 10aliphatic, wherein one or more methylene units of L are optionally and independently a C-C aliphatic moiety, C-C alkenylene, -C≡C-, C-C heteroaliphatic moiety, -C(R')2-, -Cy-, -B(R')-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2- by an optionally substituted group selected from -SC(O)-, -C(O)S-, -OC(O)-, and -C(O)O-; R 1 is hydrogen, R, or optionally substituted C-C 50 aliphatic, wherein one or more methylene units are optionally and independently C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, C1-C6 heteroaliphatic moiety, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2- by an optionally substituted group selected from -SC(O)-, -C(O)S-, -OC(O)-, and -C(O)O-; each R' is independently -R, -C(O)R, -CO2R, or -SO2R; or Two or more R', together with the atoms between them, form an optionally substituted C-C alkyl group selected from aryl, carbocyclyl, heterocyclyl, and heteroaryl. 14 Forming a base, -Cy- is phenylene, C3-C 14 Carbocyclylene, C 10 -C 14 Arylene, C5-C 14 Heteroarylene and C3-C 14an optionally substituted bivalent ring selected from heterocyclylene; Each R is independently hydrogen or C-C 20 Aliphatic, C3-C 20 Carbocyclyl, C6-C 20 Aryl, C5-C 20 Heteroaryl and C3-C 20 heterocyclyl, optionally substituted.

[0199] In some embodiments, [H] a -A c or [H] b -A c is an oligonucleotide having the structure of formula OI, or a salt thereof. In some embodiments, [H] a -A c or [H] b -A c is 5'-PX0-N1-PX1-N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8-N9-PX9-N10-P X10-N11-PX11-N12-PX12-N13-PX13-N14-PX14-N15-PX15-N16-PX16-N17-PX17-(N18-PX18) mz -(N19-PX19) nz -(N20-PX20) pz -(N21-PX21) rz -(N22-PX22) sz -(N23-PX23) tz -(N24-PX24) vz -(N25-PX25) wz -(N26-PX26-N27-PX27) yz -(CAP) zz -3' structure or a salt thereof.

[0200] In some embodiments, T LD The P in the middle is P * In some embodiments, the complex is c ) a -L LD ] b-R LD In some embodiments, the complex has the structure: c ) a -L LD -R LD It has the following structure.

[0201] In some embodiments, a is 1 to 100. In some embodiments, a is 1 to 50. In some embodiments, a is 1 to 40. In some embodiments, a is 1 to 30. In some embodiments, a is 1 to 20. In some embodiments, a is 1 to 15. In some embodiments, a is 1 to 10. In some embodiments, a is 1 to 9. In some embodiments, a is 1 to 8. In some embodiments, a is 1 to 7. In some embodiments, a is 1 to 6. In some embodiments, a is 1 to 5. In some embodiments, a is 1 to 4. In some embodiments, a is 1 to 3. In some embodiments, a is 1 to 2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10. In some embodiments, a is greater than 10. In some embodiments, b is 1 to 100. In some embodiments, b is 1 to 50. In some embodiments, b is 1 to 40. In some embodiments, b is 1 to 30. In some embodiments, b is 1 to 20. In some embodiments, b is 1 to 15. In some embodiments, b is 1 to 10. In some embodiments, b is 1 to 9. In some embodiments, b is 1 to 8. In some embodiments, b is 1 to 7. In some embodiments, b is 1 to 6. In some embodiments, b is 1 to 5. In some embodiments, b is 1 to 4. In some embodiments, b is 1 to 3. In some embodiments, b is 1 to 2. In some embodiments, b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, b is 6. In some embodiments, b is 7. In some embodiments, b is 8. In some embodiments, b is 9. In some embodiments, b is 10.In some embodiments, b is greater than 10. In some embodiments, the complex comprises A. c -L LD -R LD In some embodiments, A has the structure c is conjugated via one or more of its sugar, base, and / or internucleotide linkage moieties. c is conjugated via its 5'-OH (5'-O-). c is conjugated via its 3'-OH (3'-O-). c is complexed via an internucleotide bond. c is conjugated via a nucleobase. c is conjugated via a sugar. In some embodiments, prior to conjugation, A c -(H) b (b is A c In some embodiments, L is an oligonucleotide described herein, e.g., an oligonucleotide described in any one of the Tables. LD is -L-. In some embodiments, L LD comprises a phosphorothioate group. LD is -C(O)NH-(CH2)6-OP(=O)(S - )—O—. In some embodiments, the —C(O)NH terminus is R LD and the -O-terminus is linked to the oligonucleotide, e.g., via the 5'-terminus or 3'-terminus. LD is an optionally substituted C 10 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , or C 25 ~C 20 , C21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 35 , C 40 , C 45 , C 50 , C 60 , C 70 , or C 80 In some embodiments, R LD is an optionally substituted C 10-80 In some embodiments, R LD is an optionally substituted C 20-80 In some embodiments, R LD is an optionally substituted C 10-70 In some embodiments, R LD is an optionally substituted C 20-70 In some embodiments, R LD is an optionally substituted C 10-60 In some embodiments, R LD is an optionally substituted C 20-60 In some embodiments, R LD is an optionally substituted C 10-50 In some embodiments, R LD is an optionally substituted C 20-50 In some embodiments, R LD is an optionally substituted C 10-40 In some embodiments, R LD is an optionally substituted C 20-40 In some embodiments, R LD is an optionally substituted C 10-30 In some embodiments, R LD is an optionally substituted C 20-30 In some embodiments, R LD is the unsubstituted C 10 , C 15 , C 16 , C17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 , or C 25 ~C 20 , C 21 , C 22 , C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 35 , C 40 , C 45 , C 50 , C 60 , C 70 , or C 80 In some embodiments, R LD is the unsubstituted C 10-80 In some embodiments, R LD is the unsubstituted C 20-80 In some embodiments, R LD is the unsubstituted C 10-70 In some embodiments, R LD is the unsubstituted C 20-70 In some embodiments, R LD is the unsubstituted C 10-60 In some embodiments, R LD is the unsubstituted C 20-60 In some embodiments, R LD is the unsubstituted C 10-50 In some embodiments, R LD is the unsubstituted C 20-50 In some embodiments, R LD is the unsubstituted C 10-40 In some embodiments, R LD is the unsubstituted C 20-40 In some embodiments, R LD is the unsubstituted C 10-30 In some embodiments, R LDis the unsubstituted C 20-30 It is aliphatic.

[0202] In some embodiments, R LD is not hydrogen. LD is a lipid moiety. In some embodiments, R LD is a targeting moiety. In some embodiments, R LD is a targeting moiety that includes a carbohydrate moiety. In some embodiments, R LD is a GalNAc moiety.

[0203] In some embodiments, the single stranded RNAi agent is any one of the aforementioned compositions, further comprising one or more additional components.

[0204] In some embodiments, provided oligonucleotides have the ability to degrade target transcripts (eg, RNA) via both RNase H and RNAi mechanisms.

[0205] In some embodiments, conjugating a lipid moiety to an oligonucleotide improves at least one property of the oligonucleotide. In some embodiments, the improved property includes increased activity (e.g., increased ability to induce a decrease in the expression and / or level of a target gene or its gene product, and / or to induce single-stranded RNA interference, and / or to induce RNase H-mediated knockdown) and / or improved tissue distribution. In some embodiments, the tissue is muscle tissue. In some embodiments, the tissue is skeletal muscle, gastrocnemius muscle, triceps muscle, heart, or diaphragm. In some embodiments, the improved property includes decreased hTLR9 agonist activity. In some embodiments, the improved property includes hTLR9 antagonist activity. In some embodiments, the improved property includes increased hTLR9 antagonist activity.

[0206] Generally, the properties of the oligonucleotide compositions described herein can be assessed using any suitable assay.

[0207] One of ordinary skill in the art would readily be able to recognize and / or develop a suitable assay for a particular oligonucleotide composition.

[0208] definition As used herein, the following definitions apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0209] Aliphatic: As used herein, "aliphatic" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more units of unsaturation (not aromatic), or a combination thereof. In some embodiments, an aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-6 aliphatic carbon atoms. In yet other embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms, and in yet other embodiments, an aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof (such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl).

[0210] Alkenyl: As used herein, "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds.

[0211] Alkyl: As used herein, the term "alkyl" has its usual meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl has 1 to 100 carbon atoms. In certain embodiments, a straight-chain or branched alkyl has about 1 to 20 carbon atoms in its backbone (e.g., C1-C6 for straight chain). 20 , C2-C for branched chain 20), or about 1-10 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure whether the ring is monocyclic, bicyclic or polycyclic, or about 5, 6 or 7 carbons in the ring structure. In some embodiments, alkyl groups can be lower alkyl groups, which lower alkyl groups have from 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0212] Alkynyl: The term "alkynyl," as used herein, refers to an alkyl group, as defined herein, having one or more triple bonds.

[0213] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0214] Approximately: As used herein, the term "approximately" or "about" when in reference to a number is generally taken to include that number within 5%, 10%, 15%, or 20% in either direction (greater or lesser) of that number (except where such number is a value that may be less than 0% or greater than 100%) unless otherwise stated or otherwise apparent from the context. In some embodiments, the use of the term "about" when in reference to dosage means ±5 / mg / kg / day.

[0215] Aryl: As used herein, the term "aryl," used alone or as part of a larger moiety within "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, wherein at least one ring within the system is aromatic. In some embodiments, an aryl group is a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 14 ring members, wherein at least one ring within the system is aromatic, and wherein each ring within the system contains 3 to 7 ring members. In some embodiments, an aryl group is a biaryl group. "Aryl" may be used interchangeably with "aryl ring." In some embodiments of the present disclosure, "aryl" refers to an aromatic ring system, including, but not limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl, etc., which may bear one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthymidyl, phenanthridinyl, or tetrahydronaphthyl.

[0216] Characteristic portion: As used herein, the phrase "characteristic portion" of a protein or polypeptide refers to a portion comprising consecutive amino acids, or a collection of consecutive amino acids, both of which are characteristic of the protein or polypeptide. Each such consecutive stretch will generally contain at least two amino acids. Moreover, those skilled in the art will recognize that in many cases, at least 5, 10, 15, 20, or more amino acids are required to be characteristic of a protein. Generally, a characteristic portion is one that shares at least one functional characteristic with the related intact protein, in addition to the sequence identity specified above.

[0217] Characteristic structural element: The term "characteristic structural element" or "structural element" refers to a distinct structural element that is present in all members of a family of polypeptides, small molecules, or nucleic acids, and can therefore be used by those skilled in the art to define the members of that family. In some embodiments, structural elements of single-stranded RNAi agents include, but are not limited to, a 5'-end structure, a 5'-end region, a 5'-nucleotide portion, a seed region, a post-seed region, a 3'-end region, a 3'-end dinucleotide, a 3'-cap, a modification pattern, a stereochemical pattern in the backbone, an additional chemical moiety, etc.

[0218] Equivalent: The term "equivalent" is used herein to describe two (or more) sets of conditions or circumstances that are sufficiently similar to one another to allow for a comparison of the results obtained or the phenomena observed. In some embodiments, equivalent sets of conditions or circumstances are characterized by multiple substantially identical characteristics and one or a few altered characteristics. One skilled in the art will recognize that sets of conditions are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to reasonably justify concluding that differences in the results obtained and phenomena observed under the different sets of conditions or circumstances are caused by, or indicative of, changes in those altered characteristics.

[0219] Alicyclic: The terms "alicyclic," "carbocyclic," "carbocyclyl," "carbocyclic radical," and "carbocycle" are used interchangeably and, as used herein, refer to a saturated or partially unsaturated, but non-aromatic, cycloaliphatic monocyclic, bicyclic, or polycyclic ring system, as described herein, having 3 to 30 ring members unless otherwise specified. Alicyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, an alicyclic group has 3 to 6 carbons. In some embodiments, an alicyclic group is saturated and is cycloalkyl. The term "alicyclic" can also include an aliphatic ring fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl. In some embodiments, an alicyclic group is bicyclic. In some embodiments, an alicyclic group is tricyclic. In some embodiments, an alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to a C3-C6 monocyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule, or a C8-C6 monocyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. 10 Bicyclic hydrocarbons or C8-C 10 Polycyclic hydrocarbons, or C9-C, which are fully saturated or contain one or more unsaturated units, but are not aromatic, and have a single point of attachment to the rest of the molecule 16 Refers to polycyclic hydrocarbons.

[0220] Dosage regimen: As used herein, "dosage regimen" or "treatment regimen" often refers to a set of unit doses (often two or more) administered individually to a subject, separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may include one or more dosages. In some embodiments, a dosing regimen includes multiple doses, each separated from the other by a period of equal length. In some embodiments, a dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose. In some embodiments, different doses within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first administration at a first dosage, followed by one or more additional doses at a second dosage that is different from the first dosage. In some embodiments, a dosing regimen includes a first administration at a first dosage, followed by one or more additional doses at a second dosage that is the same as the first dosage.

[0221] Equivalent Agents: Upon reading this disclosure, those skilled in the art will recognize that the scope of useful agents within the context of this disclosure is not limited to those specifically mentioned or exemplified herein. Specifically, those skilled in the art will recognize that active agents often have a structure consisting of a core and pendant moieties attached thereto, and will therefore understand that simple alterations to such core and / or pendant moieties may not significantly alter the activity of the agent. For example, in some embodiments, replacement of one or more pendant moieties with groups of equivalent three-dimensional structure and / or chemical reactivity properties may produce a substituted compound or moiety equivalent to the original reference compound or moiety. In some embodiments, addition or removal of one or more pendant moieties may produce a substituted compound equivalent to the original reference compound. In some embodiments, alteration of the core structure, for example, by addition or removal of a small number of bonds (often no more than 5, 4, 3, 2, or 1 bond, and often only a single bond), may produce a substituted compound equivalent to the original reference compound. In many embodiments, equivalent compounds can be synthesized by the methods shown in the following general reaction schemes, or modifications thereof, using, for example, readily available starting materials, reagents, and conventional or provided synthetic procedures. In these reactions, variants that are known per se, but not mentioned herein, can also be used.

[0222] Equivalent Dosage: The term "equivalent dosage" is used herein to compare dosages of different pharmaceutically active agents that produce the same biological result. Doses of two different agents are considered "equivalent" to one another according to the present disclosure if they achieve a comparable level or degree of biological result. In some embodiments, equivalent dosages of different agents for uses according to the present disclosure are determined using the in vitro and / or in vivo assays described herein. In some embodiments, one or more lysosome activators for uses according to the present disclosure are utilized at dosages equivalent to the dosage of a reference lysosome activator. In some embodiments, the reference lysosome activator for such purposes is selected from the group consisting of small molecule allosteric activators (e.g., pyrazole pyrimidines), iminosugars (e.g., isofagomine), antioxidants (e.g., n-acetyl-cysteine), and cell trafficking regulators (e.g., Rab1a polypeptides).

[0223] Heteroaliphatic: The term "heteroaliphatic" is given its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH, and CH are independently replaced with one or more heteroatoms (including oxidized and / or substituted forms thereof). In some embodiments, a heteroaliphatic group is a heteroalkyl. In some embodiments, a heteroaliphatic group is a heteroalkenyl.

[0224] Heteroalkyl: As used herein, the term "heteroalkyl" is given its ordinary meaning in the art and refers to an aliphatic group, as described herein, in which one or more carbon atoms are independently replaced with one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0225] Heteroaryl: As used herein, the terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy," refer to a monocyclic, bicyclic, or polycyclic ring system having a total of 5 to 30 ring members, in which at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. Heteroaryl groups, in some embodiments, are groups having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), in some embodiments, 5, 6, 9, or 10 ring atoms. In some embodiments, heteroaryl groups have 6, 10, or 14 pi electrons shared in the cyclic arrangement and having 1 to 5 heteroatoms in addition to the carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl is a heterobiaryl group, such as bipyridyl. As used herein, the terms "heteroaryl" and "heteroaralkyl" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups may be monocyclic, bicyclic, or polycyclic."Heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, where the alkyl and heteroaryl portions independently may be substituted.

[0226] Heteroatom: As used herein, the term "heteroatom" refers to an atom that is neither carbon nor hydrogen. In some embodiments, a heteroatom is any oxidized form of nitrogen, sulfur, phosphorus, or silicon, any basic nitrogen or quaternized form of a substitutable nitrogen of a heterocyclic ring (e.g., N in 3,4-dihydro-2H-pyrrolyl), NH (in pyrrolidinyl), or NR (in N-substituted pyrrolidinyl). + The element is oxygen, sulfur, nitrogen, phosphorus, or silicon, including the like.

[0227] Heterocycle: As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic ring moiety (e.g., 3-30 members) that is saturated or partially unsaturated and has one or more heteroatom ring atoms. In some embodiments, a heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety, either saturated or partially unsaturated, having, in addition to carbon atoms, one or more heteroatoms, preferably 1 to 4 heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 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 N-substituted pyrrolidinyl. +It can be NR. The heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom can be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein and include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. Heterocyclyl groups may be monocyclic, bicyclic, or polycyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, where the alkyl and heterocyclyl portions are optionally independently substituted.

[0228] Intraperitoneal: As used herein, the phrases "intraperitoneal administration" and "administered intraperitoneally" have their art-recognized meaning of referring to administration of a compound or composition into the peritoneal membrane of a subject.

[0229] In vitro: As used herein, the term "in vitro" refers to events that occur not within a living organism (e.g., an animal, plant, and / or microorganism) but in an artificial environment, e.g., in a test tube or reactor, in a cell culture, etc.

[0230] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, a plant, and / or a microorganism).

[0231] Lower alkyl: The term "lower alkyl" refers to a C 1-4 Examples of lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0232] Lower haloalkyl: The term "lower haloalkyl" refers to a C alkyl group substituted with one or more halogen atoms. 1-4 refers to a linear or branched alkyl group of the formula:

[0233] Optionally substituted: As described herein, for example, oligonucleotides of the present disclosure may contain optionally substituted and / or substituted moieties. Generally, "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety have been replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure can be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at every position. In some embodiments, an optionally substituted group is unsubstituted. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is not substantially altered when subjected to conditions that allow for the production, detection, and, in some embodiments, recovery, purification, and use of the compounds for one or more purposes disclosed herein.

[0234] Suitable monovalent substituents for substitutable atoms (e.g., appropriate carbon atoms) are independently halogen; —(CH) 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o , -O-(CH2) 0-4 C(O)OR°;-(CH2)0-4 CH(OR o )2; optionally substituted with R° -(CH2) 0-4 Ph; optionally substituted with R° -(CH2) 0-4 O(CH2) 0-1 Ph; optionally substituted with R° -CH=CHPh; optionally substituted with R° -(CH2) 0-4 O(CH2) 0-1 -pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2) 0-4 N(R o )C(O)NR o 2;-N(R o )C(S)NR o 2;-(CH2) 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 ;-(CH2) 0-4 C(O)R o ;-C(S)R o ;-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 C(O)SR o ;-(CH2) 0-4 C(O)OSiR o 3;-(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0-4 SR,-SC(S)SR°;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NR o 2;-C(S)NRo 2;-C(S)SR°;-SC(S)SR°,-(CH2) 0-4 OC(O)NR o 2;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR o )R o ;-(CH2) 0-4 SSR o ;-(CH2) 0-4 S(O)2R o ;-(CH2) 0-4 S(O)2OR o ;-(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2;-(CH2) 0-4 S(O)R o ;-N(R o )S(O)NR o 2;-N(R o )S(O)2R o ;-N(OR o )R o ;-C(NH)NR o 2;-Si(R)3;-OSi(R)3;-B(R)2;-OB(R)2;-OB(OR)2;-P(R)2;-P(OR)2;-OP(R)2;-OP(OR)2;-P(O)(R)2;-P(O)(OR)2;-OP(O)(R)2;-OP(O)(OR)2;-OP(O )(OR)(SR);-SP(O)(R)2;-SP(O)(OR)2;-N(R)P(O)(R)2;-N(R)P(O)(OR)2;-P(R)2[B(R)3];-P(OR)2[B(R)3];-OP(R)2[B(R)3];-OP(OR)2[B(R)3];-(C 1-4 Linear or branched alkylene)ON(R o )2; or -(C 1-4 Straight or branched chain alkylene)C(O)ON(R o )2, (where each R o are optionally substituted as defined below and independently represent hydrogen, C 1-20C having 1 to 5 heteroatoms independently selected from aliphatic, nitrogen, oxygen, sulfur, silicon, and phosphorus 1-20 , heteroaliphatic, -CH2-(C 6-14 aryl), -O(CH2) 0-1 (aryl), (C 6-14 aryl), -CH2- (5-14 membered heteroaryl ring), a 5-20 membered monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, or two independently occurring aryl rings, regardless of the above definition. o together with their intervening atoms form a 5-20 membered, monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which may be optionally substituted as defined below.

[0235] Suitable R o The above monovalent substituents (or the ring formed by two independently occurring R° together with the intervening atoms) are independently selected from halogen, -(CH2) 0-2 R ● ,-(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2;-O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 SR ● , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● , -(CH2) 0-2 NR ● 2, -NO2, -SiR ●3. -OSiR ● 3. -C(O)SR ● 、 -(C 1-4 Straight or branched chain alkylene)C(O)OR ● , or -SSR ● , (where each R ● is unsubstituted, or, when preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph and a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents on a saturated carbon atom of R° include ═O and ═S.

[0236] Suitable divalent substituents (e.g., on the appropriate carbon atoms) are independently: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S-, (wherein each R * is hydrogen, optionally substituted as defined below, C 1-6 aliphatic and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Preferred divalent substituents attached to adjacent substitutable carbon atoms of an "optionally substituted" group are -O(CR * 2) 2-3 O-, (wherein each R * is hydrogen, optionally substituted as defined below, C 1-6aliphatic and unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0237] R * Suitable substituents on the aliphatic group are independently halogen, —R ● ,-(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, when preceded by "halo", is substituted only with one or more halogens, and independently, C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0238] Oral: As used herein, the phrases "oral administration" and "orally administered" have their art-recognized meaning to refer to administration of a compound or composition by mouth.

[0239] Parenteral: As used herein, the phrases "parenteral administration" and "parenterally administered" have their art-recognized meaning to refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0240] Partially unsaturated: As used herein, the term "partially unsaturated" refers to a moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to include rings with multiple sites of unsaturation, but is not intended to include either aryl or heteroaryl moieties as defined herein.

[0241] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those adapted for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, such as tablets targeted for buccal, sublingual, and systemic absorption, bolus administration, powders, granules, and pastes for application to the tongue; parenteral administration, such as subcutaneous, intramuscular, intravenous, or epidural injection as a sterile solution or suspension or sustained-release formulation; topical administration, such as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or buccal cavity; vaginal or rectal administration, such as a pessary, cream, or foam; sublingual administration; intraocular administration; transdermal administration; or administration to nasal, pulmonary, and other mucosal surfaces.

[0242] Pharmaceutically Acceptable: Pharmaceutically Acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0243] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0244] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in pharmaceutical contexts, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without undue harmful effects, irritation, allergic response, and the like, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include non-toxic acid addition salts, such as, but not limited to, salts of amino groups formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as, for example, acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed using other methods, such as those used with sugars, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Examples of suitable salts include, but are not limited to, sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.In some embodiments, provided compounds (e.g., oligonucleotides) contain one or more acidic groups, and pharmaceutically acceptable salts are alkali salts, alkaline earth metal salts, or ammonium salts (e.g., ammonium salts of N(R)3, where each R is independently defined and described in this disclosure). Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, where appropriate. In some embodiments, provided compounds contain multiple acidic groups, e.g., provided oligonucleotides may contain two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts or common salts of such compounds contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or common salt), all ionizable hydrogens in acidic groups are replaced with cations. In some embodiments, a pharmaceutically acceptable salt is a sodium salt of a provided oligonucleotide. In some embodiments, a pharmaceutically acceptable salt is a sodium salt of a provided oligonucleotide, in which each acidic phosphate group is present as a salt form (all sodium salts), such as Na. + Contains 19 sodium salts of WV-2555, or Na + It is the sodium salt of WV-2555, which contains 23 units.

[0245] Prodrug: A typical "prodrug," as the term is used herein and understood in the art, is an entity that, when administered to an organism, is metabolized within the body to deliver the desired active agent (e.g., a therapeutic or diagnostic agent). Typically, such metabolism removes at least one "prodrug moiety" to form the active agent. Various forms of "prodrugs" are known in the art. For examples of such prodrug moieties, see below: a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, 42:309-396, edited by K. Widder, et al. (Academic Press, 1985), b)Prodrugs and Targeted Delivery, edited by by J. Rautio (Wiley, 2011), c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen, d)Bundgaard,Chapter 5 “Design and Application of Prodrugs”, by H.Bundgaard,p.113-191(1991), e) Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992), f) Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988), and g) Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984).

[0246] As with other compounds described herein, the prodrug may be provided in any of a variety of forms (e.g., crystalline form, salt form, etc.) In some embodiments, the prodrug is provided as a pharmaceutically acceptable salt thereof.

[0247] Protecting group: As used herein, the term "protecting group" is known in the art and is described in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3 rdIncluded are protecting groups detailed in the "Protective Groups for Nucleoside and Nucleotide Chemistry" series, edited by Serge L. Beaucage et al., 1999, John Wiley & Sons, 1999, which is incorporated herein by reference in its entirety. Also included are protecting groups specifically adapted for nucleoside and nucleotide chemistry described in "Current Protocols in Nucleic Acid Chemistry," edited by Serge L. Beaucage et al., 06 / 2012, Chapter 2 of which is incorporated herein by reference in its entirety. Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), and carbamic acid esters such as methyl methyl carbamate (Methyl methyl carbamate). 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-Dicyclohexylcarboxamido)ethyl, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrocarbamate Tobenzyl, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiobenzyl carbamate Phenyl (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate , 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-Dimethoxycarbonylvinyl, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborinyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1- Methylcyclohexyl, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylcarbamate benzyl, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl) Propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-Tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, tetra ... Ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methionine N-hydroxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenyl Boronic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte) , 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0248] Suitable protected carboxylic acids include, but are not limited to, silyl-, alkyl-, alkenyl-, aryl-, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, and tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl. Examples of suitable arylalkyl groups include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.

[0249] Suitable hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl ( MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl] -4-Methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxymethyl, 1-(2-chloroethoxy)methyl, 1-methyl-1-methoxymethyl, 1-methyl-1-benzyloxymethyl, 1-methyl-1-benzyloxy-2-fluoromethyl, 2,2,2-trimethyl- methyl, 2-trimethylsilylmethyl, 2-(phenylselenyl)methyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl )methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), trimethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), dimethylisopropylsilyl isopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoyl formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxy oxyacetate, p-chlorophenoxyacetate, 3-phenyl propionate, 4-oxovalerate (levulinate), 4,4-(methylenedithio)valerate (levulinoyl dithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenyl benzoate, 2,4,6-trimethyl benzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl methyl carbonate, 2,2,2-Trichloromethyl alkyl carbonate (Troc), 2-(trimethylsilyl)methyl carbonate (TMSEC), 2-(phenylsulfonyl)methyl carbonate (Psec), 2-(triphenylphosphonio)methyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoic acid, 4-azidobutyrate, 4-nitro-4-methyl valerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)methyl, 4- (methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butanoate, o-(methoxycarbonyl)benzoate, α-naphthoic acid, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzyl sulfonate, and tosylate (Ts). For the protection of 1,2- or 1,3-diols, protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,Examples of suitable boronic acid derivatives include 4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronate esters, ethyl borate, and phenyl borate.

[0250] In some embodiments, the hydroxyl protecting group is acetyl, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl, p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl, triphenylmethyl(trityl), 4,4′-dimethoxytrityl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, 9-fluorenylmethyl carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl (MMTr), 4,4′-dimethoxytrityl, (D MTr) and 4,4',4''-trimethoxytrityl (TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE), 2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl 2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl, 3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl, 4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl, butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl, diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In some embodiments, each of the hydroxyl protecting groups is independently selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, and 4,4'-dimethoxytrityl. In some embodiments, the hydroxyl protecting groups are selected from the group consisting of trityl, monomethoxytrityl, and 4,4'-dimethoxytrityl groups.

[0251] In some embodiments, the phosphite linkage protecting group is a group that is added to a phosphite linkage (e.g., an internucleotide linkage) throughout oligonucleotide synthesis. In some embodiments, the protecting group is added to the sulfur atom of a phosphorothioate group. In some embodiments, the protecting group is added to the oxygen atom of an internucleotide phosphorothioate linkage. In some embodiments, the protecting group is added to the oxygen atom of an internucleotide phosphate linkage. In some embodiments, the protecting group is 2-cyanoethyl (CE or Cne), 2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl, benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe), 2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl, 4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl, 4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,N-methyl)aminoethyl, or 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.

[0252] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). In some embodiments, a protein contains only naturally occurring amino acids. In some embodiments, a protein contains one or more non-naturally occurring amino acids (e.g., a moiety that forms one or more peptide bonds with adjacent amino acids). In some embodiments, one or more residues in a protein chain contain a non-amino acid moiety (e.g., a glycan, etc.). In some embodiments, a protein contains two or more polypeptide chains linked, for example, by one or more disulfide bonds or associated by other means. In some embodiments, a protein contains l-amino acids, d-amino acids, or both. In some embodiments, a protein contains one or more amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0253] RNA interference: As used herein, the term "RNA interference" or "RNAi" refers to the post-transcriptional target gene silencing process involving RISC (RNA-induced silencing complex). The process of RNAi reportedly occurs naturally when longer dsRNA is cleaved by ribonuclease III (Dicer) and converted into shorter fragments called siRNAs. Naturally occurring siRNAs (small interfering RNAs) are typically about 21-23 nucleotides in length, have a duplex of about 19 base pairs and two single-stranded overhangs, and are typically RNA. These RNA segments then reportedly induce the degradation of target nucleic acids (such as mRNA or pre-mRNA). Dicer is also reportedly involved in excising 21- and 22-nucleotide small RNAs (stRNAs) from precursor RNAs with conserved structures involved in translational control. (Hutvagner et al. 2001, Science, 293, 834). Those skilled in the art know that RNAi can be mediated by single-stranded or double-stranded oligonucleotides comprising a sequence complementary or substantially complementary to a target sequence (e.g., in a target mRNA). Thus, in some embodiments of the present disclosure, the single-stranded oligonucleotides described herein can act as RNAi agents, and in some embodiments, the double-stranded oligonucleotides described herein can act as RNAi agents. In some embodiments, the RNAi response involves an endonuclease complex commonly referred to as an RNA-induced silencing complex (RISC), where RISC induces cleavage of single-stranded mRNA complementary to the antisense strand of siRNA. In some embodiments, RISC induces cleavage of target RNA complementary to the provided oligonucleotide that can function as a single-stranded RNAi agent. In some embodiments, cleavage of target RNA occurs in the middle of the region complementary to the antisense strand of an siRNA duplex RNAi agent or an siRNA single-stranded RNAi agent. In some embodiments, RNA interference is induced by a single-stranded oligonucleotide that acts as a single-stranded RNAi agent, which can induce RNA interference through a mechanism involving the RISC pathway.

[0254] RNAi agent: As used herein, the terms "RNAi agent," "iRNA agent," and the like refer to an oligonucleotide that, when administered to a system in which a target gene product (e.g., a transcript of a target gene, such as a pre-mRNA (pre-mRNA) or mRNA) is expressing, reduces the level and / or activity (e.g., translation) of that target gene product. In some embodiments, an RNAi agent may be or comprise a single-stranded or double-stranded oligonucleotide. In some embodiments, an RNAi agent may have a structure recognized in the art as an siRNA (short inhibitory RNA), shRNA (short or small hairpin RNA), dsRNA (double-stranded RNA), microRNA, or the like. In some embodiments, an RNAi agent may specifically bind to an RNA target (e.g., a transcript of a target gene). In some embodiments, an RNAi agent, upon binding to its target, is incorporated into RISC (RNA-induced silencing complex). In some embodiments, an RNAi agent induces degradation of its target and / or inhibits translation of its target, which induction and inhibition, in some embodiments, occurs via a mechanism involving the RISC (RNA-induced silencing complex) pathway. In some embodiments, an RNAi agent is an oligonucleotide that activates the RISC complex / pathway. In some embodiments, an RNAi agent comprises an antisense strand sequence. In some embodiments, an RNAi agent comprises only one oligonucleotide strand (e.g., a single-stranded oligonucleotide). In some embodiments, a single-stranded RNAi agent oligonucleotide can be or comprise a sense or antisense strand sequence, as described by Sioud 2005 J. Mol. Biol. 348:1079-1090. In some embodiments, an RNAi agent is a compound capable of inducing RNA interference. In some embodiments, an RNAi agent can have a structure or format found in a "canonical" siRNA structure. In some embodiments, an RNAi agent can have a structure that differs from a "canonical" siRNA structure.To name just a few examples, in some embodiments, an RNAi agent may be longer or shorter than its canonical counterpart, may have blunt ends, and / or may include one or more modifications, mismatches, gaps, and / or nucleotide substitutions. In some embodiments, an RNAi agent comprises a 3'-end cap as described herein. While not wishing to be bound by any particular theory, Applicant proposes that in some embodiments, a 3'-end cap may enable both of the following two functions: (1) achieving RNA interference, and (2) increasing the duration of activity and / or biological half-life (which may be achieved, for example, by increasing binding to the PAZ domain of Dicer and / or the PAZ domain of one or more Ago proteins, and / or reducing or preventing degradation of the RNAi agent (e.g., by nucleases, such as those in serum or intestinal fluids)). In some embodiments, an RNAi agent of the present disclosure targets (e.g., binds to, anneals to, etc.) a target mRNA. In some embodiments, exposure of an RNAi agent to its target reduces the activity, level, and / or expression of the target, e.g., "knocks down" or "knocks out." Specifically, in some embodiments, when a disease, disorder, and / or condition is characterized by overexpression and / or increased activity of a target gene, administering the RNAi agent to a cell, tissue, or subject knocks down the target gene sufficiently to restore activity to normal levels or to reduce activity to a level that is capable of alleviating, ameliorating, mitigating, suppressing, preventing, delaying the onset of, reducing the severity of, and / or reducing the incidence of, one or more symptoms or features of the disease, disorder, and / or disease state, disorder state, and / or condition. In some embodiments, the RNAi agent is double-stranded, comprising an antisense strand, which is a single-stranded RNAi agent described herein, in combination with a sense strand, wherein the antisense strand is capable of inducing RNA interference in combination with the sense strand.

[0255] Sample: As used herein, a "sample" refers to a particular organism or a substance obtained therefrom. In some embodiments, the sample is a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest includes an organism, such as an animal or a human. In some embodiments, the biological sample includes biological tissue or bodily fluid. In some embodiments, the biological sample is or includes bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing bodily fluid; suspended nucleic acid; sputum; saliva; urine; cerebrospinal fluid, ascites; pleural effusion; feces; lymphatic fluid; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washings or lavage fluids, such as ductal lavage or bronchoalveolar lavage; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other bodily fluids, secretions, and / or excretions; and / or cells derived therefrom. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like. In some embodiments, as is clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components thereof and / or adding one or more agents thereto), such as by filtration using a semipermeable membrane. Such a "processed sample" may contain, for example, nucleic acids or proteins extracted from the sample, or nucleic acids or proteins obtained by subjecting the primary sample to techniques such as mRNA amplification or reverse transcription, isolation and / or purification of certain components, etc. In some embodiments, the sample is an organism. In some embodiments, the sample is a plant. In some embodiments, the sample is an animal. In some embodiments, the sample is a human. In some embodiments, the sample is a non-human organism.

[0256] Single-stranded RNA interference: As used herein, " single-stranded RNAi " or " single-stranded RNA interference " or the like refers to the process or method of gene silencing that is at least partially induced by administering single-stranded RNAi agent to the system (for example, cell, tissue, organ, subject, etc.) that RNAi should be induced by this agent, and that requires RISC pathway.These terms can be used in certain cases herein to distinguish from " double-stranded RNAi " or " double-stranded RNA interference ", in which double-stranded RNAi agent is administered to system, and can be further processed, for example, one of its two strands is incorporated into RISC, resulting in, for example, translational repression, target RNA cleavage, etc.

[0257] Single-stranded RNAi agent: As used herein, " single-stranded RNAi agent " refers to the single-stranded oligonucleotide that can induce single-stranded RNA interference (RNAi or iRNA) or gene silencing through RISC pathway.Single-stranded RNAi agent can comprise one or more single-stranded nucleotide polymers.

[0258] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0259] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or nearly all extent or degree of a characteristic or property of interest. A base sequence that is substantially complementary to a second sequence is not identical to the second sequence, but is mostly or nearly identical to the second sequence. Furthermore, those skilled in the art of biology will understand that biological and chemical phenomena rarely go to completion and / or proceed to completion, or achieve or avoid absolute consequences. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0260] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0261] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition is an individual who is at a higher risk than individuals in the general population of developing the disease, disorder, and / or condition. 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.

[0262] Systemic: As used herein, the terms "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" have their art-recognized meaning of referring to administering a compound or composition so that it enters the recipient's entire body.

[0263] Tautomers: As used herein, the term "tautomers" is used to describe different isomeric forms of an organic compound that are readily interconvertible. Tautomers may be characterized by the formal migration of a hydrogen atom or a proton accompanied by the interchange of a single bond and an adjacent double bond. In some embodiments, tautomers may result from prototropic tautomerism (i.e., relocation of a proton). In some embodiments, tautomers may result from valence tautomerism (i.e., rapid reorganization of bond electrons). All such tautomers are intended to be included within the scope of this disclosure. In some embodiments, tautomers of a compound exist in mobile equilibrium with each other, resulting in the formation of mixtures when attempting to prepare separate substances. In some embodiments, tautomers of a compound are separable and isolatable compounds. In some embodiments of the present disclosure, chemical compositions may be provided that are or include pure preparations of a single tautomer of a compound. In some embodiments of the present disclosure, chemical compositions may be provided as mixtures of two or more tautomers of a compound. In certain embodiments, such mixtures contain equal amounts of different tautomers. In certain embodiments, such mixtures contain different amounts of at least two tautomers of a compound. In some embodiments of the present disclosure, a chemical composition may contain all of the tautomers of a compound. In some embodiments of the present disclosure, a chemical composition may not contain all of the tautomers of a compound. In some embodiments of the present disclosure, a chemical composition may contain one or more tautomers of a compound in amounts that vary over time as a result of interconversion. In some embodiments of the present disclosure, the tautomer is keto-enol tautomerism. Those skilled in the chemical arts will recognize that keto-enol tautomers can be "captured" (i.e., chemically modified to retain the "enol" form) using any suitable reagent known in the chemical arts to generate the enol derivative, which can then be isolated using one or more suitable techniques known in the art. Unless otherwise specified, the present disclosure encompasses all tautomers of the relevant compounds, whether in pure form or in mixture with each other.

[0264] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0265] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that, when administered as part of a treatment regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance being delivered, and the target cell or tissue. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose. In some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0266] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to subjects who show only early signs of the disease, disorder, and / or condition, for example, to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0267] Unsaturated: As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0268] Unit dose: As used herein, the phrase "unit dose" refers to an amount administered as a single administration of a pharmaceutical composition and / or administered in a physically discrete unit. In many embodiments, a unit dose contains a predetermined amount of an active agent. In some embodiments, a unit dose contains an entire single dose of an agent. In some embodiments, two or more unit doses are administered to achieve the entire single dose. In some embodiments, administration of multiple unit doses is required or anticipated to be required to achieve the intended effect. A unit dose may be, for example, a predetermined amount of one or more therapeutic agents, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more solid therapeutic agents, or a sustained-release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents. It will be appreciated that a unit dose may be present in a formulation containing any of a variety of ingredients in addition to a therapeutic agent. For example, acceptable carriers (e.g., pharmaceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc. may be included, as described below. It will be understood by those skilled in the art that in many embodiments, an appropriate total daily dosage of a particular therapeutic agent may comprise a fraction or multiple of a unit dose, e.g., as may be determined by an attending physician within the scope of sound medical judgment. In some embodiments, the individual effective dosage level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the individual active compound employed, the individual composition employed, the age, weight, general health, sex, and diet of the subject, the time of administration and the rate of excretion of the individual active compound employed, the duration of treatment, drugs and / or therapies used in combination with or concurrently with the individual compound employed, and similar factors known in the medical arts.

[0269] Wild-type: As used herein, the term "wild-type" has its art-recognized meaning of referring to an entity having a structure and / or activity that occurs in nature in a "normal" state or situation (as opposed to mutant, diseased, altered, etc.). Those of skill in the art will recognize that wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0270] Nucleic Acid: As used herein, the term "nucleic acid" includes any nucleotide and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, by equivalents, analogs of either 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. The term encompasses polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from nucleobases and / or N- or C-glycosides of modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotide linkages. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified internucleotide linkages. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix poly- refers to a nucleic acid containing from 2 to about 10,000 nucleotide monomer units, and the prefix oligo- refers to a nucleic acid containing from 2 to about 200 nucleotide monomer units.

[0271] 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. Natural bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, although natural and unnatural base analogs are also understood to be included. Natural sugars are the pentoses (five-carbon sugars) deoxyribose (forming DNA) or ribose (forming RNA), although natural and unnatural sugar analogs are also understood to be included. Nucleotides are linked via internucleotide linkages to form nucleic acids, or polynucleotides. Many internucleotide linkages are known in the art, including, but not limited to, phosphate, phosphorothioate, and boranophosphate. Artificial nucleic acids include PNA (peptide nucleic acid), phosphotriesters, phosphorothioates, H-phosphonates, phosphoramidates, boranophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, natural nucleotides contain naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also encompasses structural analogs (such as modified nucleotides and nucleotide analogs) that are used in place of natural or naturally occurring nucleotides.

[0272] Modified Nucleotide: The term "modified nucleotide" includes any chemical moiety that is structurally different from a naturally occurring nucleotide but that is capable of performing at least one function of a naturally occurring nucleotide. In some embodiments, a modified nucleotide comprises a modification to the sugar, base, and / or internucleotide linkage. In some embodiments, a modified nucleotide comprises a modified sugar, a modified nucleobase, and / or a modified internucleotide linkage. In some embodiments, a modified nucleotide is capable of performing at least one function of a nucleotide, such as forming a subunit of a polymer that is capable of base pairing with a nucleic acid that comprises at least a complementary base sequence.

[0273] Analog: The term "analog" includes any chemical moiety that is structurally different from a reference chemical moiety or a reference class of moieties, but has the ability to perform at least one function of such reference chemical moiety or a reference class of moieties. Non-limiting examples include nucleotide analogs that are structurally different from nucleotides but perform at least one function of nucleotides, and nucleobase analogs that are structurally different from nucleobases but perform at least one function of nucleobases, etc.

[0274] Nucleoside: The term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently linked to a sugar or modified sugar.

[0275] Modified nucleoside: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a naturally occurring nucleoside, but containing a chemical modification that renders the moiety distinct from a naturally occurring nucleoside. Non-limiting examples of modified nucleosides include those containing modifications to the base and / or sugar. Non-limiting examples of modified nucleosides include those with 2' modifications to the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (those lacking a nucleobase). In some embodiments, the modified nucleoside is capable of performing at least one function of a nucleoside, such as forming a portion of a polymer capable of base pairing with a nucleic acid containing at least a complementary base sequence.

[0276] Nucleoside Analog: The term "nucleoside analog" refers to a chemical moiety that is chemically different from a naturally occurring nucleoside but is capable of performing at least one function of a nucleoside. In some embodiments, nucleoside analogs include sugar analogs and / or nucleobase analogs. In some embodiments, modified nucleosides are capable of performing at least one function of a nucleoside, such as forming a portion of a polymer that is capable of base pairing with a nucleic acid containing a complementary base sequence.

[0277] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in a closed and / or open state. In some embodiments, a sugar is a monosaccharide. In some embodiments, a sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentofuranose, pentopyranose, and hexopyranose moieties. As used herein, the term "sugar" also encompasses structural analogs, such as glycols, that are used in place of traditional sugar molecules, polymers of which form the backbone, such as the nucleic acid analog glycol nucleic acid (GNA). As used herein, the term "sugar" also encompasses structural analogs, such as modified sugars and nucleotide sugars, that are used in place of natural or naturally occurring nucleotides.

[0278] Modified sugar: The term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic state, or some other physicochemical property of the sugar.

[0279] Nucleobase: The term "nucleobase" refers to the portion of a nucleic acid that participates in hydrogen bonding to connect 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, a naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, a nucleobase is a "modified nucleobase," e.g., a nucleobase other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, a modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, modified nucleobase mimics mimic the spatial arrangement, electronic state, or some other physicochemical properties of nucleobases, and retain the hydrogen bonding properties that bind one nucleic acid strand to another in a sequence-specific manner. In some embodiments, modified nucleobases can pair with all five natural bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of oligonucleotide duplexes. As used herein, the term "nucleobase" also encompasses structural analogs (such as modified nucleobases and nucleobase analogs) that are used in place of natural or naturally occurring nucleotides.

[0280] Modified nucleobase: The terms "modified nucleobase," "modified base," and the like 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 that includes a modification. In some embodiments, a modified nucleobase is capable of performing at least one function of a nucleobase, such as forming a portion of a polymer that is capable of base pairing with a nucleic acid that includes at least a complementary base sequence.

[0281] 3'-end cap: The term "3'-end cap" refers to a non-nucleotide chemical moiety attached to the 3'-end of an oligonucleotide (e.g., an RNAi agent). In some embodiments, a 3'-terminal dinucleotide can be replaced by a 3'-end cap. In some embodiments, the 3'-end cap of an oligonucleotide performs at least one of the following functions: achieving RNA interference induced by the oligonucleotide, protecting the oligonucleotide from or reducing the amount or rate of degradation of the oligonucleotide (e.g., by nucleases), reducing off-target effects of the sense strand, or enhancing the activity, duration, or efficiency of RNA interference induced by the oligonucleotide. When a 3'-end cap is described as "non-nucleotide," it is intended that the 3'-end cap is linked to the remaining sugar moiety of the oligonucleotide rather than to a nucleotide or oligonucleotide moiety, and that this linkage occurs as if the 3'-end cap were part of the oligonucleotide chain. Certain examples of 3'-end caps are described herein. Those skilled in the art will understand that other 3'-end caps known in the art can be utilized in accordance with the present disclosure.

[0282] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group may then be unmasked by removal of the blocking group. In some embodiments, the blocking group is a protecting group.

[0283] Moiety: The term "moiety" refers to a specific segment of a molecule's functionality. A chemical moiety is a recognized chemical entity incorporated into or attached to a molecule.

[0284] Solid Support: The term "solid support" refers to any support that allows for nucleic acid synthesis. In some embodiments, the term refers to glass or a polymer that is insoluble in the medium utilized in the reaction steps performed to synthesize nucleic acids and that has been derivatized to contain reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or controlled pore glass (CPG). In some embodiments, the solid support is controlled pore glass (CPG). In some embodiments, the solid support is a hybrid support of controlled pore glass (CPG) and highly cross-linked polystyrene (HCP).

[0285] Linker or Linking Moiety: The terms "linker," "linking moiety," and the like refer to any chemical moiety that links one chemical moiety to another. In some embodiments, a linker is a moiety that links one oligonucleotide to another in a multimer. In some embodiments, a linker is a moiety that is optionally positioned between the terminal nucleoside and the solid support, or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0286] Gene: As used herein, the terms "gene," "recombinant gene," and "gene construct" refer to a DNA molecule or portion of a DNA molecule that encodes a protein or portion thereof. In addition to including an open reading frame encoding a protein (as an exon sequence), a DNA molecule may further contain intron sequences. As used herein, the term "intron" refers to a DNA sequence present in a given gene that is not translated into protein and that is sometimes, but not always, found between exons. It may be desirable to regulate the activity or expression of a gene by operably linking it to (or by including) one or more promoters, enhancers, repressors, and / or other regulatory sequences, as is well known in the art.

[0287] Complementary DNA: As used herein, "complementary DNA" or "cDNA" includes recombinant polynucleotides synthesized by reverse transcription of mRNA, wherein intervening sequences (introns) have been removed.

[0288] Homology: "Homology" or "identity" or "similarity" refers to the sequence similarity between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence that can be positioned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base, the molecules are identical at that position. When an equivalent site is occupied by an identical or similar (e.g., similar in steric and / or electronic properties) nucleic acid residue, the molecules can be called homologous (similar) at that position. Expression as a percentage of homology / similarity or identity refers to a function of the number of identical or similar nucleic acids at a position shared by the compared sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 35% identity, less than 30% identity, or less than 25% identity with the sequences described herein. In comparing two sequences, the absence of a residue (amino acid or nucleic acid) or the presence of an extra residue also reduces identity and homology / similarity.

[0289] In some embodiments, the term "homology" describes a mathematically based comparison of sequence similarities and is used to identify genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" to perform searches against public databases to identify, for example, other family members, related sequences, or homologs. In some embodiments, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. In some embodiments, BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the present disclosure. In some embodiments, to obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

[0290] Identity: As used herein, "identity" refers to the percentage of identical nucleotide residues at corresponding positions in two or more sequences, where the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs.Computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387(1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410(1990) and Altschul et al. Nuc. Acids Res. 25:3389-3402(1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)). The well-known Smith Waterman algorithm may also be used to determine identity.

[0291] Oligonucleotide: The term "oligonucleotide" refers to a polymer or oligomer of nucleotides, which may contain any combination of natural and unnatural nucleobases, sugars, and internucleotide linkages.

[0292] An oligonucleotide may be single-stranded or double-stranded. As used herein, the term "oligonucleotide strand" encompasses single-stranded oligonucleotides. A single-stranded oligonucleotide may have a double-stranded region (formed by two portions of the single-stranded oligonucleotide), and a double-stranded oligonucleotide (comprising two oligonucleotide strands) may have a single-stranded region, e.g., in a region where the two oligonucleotide strands are not complementary to each other. In some embodiments, the oligonucleotide is capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, the oligonucleotide is capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference or a biochemical mechanism that does not involve RISC (including, but not limited to, RNase H-mediated knockdown of gene expression or steric hindrance). In some embodiments, the oligonucleotide is capable of inducing a decrease in the expression and / or level of a target gene or its gene product through RNA interference and / or RNase H-mediated knockdown. Examples of oligonucleotides include, but are not limited to, structural genes, genes including regulatory and termination regions, self-replicating systems such as viral DNA or plasmid DNA, single-stranded and double-stranded RNAi agents and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNA, microRNA mimics, supermicroRNAs, aptamers, antimicroRNAs, antagomirs, Ul adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0293] Double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as RNAi agents or iRNA agents. In some embodiments, these RNA interference-inducing oligonucleotides associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). In many embodiments, the double-stranded RNAi agent is long enough so that it can be cleaved by endogenous molecules, such as Dicer, to generate smaller oligonucleotides that can enter the RISC mechanism and participate in RISC-mediated cleavage and / or translational repression of target sequences, such as target mRNA sequences.

[0294] Oligonucleotides of the present disclosure may be of various lengths. In certain embodiments, oligonucleotides may range from about 2 to about 200 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides may range in length from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides. In some embodiments, oligonucleotides are about 10 to about 40 nucleotides in length. In some embodiments, oligonucleotides are about 9 to about 39 nucleotides in length. In some embodiments, oligonucleotides are at least 4 nucleotides in length. In some embodiments, oligonucleotides are at least 5 nucleotides in length. In some embodiments, oligonucleotides are at least 6 nucleotides in length. In some embodiments, oligonucleotides are at least 7 nucleotides in length. In some embodiments, oligonucleotides are at least 8 nucleotides in length. In some embodiments, oligonucleotides are at least 9 nucleotides in length. In some embodiments, oligonucleotides are at least 10 nucleotides in length. In some embodiments, oligonucleotides are at least 11 nucleotides in length. In some embodiments, oligonucleotides are at least 12 nucleotides in length. In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25 nucleotides in length. In some embodiments, the oligonucleotide is at least 30 nucleotides in length. In some embodiments, the oligonucleotide is a complementary duplex at least 18 nucleotides in length. In some embodiments, the oligonucleotide is a complementary duplex at least 21 nucleotides in length. In some embodiments, each nucleotide counted in length independently comprises an optionally substituted nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil.

[0295] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to the bond that links the nucleoside units of an oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkage is a phosphodiester bond (a natural phosphate bond) 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 bond is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties include, but are not limited to, =S, =Se, =NR', -SR', -SeR', -N(R'), B(R'). 3、 In some embodiments, the internucleotide linkage is selected from -S-, -Se-, and -N(R')-, where each R' is as defined and described in the present disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a nucleotide linkage. [ka] or a modified phosphorothioate triester bond.

[0296] In some embodiments, the internucleotide linkage is, for example, one of a PNA (peptide nucleic acid) linkage or a PMO (phosphorodiamidate morpholino oligomer) linkage.

[0297] Those skilled in the art will appreciate that internucleotide linkages can exist as anions or cations at a given pH, depending on the presence of acidic or basic moieties in the linkage.

[0298] Unless otherwise specified, when used in conjunction with an oligonucleotide sequence, s, s1, s2, s3, s4, s5, s6, and s7 each independently represent the following modified internucleotide linkages as depicted below: [Table 1] TIFF2026004488000009.tif220165TIFF2026004488000010.tif179165

[0299] For example, (Rp, Sp)-ATsCs1GA has 1) a phosphorothioate internucleotide bond between T and C; [ka] and 2) between C and G [ka] Unless otherwise specified, the Rp / Sp designation preceding an oligonucleotide sequence describes the configuration of the chiral phosphorus atoms in the internucleotide linkages of the oligonucleotide sequence in order from 5' to 3'. For example, in (Rp, Sp)-ATsCs1GA, the phosphorus in the "s" bond between T and C has the Rp configuration, and the "s1" bond between C and G has the Sp configuration.

[0300] In some embodiments, "All-(Rp)" or "All-(Sp)" is used to indicate that all chiral linking phosphorus atoms of an oligonucleotide have the same Rp or Sp configuration, respectively.

[0301] Oligonucleotide type: As used herein, the term "oligonucleotide type" refers to an oligonucleotide having a particular base sequence, backbone linkage pattern (i.e., the pattern of internucleotide linkage types, e.g., phosphate, phosphorothioate, etc.), backbone chiral center pattern (i.e., the pattern of bond phosphorus stereochemistry (Rp / Sp)), and backbone phosphorus modification pattern (e.g., "-XLR" in Formula I). 1 "Type" is used to define oligonucleotides having a pattern of "groups." In some embodiments, oligonucleotides of a commonly designated "type" are structurally identical to each other.

[0302] Those skilled in the art will recognize that the synthetic methods of the present disclosure provide a degree of control during oligonucleotide chain synthesis, whereby each nucleotide unit of the oligonucleotide chain can be pre-designed and / or pre-selected to have a specific stereochemistry at the binding phosphorus and / or a specific modification at the binding phosphorus, and / or a specific base and / or a specific sugar. In some embodiments, the oligonucleotide chain is pre-designed and / or pre-selected to have a specific combination of stereocenters at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or pre-determined to have a specific combination of modifications at the binding phosphorus. In some embodiments, the oligonucleotide chain is designed and / or pre-selected to have a specific combination of bases. In some embodiments, the oligonucleotide chain is designed and / or pre-selected to have a specific combination of one or more of the structural features described above. In some embodiments, the present disclosure provides compositions comprising or consisting of a plurality of oligonucleotide molecules (e.g., chiral controlled oligonucleotide compositions). In some embodiments, all of the molecules are the same type of molecule (i.e., all such molecules are structurally identical to one another). However, in many embodiments, the provided compositions comprise a plurality of oligonucleotides of different types, typically in predetermined relative amounts.

[0303] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical assignment of the chiral linkage phosphorus in a chiral internucleotide linkage within an oligonucleotide. In some embodiments, the control is achieved through chiral elements that are absent from the sugar and base moieties of the oligonucleotide. For example, in some embodiments, the control is achieved using one or more chiral auxiliary groups during oligonucleotide preparation, as exemplified in the present disclosure; these chiral auxiliary groups are often part of the chiral phosphoramidites used during oligonucleotide preparation. In contrast to chiral control, those skilled in the art will recognize that conventional oligonucleotide synthesis without the use of chiral auxiliary groups does not provide stereochemical control at the chiral internucleotide linkage when the chiral internucleotide linkage is formed using such conventional oligonucleotide synthesis. In some embodiments, the stereochemical assignment of each chiral linkage phosphorus in a chiral internucleotide linkage within an oligonucleotide is controlled.

[0304] Chirality-controlled oligonucleotide composition: As used herein, the terms "chirality-controlled oligonucleotide composition," "chirality-controlled nucleic acid composition," and the like refer to a composition comprising multiple oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common pattern of backbone linkages, and 3) a common pattern of backbone phosphorus modifications, wherein the multiple oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chirality-controlled internucleotide linkages), and the level of the multiple oligonucleotides (or nucleic acids) in the composition is predetermined (e.g., via chirality-controlled oligonucleotide preparation to form one or more chiral internucleotide linkages). In some embodiments, about 1% to 100% (e.g., about 5% to 100%, about 10% to 100%, about 20% to 100%, about 30% to 100%, about 40% to 100%, about 50% to 100%, about 60% to 100%, about 70% to 100%, about 80 to 100%, about 90 to 100%, about 95 to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, About 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said plurality of oligonucleotides.In some embodiments, about 1% to 100% (e.g., about 5% to 100%, about 10% to 100%, about 20% to 100%, about 30% to 100%, about 40% to 100%, about 50% to 100%, about 60% to 100%, about 70% to 100%, about 80 to 100%, about 90 to 100%, about 95 to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 91%) of all oligonucleotides sharing a common base sequence in a chiral controlled oligonucleotide composition. , about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said plurality of oligonucleotides.In some embodiments, about 1% to 100% (e.g., about 5% to 100%, about 10% to 100%, about 20% to 100%, about 30% to 100%, about 40% to 100%, about 50% to 100%, about 60% to 100%, about 70% to 100%, about 80% to 100%, about 90% to 100%, about 95% to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are said plurality of oligonucleotides.In some embodiments, the predetermined level is between about 1% and 100% (e.g., between about 5% and 100%, between about 10% and 100%, between about 20% and 100%, between about 30% and 100%, between about 40% and 100%, between about 50% and 100%, between about 60% and 65% of all oligonucleotides in the composition, or all oligonucleotides in the composition that share a common base sequence (e.g., of a plurality of oligonucleotides or of a single oligonucleotide type), or all oligonucleotides in the composition that share a common base sequence, a common pattern of backbone linkages, and a common pattern of backbone phosphorus modifications, or all oligonucleotides in the composition that share a common base sequence, a common pattern of base modifications, a common pattern of sugar modifications, a common pattern of internucleotide linkage types, and / or a common pattern of internucleotide linkage modifications). % to 100%, about 70% to 100%, about 80 to 100%, about 90 to 100%, about 95 to 100%, about 50% to 90%, or about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or at least 5%, at least 10%, or at least At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the plurality of oligonucleotides.In some embodiments, the plurality of oligonucleotides is 1 to 50 (e.g., about 1 to 10, about 1 to 20, about 5 to 10, about 5 to 20, about 10 to 15, about 10 to 20, about 10 to 25, about 10 to 30, about or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20), or at least and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides comprises about 1% to 100% (e.g., about 5% to 100%, about 10% to 100%, about 20% to 100%, about 30% to 100%, about 40% to 100%, about 50% to 100%, about 60% to 100%, about 70% to 100%, about 80% to 100%, about 90% to 100%, about 95% to 100%, about 50% to 90%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, or at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% share the same stereochemistry. In some embodiments, each chiral internucleotide linkage is a chiral controlled internucleotide linkage, and the composition is a completely chiral controlled oligonucleotide composition.In some embodiments, less than all chiral internucleotide linkages are chiral-controlled internucleotide linkages, and the composition is a partially chiral-controlled oligonucleotide composition. In some embodiments, the chiral-controlled oligonucleotide composition comprises a predetermined level of an individual oligonucleotide type or nucleic acid type. For example, in some embodiments, the chiral-controlled oligonucleotide composition comprises one oligonucleotide type. In some embodiments, the chiral-controlled oligonucleotide composition comprises two or more oligonucleotide types. In some embodiments, the chiral-controlled oligonucleotide composition comprises multiple oligonucleotide types. In some embodiments, the chiral-controlled oligonucleotide composition is a composition of oligonucleotides of one oligonucleotide type, and the composition comprises a predetermined level of multiple oligonucleotides of that oligonucleotide type.

[0305] Chirally homogeneous: As used herein, the term "chirally homogeneous" is used to describe an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the bond phosphorus. For example, an oligonucleotide whose nucleotide units all have Rp stereochemistry at the bond phosphorus is chirally homogeneous. Similarly, an oligonucleotide whose nucleotide units all have Sp stereochemistry at the bond phosphorus is chirally homogeneous.

[0306] Predetermined: Predetermined (or predetermined) means intentionally selected, as opposed to, for example, randomly occurring or realized without control. Those skilled in the art will understand, upon reading this specification, that the present disclosure provides techniques that allow for the selection of specific chemistry and / or stereochemistry incorporated into oligonucleotide compositions and further provide techniques that allow for the controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical properties. Such provided compositions are "predetermined" as described herein. A composition that may contain a certain oligonucleotide is one that happens to be produced through a process that is not controlled to intentionally produce a specific chemistry and / or stereochemistry; it is not a "predetermined" composition. In some embodiments, a predetermined composition is one that can be intentionally reproduced (e.g., through repetition of a controlled process). In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition means that the absolute and / or relative amounts (ratios, proportions, etc.) of the plurality of oligonucleotides in the composition are controlled. In some embodiments, a predetermined level of a plurality of oligonucleotides in a composition is achieved through chiral-controlled oligonucleotide preparation.

[0307] Bound phosphorus: As defined herein, the phrase "bound phosphorus" is used to indicate that the particular phosphorus atom being referred to is a phosphorus atom present in an internucleotide linkage, which phosphorus atom corresponds to the phosphodiester phosphate atom of the internucleotide linkage present in natural DNA and RNA. In some embodiments, the bound phosphorus atom is in a modified internucleotide linkage, wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, the bound phosphorus atom is represented by P of Formula I L In some embodiments, the bound phosphorus atom is chiral.

[0308] P-modification: As used herein, the term "P-modification" refers to any modification at the bound phosphorus other than a stereochemical modification. In some embodiments, a P-modification includes the addition, substitution, or removal of a pendant moiety covalently attached to the bound phosphorus. In some embodiments, a "P-modification" refers to a modification of -XLR 1 wherein X, L and R 1 are each independently as defined and described in this disclosure.

[0309] Blockmir: As used herein, the term "blockmir" refers to an oligonucleotide chain in which the pattern of structural features characterizing each individual nucleotide unit thereof is characterized by the presence of at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus bond. A common structural feature refers to a common stereochemistry at the bond phosphorus or a common modification at the bond phosphorus. In some embodiments, at least two consecutive nucleotide units that share a common structural feature at the internucleotide phosphorus bond are referred to as a "block." In some embodiments, provided oligonucleotides are blockmirs.

[0310] In some embodiments, the blockmir is a "stereoblockmir," e.g., at least two consecutive nucleotide units have the same stereochemistry at the bound phosphorus. Such at least two consecutive nucleotide units form a "stereoblock."

[0311] In some embodiments, a blockmir is a "P-modified blockmir," e.g., at least two consecutive nucleotide units have the same modification at the linked phosphorus. Such at least two consecutive nucleotide units form a "P-modified block." For example, (Rp, Sp)-ATsCsGA is a P-modified blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same P-modification (i.e., both are phosphorothioate diester). In the same oligonucleotide, (Rp, Sp)-ATsCsGA, TsCs form a block, which is a P-modified block.

[0312] In some embodiments, the blockmir is a "linked blockmir," e.g., at least two consecutive nucleotide units have the same stereochemistry and the same modification at the linking phosphorus. At least two consecutive nucleotide units form a "linking block." For example, (Rp, Rp)-ATsCsGA is a linked blockmir because at least two consecutive nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P-modification (both phosphorothioate). In the same oligonucleotide, (Rp, Rp)-ATsCsGA, TsCs form a block, which is a linking block.

[0313] In some embodiments, the blockmir comprises one or more blocks independently selected from a stereoblock, a P-modified block, and a conjugated block, hi some embodiments, the blockmir is a stereoblockmir with respect to one block, and / or a P-modified blockmir with respect to another block, and / or a conjugated blockmir with respect to yet another block.

[0314] Altmer: As used herein, the term "altmer" refers to an oligonucleotide in which the pattern of structural features that characterize each individual nucleotide unit thereof is characterized in that no two consecutive nucleotide units of the oligonucleotide chain share a particular structural feature at the internucleotide phosphorus bond. In some embodiments, an altmer is designed to contain a repeating pattern. In some embodiments, an altmer is designed to not contain a repeating pattern. In some embodiments, a provided oligonucleotide is an altmer.

[0315] In some embodiments, the altmer is a "stereoaltomer," eg, no two consecutive nucleotide units have the same stereochemistry at the bond phosphorus.

[0316] In some embodiments, the altmer is a "P-modified altmer," e.g., no two consecutive nucleotide units have the same modification at the linked phosphorus, e.g., all-(Sp)-CAs1GsT, in which each linked phosphorus has a different P-modification than the others.

[0317] In some embodiments, the altmer is a "linked altmer," eg, no two consecutive nucleotide units have the same stereochemistry or the same modification at the linking phosphorus.

[0318] Unimer: As used herein, the term "unimer" refers to an oligonucleotide in which the pattern of structural features that characterize each individual nucleotide unit is such that all nucleotide units in the strand share at least one common structural feature at the internucleotide phosphorus linkage. A common structural feature refers to a common stereochemistry at the linking phosphorus or a common modification at the linking phosphorus. In some embodiments, a provided oligonucleotide is a unimer.

[0319] In some embodiments, the unimers are "stereounimers," eg, all nucleotide units have the same stereochemistry at the bond phosphorus.

[0320] In some embodiments, the unimers are "P-modified unimers," eg, all nucleotide units have the same modification at the bound phosphorus.

[0321] In some embodiments, the unimers are "linked unimers," eg, all nucleotide units have the same stereochemistry and the same modification at the linking phosphorus.

[0322] Gapmer: As used herein, the term "gapmer" refers to an oligonucleotide chain characterized in that at least one internucleotide phosphorus bond of the oligonucleotide chain is a phosphodiester bond, such as those found in naturally occurring DNA or RNA. In some embodiments, two or more internucleotide phosphorus bonds of the oligonucleotide chain are phosphodiester bonds, such as those found in naturally occurring DNA or RNA. In some embodiments, the provided oligonucleotide is a gapmer.

[0323] Skipmer: As used herein, the term "skipmer" refers to a gapmer type in which every other internucleotide phosphorus bond of an oligonucleotide strand is a phosphodiester bond, such as those present in natural DNA or RNA, and the other internucleotide phosphorus bonds of the oligonucleotide strand are modified internucleotide bonds. In some embodiments, provided oligonucleotides are skipmers.

[0324] For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, inside cover of Handbook of Chemistry and Physics, 67th Ed., 1986-87.

[0325] The methods and structures described herein for the compounds and compositions of the present disclosure also apply to the pharmaceutically acceptable acid or base addition salts and all stereoisomers of these compounds and compositions. [Brief explanation of the drawings]

[0326] [Figure 1A] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1B] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1C] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1D] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1E] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1F] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1G] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1H] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1I] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1J] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1K] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 1L] Schematic representation of various ssRNAi and hybrid formats is shown. [Figure 2] Schematic representation of various antisense oligonucleotide formats. [Figure 3A] Examples of multimeric formats are shown. The oligonucleotides can be linked directly and / or via a linker. As shown, the multimers can contain oligonucleotide monomers of the same or different structures / types. In some embodiments, the monomers of the multimer are ssRNAi agents. In some embodiments, the monomers of the multimer are RNase H-dependent antisense oligonucleotides (ASOs). The monomers can be linked through various positions, for example, through the 5' end, the 3' end, or positions intervening therebetween. [Figure 3B] Examples of chemical methods for linking monomers to form multimers are provided, and these monomers can exert their function through various pathways. DETAILED DESCRIPTION OF THE INVENTION

[0327] Synthetic oligonucleotides provide useful molecular tools in a variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and novel nanomaterial applications. The applications of naturally occurring nucleic acids (e.g., unmodified DNA or unmodified RNA) are limited, for example, by their susceptibility to endonuclease and exonuclease activity. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks. These synthetic counterparts include synthetic oligonucleotides containing chemical modifications (e.g., base, sugar, backbone, etc.) that, among other things, reduce the susceptibility of these molecules to degradation and improve other properties of the oligonucleotide. From a structural perspective, in addition to the introduction of chirality by modifying the internucleotide phosphate linkages, the arrangement of the phosphorus atoms forming the oligonucleotide backbone can affect certain properties of the oligonucleotide. For example, in vitro studies have shown that the properties of antisense oligonucleotides (e.g., binding affinity, sequence-specific binding to complementary RNA, stability against nucleases, etc.) are affected, among other things, by the chirality of the backbone phosphorus atoms.

[0328] Among other things, the present disclosure encompasses the recognition that structural elements of an oligonucleotide, such as chemical modification (e.g., sugar modification, base modification, and / or internucleotide linkage modification) or pattern thereof, conjugation to lipids or other moieties, and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or pattern thereof), can significantly impact its properties and activity (e.g., stability, specificity, selectivity, activity for reducing levels of target gene products (transcripts and / or proteins)), in some embodiments, oligonucleotide properties can be tailored by optimizing chemical modification (e.g., base modification, sugar modification, and / or internucleotide linkage modification), chemical modification pattern, stereochemistry, and / or stereochemical pattern.

[0329] In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements (e.g., controlled chemical modifications and / or controlled backbone stereochemical patterns) confer unexpected properties and activities (including, but not limited to, those described herein). In some embodiments, provided compositions comprising oligonucleotides with chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.) or patterns thereof have improved properties and activities. Non-limiting examples of such improved properties include inducing a decrease in the expression and / or level of a target gene or its gene product, and / or inducing RNA interference and / or inducing RNase H-mediated knockdown. In some embodiments, the present disclosure provides techniques (e.g., oligonucleotides, compositions, methods, etc.) for single-stranded RNAi. In some embodiments, the provided oligonucleotides are ssRNAi agents.

[0330] In some embodiments, RNA interference is reportedly a post-transcriptional targeted gene silencing technique in which an RNAi agent is used to target RNA (e.g., a gene transcript such as messenger RNA (mRNA)) containing a sequence complementary to the RNAi agent, resulting in cleavage mediated by the RISC (RNA-induced silencing complex) pathway. In nature, a form of RNAi reportedly occurs when long dsRNA (double-stranded RNA) (e.g., exogenous dsRNA introduced into mammalian cells) is cleaved by ribonuclease III (Dicer) and converted into shorter fragments called siRNAs. siRNAs (small interfering RNAs or short inhibitory RNAs) typically have a length of about 21-23 nucleotides and contain a duplex of about 19 base pairs. The smaller RNA segments then reportedly mediate degradation of the target mRNA. The RNAi response reportedly also features an endonuclease complex commonly referred to as the RNA-induced silencing complex (RISC), which induces cleavage of single-stranded mRNA complementary to the antisense strand of the siRNA. Cleavage of the target RNA reportedly occurs in the center of the region complementary to the antisense strand of the siRNA duplex. The use of an RNAi agent against a target transcript reportedly reduces gene activity, levels, and / or expression, e.g., "knocks down" or "knocks out" a target gene or sequence. Artificial siRNAs are useful for both therapeutic and experimental applications.

[0331] In one embodiment, the RNA interference agent comprises a single-stranded RNA that interacts with a target RNA sequence and induces cleavage of the target RNA. Without wishing to be bound by theory, it has been reported that long double-stranded RNA introduced into plant and invertebrate cells is converted into siRNAs through degradation by a type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease III-like enzyme, reportedly processes dsRNA, converting it into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Reportedly, siRNA is then incorporated into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107:309).When one or more endonucleases in RISC bind to appropriate target mRNA, they cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev.15:188).Therefore, in one aspect, the present disclosure relates to a single-stranded RNA that promotes the formation of RISC complex and causes target gene silencing.

[0332] In some embodiments, suitable RNAi agents can be selected by any process known in the art or conceivable to those skilled in the art in accordance with the present disclosure. For example, selection criteria can include one or more of the following steps: initial analysis of the target gene sequence and design of the RNAi agent (which can take into account sequence similarity across species (human, cynomolgus monkey, mouse, etc.) and differences to other (non-target) genes); screening in vitro (e.g., at 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 (in some embodiments, it is desired that the RNAi agent against the target does not inhibit the viability of such cells), testing with human PBMCs (peripheral blood mononuclear cells) (e.g., testing the levels of TNF-alpha to estimate immunogenicity, in which immunostimulatory sequences are generally less desirable), testing in human whole blood assays (in which fresh human blood is treated with the RNAi agent and the levels of cytokines / chemokines [e.g., TNF-alpha (tumor necrosis factor-alpha) and / or MCP1 (monocyte chemotactic protein 1)] are determined, in which immunostimulatory sequences are generally less desirable), determining gene knockdown in vivo using cells or tumors in test animals, and optimizing specific modifications of the RNAi agent.

[0333] The so-called canonical siRNA structure is reportedly a double-stranded RNA molecule, with each strand being approximately 21 nucleotides in length. These two strands are reportedly an antisense (or "guide") strand (which recognizes and binds to a complementary sequence in the target transcript) and a sense (or "passenger") strand (which is complementary to the antisense strand). The sense and antisense strands are reportedly largely complementary and typically form two 3' overhangs of two nucleotides at each end.

[0334] Although canonical siRNA structures are reportedly double-stranded, RNAi agents can also be single-stranded. In some embodiments, a single-stranded RNAi agent corresponds to the antisense strand of a double-stranded siRNA, and such a single-stranded RNAi agent does not include a corresponding passenger strand.

[0335] However, it has been reported that not all structural elements tested for single-stranded RNAi agents are effective, and some structural elements introduced into oligonucleotides can reportedly interfere with single-stranded RNA interference activity.

[0336] In some embodiments, the present disclosure provides oligonucleotides and compositions useful as RNAi agents. In some embodiments, the present disclosure provides oligonucleotides and compositions useful as single-stranded RNAi agents. The present disclosure provides, inter alia, single-stranded oligonucleotides with novel structures capable of inducing RNA interference. Without wishing to be bound by any particular theory, the present disclosure provides that single-stranded RNAi agents have advantages over double-stranded RNAi agents. For example, single-stranded RNAi agents have lower cost because only one strand needs to be constructed. Additionally or alternatively, only one strand (the antisense strand) is administered to target the target transcript. While the source of off-target effects induced by dsRNA is the incorporation of the sense strand into RISC, which binds to and knocks down undesired targets (Jackson et al. 2003 Nat. Biotech. 21:635-637), single-stranded RNAi agents may induce fewer off-target effects compared to corresponding double-stranded RNAi agents. Furthermore, some single-stranded RNAi agents, including some disclosed herein, can target specific sequences that have not previously been successfully targeted using double-stranded RNAi agents (e.g., some single-stranded RNAi agents can reduce the level of a sequence and / or the level of its products (transcripts and / or proteins) more significantly than double-stranded RNAi agents). The present disclosure provides, among other things, novel formats (modifications, stereochemistry, combinations thereof, etc.) of oligonucleotides capable of inducing single-stranded RNA interference.

[0337] Oligonucleotides In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product. In some embodiments, provided oligonucleotides are capable of inducing a reduction in the level of a target product. In some embodiments, provided oligonucleotides are capable of reducing the level of a transcription product of a target gene. In some embodiments, provided oligonucleotides are capable of reducing the level of an mRNA of a target gene. In some embodiments, provided oligonucleotides are capable of reducing the level of a protein encoded by a target gene. In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product via RNA interference. In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product via RNA interference or a biochemical mechanism that does not involve RISC (including, but not limited to, RNase H-mediated knockdown of gene expression or steric hindrance). In some embodiments, provided oligonucleotides are capable of inducing a decrease in the expression and / or level of a target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. In some embodiments, provided oligonucleotides can induce a decrease in the expression and / or levels of a target gene or gene product by steric blocking of translation after binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing, and / or by exon inclusion or exon exclusion.In some embodiments, oligonucleotides provided in accordance with the present disclosure include one or more structural elements described herein or known in the art, such as base sequence, modification, stereochemistry, internucleotide linkage pattern, GC content, long GC stretches, backbone linkage pattern, backbone chiral center pattern, backbone phosphorus modification pattern, additional chemical moieties (including, but not limited to, one or more targeting moieties, lipid moieties, and / or carbohydrate moieties), seed region, post-seed region, 5'-end structure, 5'-end region, 5'-nucleotide moiety, 3'-end region, 3'-end dinucleotide, 3'-end cap, etc. In some embodiments, the seed region of the oligonucleotide is or includes the second to eighth nucleotides, the second to seventh nucleotides, the second to sixth nucleotides, the third to eighth nucleotides, the third to seventh nucleotides, the third to seventh nucleotides, or the fourth to eighth nucleotides or the fourth to seventh nucleotides, counting from the 5' end, and the post-seed region of the oligonucleotide is the region immediately adjacent to the 3' side of the seed region and is interposed between the seed region and the 3' end region.

[0338] In some embodiments, provided compositions comprise an oligonucleotide, hi some embodiments, provided compositions comprise one or more lipid moieties, one or more carbohydrate moieties (other than the sugar moieties of the nucleoside units that form the oligonucleotide chain at the internucleotide linkages, unless otherwise specified), and / or one or more targeting components.

[0339] In some embodiments, the present disclosure provides a compound of formula OI: [ka] or a salt thereof, wherein: R E is the 5'-terminal group, Each BA is independently 1-30 Alicyclic, C6-30 C having 1 to 10 heteroatoms independently selected from aryl, oxygen, nitrogen, sulfur, phosphorus, and silicon 5-30 C having 1 to 10 heteroatoms independently selected from heteroaryl, oxygen, nitrogen, sulfur, phosphorus, boron, and silicon 3-30 an optionally substituted group selected from heterocyclyl, natural nucleobase moieties, and modified nucleobase moieties; R s are each independently -F, -Cl, -Br, -I, -CN, -N3, -NO, -NO2, -L-R', -L-OR', -L-SR', -LN(R')2, -OL-OR', -OL-SR', or -OLN(R')2; s is 0 to 20, Each L is independently a covalent bond or C 1-30 C having an aliphatic group and 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon. 1-30 an optionally substituted linear or branched divalent radical selected from heteroaliphatic groups, in which one or more methylene units are C 1-6 Alkylene, C 1-6Alkenylene, -C≡C-, -C(R')2-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -S( O)-, -S(O)2-, -S(O)2N(R')-, -C(O)S-, -C(O)O-, -P(O)(OR')-, -P(O)(SR')-, -P(O)(R')-, -P(O)(NR')-, -P(S)(OR')-, -P(S)(SR')-, one or more carbon atoms are optionally and independently replaced by -P(S)(R')-, -P(S)(NR')-, -P(R')-, -P(OR')-, -P(SR')-, -P(NR')-, -P(OR')[B(R')3]-, -OP(O)(OR')O-, -OP(O)(SR')O-, -OP(O)(R')O-, -OP(O)(NR')O-, -OP(OR')O-, -OP(SR')O-, -OP(NR')O-, -OP(R')O-, or -OP(OR')[B(R')3]O-; L are optionally and independently replaced by Cy L are each independently, C 3-20 Alicyclic ring, C 6-20 an optionally substituted tetravalent group selected from an aryl ring, a 5-20 membered heteroaryl ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and a 3-20 membered heterocyclyl ring having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon...

Claims

[Claim 1] 1. A composition comprising an oligonucleotide that is a single stranded RNAi agent, wherein the single stranded RNAi agent is complementary or substantially complementary to a target RNA sequence; having a length of about 15 to about 49 nucleotides; having the ability to induce target-specific RNA interference; The composition, wherein the single stranded RNAi agent comprises at least one unnatural base, sugar, and / or internucleotide linkage.

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