Oligonucleotide compositions and methods of use thereof

Abstract

The present disclosure provides, inter alia, designed oligonucleotides, compositions, and methods thereof. In some embodiments, the provided oligonucleotide compositions provide improved single stranded RNA interference and / or RNase H mediated knock-down. The present disclosure encompasses, among other things, the recognition of structural elements of oligonucleotides, such as base sequences, chemical modifications (e.g., modifications of sugars, bases and / or inter-nucleotide linkages), or patterns thereof, conjugation to additional chemical moieties and / or stereochemistry [e.g., stereochemistry of the backbone chiral center (chiral inter-nucleotide linkages)], and / or patterns thereof, the oligonucleotide has significant influence on properties and activity of the oligonucleotide, such as RNA interference (RNAi) activity, stability, delivery and the like. In some embodiments, the disclosure provides methods of treating disease using the provided oligonucleotide compositions (e.g., in RNA interference and / or RNase H mediated knock-down).

Description

[0001] This application is a divisional application of Chinese patent application No. 201880048436.4, filed on June 1, 2018, entitled "Oligonucleotide Composition and Method of Using the Same".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application Nos. 62 / 514,769, filed June 2, 2017; U.S. Provisional Application Nos. 62 / 514,771, filed April 12, 2018; U.S. Provisional Application Nos. 62 / 656,949, filed May 11, 2018; and U.S. Provisional Application Nos. 62 / 670,686, filed May 11, 2018, and 62 / 670,709, each of which is incorporated herein by reference in its entirety. Background Technology

[0004] Oligonucleotides targeting genes can be used in a variety of applications, such as therapeutic, diagnostic, research, and nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) can be limited, for example, by their susceptibility to endonucleases and exonucleases. Summary of the Invention

[0005] This disclosure particularly covers the understanding that controlling structural elements of oligonucleotides, such as chemical modifications (e.g., modifications to sugar, base, and / or internucleotide linkages) or their patterns, stereochemical alterations (e.g., stereochemistry of backbone chiral nucleotide linkages) or their patterns, and / or conjugations with additional chemical moieties (e.g., lipid moieties, targeting moieties, carbohydrate moieties, moieties that bind to desialylate glycoprotein receptors or ASGPR, such as the GalNAc moieties), can significantly influence the properties and / or activities of oligonucleotides. In some embodiments, said properties and / or activities include, but are not limited to, involvement in guiding the reduction of expression, activity, or level of genes or their gene products mediated by, for example, RNA interference (RNAi interference), single-stranded RNA interference (ssRNAi), RNase H-mediated knockdown, steric hindrance of translation, etc.

[0006] In some embodiments, this disclosure demonstrates that compositions comprising oligonucleotides (and particularly single-stranded oligonucleotides) having controlled structural elements provide unexpected properties and / or activities.

[0007] In some embodiments, this disclosure covers the understanding that stereochemistry, particularly the stereochemistry of the main-chain chiral center, can unexpectedly improve the properties of oligonucleotides. Many previous observations have shown that some structural elements that increase stability can also decrease activity, such as RNA interference; conversely, this disclosure demonstrates that stereochemical control can surprisingly increase stability without significantly reducing activity.

[0008] In some embodiments, this disclosure provides techniques (e.g., compounds, methods, etc.) for improving the stability of oligonucleotides while maintaining or increasing their activity, including compositions of stable oligonucleotides.

[0009] In some embodiments, this disclosure provides oligonucleotides having certain 5′-terminal structures.

[0010] This disclosure demonstrates, in particular, that such oligonucleotides can possess the desired properties.

[0011] In some embodiments, this disclosure provides a 5′-terminal structure of an oligonucleotide that can provide high biological activity (e.g., RNAi activity) when used according to this disclosure.

[0012] It has been reported in the literature that, in many cases, RNAi activity requires the presence of a 5′-phosphate (or modified phosphate) moiety; in some embodiments, this disclosure demonstrates, surprisingly, that oligonucleotides having an unmodified 5′-terminus (i.e., having a 5′-OH) can yield RNAi activity comparable to that of oligonucleotides containing the same 5′-phosphate (or modified phosphate) moiety in other aspects. Therefore, in some embodiments, this disclosure particularly provides oligonucleotides whose sequences are targeted to RNAi target sites, said oligonucleotides may contain one or more other structural features useful (or harmless) to RNAi activity, as described herein and / or known in other literature in the art, wherein said oligonucleotide has a 5′-OH moiety.

[0013] In some embodiments, this disclosure covers the understanding that various additional chemical moieties, such as lipid and / or carbohydrate moieties, when incorporated into an oligonucleotide, can improve one or more oligonucleotide properties, such as knockdown of a target gene or its gene product. In some embodiments, the additional chemical moieties are optional. In some embodiments, the oligonucleotide may contain more than one additional chemical moieties. In some embodiments, the oligonucleotide may contain two or more additional chemical moieties, wherein said additional chemical moieties are the same or different, or belong to the same category (e.g., target moieties, carbohydrate moieties, ASPGR-binding moieties, lipid moieties, etc.) or do not belong to the same category. In some embodiments, certain additional chemical moieties facilitate the delivery of the oligonucleotide to desired cells, tissues, and / or organs. In some embodiments, certain additional chemical moieties facilitate the internalization of the oligonucleotide and / or increase its stability.

[0014] In some embodiments, this disclosure provides techniques for incorporating various additional chemical moieties into oligonucleotides. In some embodiments, this disclosure provides reagents and methods, for example, for incorporating additional chemical moieties via nucleobases (e.g., via covalent linkage, optionally via a linker, into sites on the nucleobases).

[0015] In some implementations, this disclosure demonstrates that oligonucleotides whose structures contain one or more of the features described herein can achieve surprisingly high target specificity.

[0016] In some embodiments, this disclosure provides techniques for achieving allele-specific repression, such as oligonucleotide compositions and methods thereof, wherein transcripts of one allele from a specific target gene are selectively knocked down relative to at least one other allele of the same gene.

[0017] This disclosure particularly provides structural elements, techniques, and / or features that can be incorporated into oligonucleotides and can impart or modulate one or more properties thereto (e.g., oligonucleotides identical to other aspects lacking the relevant techniques or features). In some embodiments, this disclosure describes the efficient incorporation of one or more of the provided techniques and / or features into oligonucleotides of various sequences.

[0018] In some embodiments, this disclosure demonstrates that certain structural elements, techniques, and / or features provided are particularly useful for oligonucleotides (e.g., RNAi agents) that participate in and / or guide RNAi mechanisms. However, the doctrine of this disclosure is not limited to oligonucleotides that participate in or function through any particular mechanism. In some embodiments, this disclosure relates to any oligonucleotide that can be used for any purpose, that can function through any mechanism, and that comprises any sequence, structure, or format (or portion thereof) described herein. In some embodiments, this disclosure provides oligonucleotides that can be used for any purpose, the oligonucleotides acting by any mechanism and comprising any sequence, structure, or format (or part thereof) described herein, including but not limited to any 5′-terminal structure; a 5′-terminal region; a first region (including but not limited to a seed region); a second region (including but not limited to a post-seed region); and a 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 moieties, carbohydrate moieties, APGR-binding moieties, and lipid moieties); stereochemical or stereochemical patterns; modifications or modified patterns; internucleotide linkages or internucleotide linkage patterns; sugar modifications or sugar modified patterns; base modifications or base modified patterns. In some embodiments, the provided oligonucleotides may participate in (e.g., guide) RNAi mechanisms. In some embodiments, the provided oligonucleotides may participate in RNase H (ribonuclease H) mechanisms. In some embodiments, the provided oligonucleotides may act as translation inhibitors (e.g., providing steric hindrance for translation).

[0019] In some embodiments, the provided oligonucleotide can participate in exon skipping mechanisms. In some embodiments, the provided oligonucleotide can be an aptamer. In some embodiments, the provided oligonucleotide can bind to and inhibit the function of proteins, small molecules, nucleic acids, or cells. In some embodiments, the provided oligonucleotide can participate in the formation of triple helices with double-stranded nucleic acids in cells. In some embodiments, the provided oligonucleotide can bind to genomic (e.g., chromosome) nucleic acids. In some embodiments, the provided oligonucleotide can bind to genomic (e.g., chromosome) nucleic acids, thereby preventing or reducing the expression of said nucleic acids (e.g., by preventing or reducing transcription, transcriptional enhancement, modification, etc.). In some embodiments, the provided oligonucleotide can bind to DNA quadruplexes. In some embodiments, the provided oligonucleotide can be immunomodulatory. In some embodiments, the provided oligonucleotide can be immunostimulatory. In some embodiments, the provided oligonucleotide can be immunostimulatory and may contain a CpG sequence. In some embodiments, the provided oligonucleotide can be immunostimulatory, may contain a CpG sequence, and can be used as an adjuvant. In some embodiments, the provided oligonucleotide can be immunostimulatory, may contain a CpG sequence, and can be used as an adjuvant for treating diseases (e.g., infectious diseases or cancer). In some embodiments, the provided oligonucleotide may be therapeutic. In some embodiments, the provided oligonucleotide may be non-therapeutic. In some embodiments, the provided oligonucleotide may be either therapeutic or non-therapeutic. In some embodiments, the provided oligonucleotide may be used for therapeutic applications, diagnostic applications, research applications, and / or nanomaterial applications. In some embodiments, the provided oligonucleotide may be used for experimental purposes. In some embodiments, the provided oligonucleotide may be used for experimental purposes, such as as a probe, for microarrays, etc. In some embodiments, the provided oligonucleotide may participate in more than one biological mechanism; for example, in some such embodiments, the provided oligonucleotide may participate in both RNAi and RNase H mechanisms simultaneously.

[0020] In some embodiments, the provided oligonucleotides target a target (e.g., a target sequence, target RNA, target mRNA, target premRNA, target gene, etc.). A target gene is a gene intended to alter the expression and / or activity of one or more gene products (e.g., RNA and / or protein products). In many embodiments, the target gene is intended to be repressed. Therefore, when the oligonucleotides described herein act on a specific target gene, the presence and / or activity of one or more gene products of the gene are altered when the oligonucleotides are present, compared to the absence of the oligonucleotides.

[0021] In some embodiments, a target is a specific allele intended to alter the expression and / or activity of one or more products (e.g., RNA and / or protein products) relative to the target. In many embodiments, a target allele is an allele whose presence and / or expression are associated (e.g., correlated) with the presence, incidence, and / or severity of one or more diseases and / or symptoms. Alternatively or in addition, in some embodiments, a target allele is an allele whose alteration of the level and / or activity of one or more gene products is associated with improvement in one or more aspects of the disease and / or symptom (e.g., delayed onset, reduced severity, responsiveness to other therapies, etc.).

[0022] In some embodiments, where the presence and / or activity of a specific allele (disease-associated allele) is associated (e.g., correlated) with the presence, incidence, and / or severity of one or more conditions, diseases, and / or symptoms, different alleles of the same gene may be present but not so associated or less associated (e.g., exhibiting less significant or statistically insignificant correlation). In some such embodiments, oligonucleotides and methods thereof as described herein can preferentially or specifically target associated alleles relative to one or more less associated / unassociated alleles, thereby mediating allele-specific repression.

[0023] In some embodiments, the target sequence is a sequence that binds to an oligonucleotide as described herein. In many embodiments, the target sequence is identical to or precisely complementary to the sequence of consecutive residues in the provided oligonucleotide or therein (e.g., the provided oligonucleotide includes a target-binding sequence that is identical to or precisely complementary to the target sequence). In some embodiments, the target-binding sequence is precisely complementary to a target sequence of a transcript (e.g., pre-mRNA, mRNA, etc.). The target-binding sequence / target sequence can have various lengths to provide an oligonucleotide with the desired activity 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 more). In some embodiments, minor differences / mismatches between the oligonucleotide (the relevant portion) and its target sequence (including, but not limited to, the 5′- and / or 3′-terminal regions of the target and / or oligonucleotide sequence) are permitted. In many embodiments, the target sequence is present within the target gene. In many embodiments, the target sequence is present in transcripts (e.g., mRNA and / or pre-mRNA) produced by the target gene.

[0024] In some embodiments, the target sequence includes one or more allelic sites (i.e., locations within the target gene where allelic variations occur). In some embodiments, the allelic site is a mutation. In some embodiments, the allelic site is an SNP. In some such embodiments, the provided oligonucleotide preferentially or specifically binds to one allele relative to one or more other alleles. In some embodiments, the provided oligonucleotide preferentially binds to disease-associated alleles. For example, in some embodiments, the oligonucleotide provided herein (or its target-binding sequence portion) has a sequence that is completely or at least partially identical or precisely complementary to a specific allelic form of the target sequence.

[0025] In some embodiments, the oligonucleotides provided herein (or their target-binding sequence portions) have a sequence that is identical to or precisely complementary to a target sequence comprising an allele or an allele of a disease-related allele. In some embodiments, the oligonucleotides provided herein have a target-binding sequence precisely complementary to a target sequence comprising an allele of a transcript containing an allele (in many embodiments, a disease-related allele), wherein the allele is a mutation. In some embodiments, the oligonucleotides provided herein have a target-binding sequence precisely complementary to a target sequence comprising an allele of a transcript containing an allele (in many embodiments, a disease-related allele), wherein the allele is an SNP. In some embodiments, the sequence is any sequence disclosed herein.

[0026] Unless otherwise indicated, all sequences (including, but not limited to, base sequences and chemical, modified and / or stereochemical patterns) are presented in 5′ to 3′ order.

[0027] In some embodiments, this disclosure provides compositions and methods relating to oligonucleotides that are specific to a target and have any format, structural elements, or base sequence of any oligonucleotide disclosed herein.

[0028] In some embodiments, this disclosure provides compositions and methods relating to oligonucleotides that are target-specific and have or contain a region of at least 15 consecutive nucleotides of the base sequence of any oligonucleotide disclosed herein, wherein the first nucleotide of the base sequence or the first nucleotide of the at least 15 consecutive nucleotides may optionally be replaced by T or DNA T. In some embodiments, the oligonucleotide is capable of directing ssRNAi.

[0029] In some embodiments, this disclosure provides compositions and methods for RNA interference directed by single-stranded RNAi agents. In some embodiments, the oligonucleotides of such compositions may have the oligonucleotide format, structural elements, or base sequences disclosed herein.

[0030] In some embodiments, this disclosure provides compositions and methods for RNase H-mediated knockdown of target gene RNA directed by oligonucleotides (e.g., antisense oligonucleotides).

[0031] The provided oligonucleotides and oligonucleotide compositions may have any format, structural element, or base sequence of any oligonucleotide disclosed herein. In some embodiments, the structural element is a 5′-terminal structure, a 5′-terminal region, a 5′-nucleotide, a seed region, a post-seed region, a 3′-terminal region, a 3′-terminal dinucleotide, a 3′-terminal cap, or any portion of any of these structures, GC content, long GC extension and / or any modification, chemical, stereochemical, pattern of modification, chemical or stereochemical, or chemical motif (e.g., including but not limited to a targeting motif, a lipid motif, a GalNAc motif, a carbohydrate motif, etc.), any component, or any combination of the foregoing.

[0032] In some embodiments, this disclosure provides compositions and methods of using oligonucleotides.

[0033] In some embodiments, this disclosure provides compositions and methods of using oligonucleotides that can simultaneously direct RNA interference and RNase H-mediated knockdown of target gene RNA. In some embodiments, the oligonucleotides of such compositions may have the format, structural elements, or base sequences of the oligonucleotides disclosed herein.

[0034] In some embodiments, oligonucleotides that direct specific events or activities are involved in said specific events or activities, such as a decrease in the expression, level, or activity of a target gene or its gene product. In some embodiments, an oligonucleotide is considered to "direct" said specific event or activity when its presence in a system in which the specific event or activity may occur is associated with an increase in the detectable occurrence, frequency, intensity, and / or level of said event or activity.

[0035] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of the oligonucleotide as described herein, such as: a base sequence (or a portion of at least 15 consecutive bases thereof); a pattern of internucleotide linkages (or a portion of at least 5 consecutive internucleotide linkages thereof); a stereochemical pattern of internucleotide linkages (or a portion of at least 5 consecutive internucleotide linkages 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; and in some embodiments, at least one structural element comprises a chiral-controlled chiral center. In some embodiments, the 3′-terminal dinucleotide may comprise a total of two nucleotides. In some embodiments, the oligonucleotide also comprises, as non-limiting examples, a chemical moieties selected from: a targeting moieties, a carbohydrate moieties, a GalNAc moieties, a lipid moieties, and any other chemical moieties described herein or known in the art. In some embodiments, the APGR-binding moieties are GalNAc moieties as described herein and / or known in the art, or variants, derivatives, or modified forms thereof. In some embodiments, the oligonucleotide is a single-stranded RNAi agent. In some implementations, the first region is the seed region. In some implementations, the second region is the post-seed region.

[0036] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of a single-stranded RNAi agent as described herein, such as a 5′-terminal structure; or a 5′-terminal region; a seed region; a post-seed region (the region between the seed region and the 3′-terminal region); and a 3′-terminal region (which may be a 3′-terminal dinucleotide and / or a 3′-terminal cap); and optional additional chemical moieties; and in some embodiments, at least one structural element comprises a chiral-controlled chiral center. In some embodiments, the 3′-terminal dinucleotide may comprise a total of two nucleotides. In some embodiments, the oligonucleotide also comprises, as non-limiting examples, a chemical moieties selected from: a targeting moieties, carbohydrate moieties, GalNAc moieties, and lipid moieties. In some embodiments, the APGR-binding moieties are any GalNAc or its variants, derivatives, or modifications as described herein or known in the art.

[0037] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of an oligonucleotide as described herein, such as 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-controlled chiral center. In some embodiments, the oligonucleotide comprises a span of at least five nucleotides without 2′-modification. In some embodiments, the oligonucleotide further comprises, as non-limiting examples, additional chemical moieties selected from: a targeting moieties, a carbohydrate moieties, a GalNAc moieties, and lipid moieties. In some embodiments, the provided oligonucleotide is capable of directing RNA interference. In some embodiments, the provided oligonucleotide is capable of directing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotide is capable of simultaneously directing 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.

[0038] In some embodiments, the provided oligonucleotide comprises any one or more structural elements of a single-stranded RNAi agent, such as a 5′-terminal structure, a 5′-terminal region, a seed region, a post-seed region, and a 3′-terminal region, and optionally additional chemical moieties, wherein at least one structural element comprises a chiral center controlled by chirality; and in some embodiments, the oligonucleotide is also capable of directing RNase H-mediated knockdown of the target gene RNA. In some embodiments, the oligonucleotide comprises a span of at least five 2′-deoxynucleotides in total. In some embodiments, the oligonucleotide further comprises, as non-limiting examples, a chemical moieties selected from: a targeting moieties, a carbohydrate moieties, a GalNAc moieties, and a lipid moieties, and any other additional chemical moieties described herein.

[0039] In some embodiments, this disclosure demonstrates that oligonucleotide properties can be modulated by chemical modifications. In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more nucleotide inter-linkings, sugars, and / or base modifications. In some embodiments, this disclosure provides an oligonucleotide composition capable of directing single-stranded RNA interference and comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more nucleotide inter-linkings, and / or one or more sugars and / or one or more base modifications. In some embodiments, the oligonucleotide or oligonucleotide composition is also capable of directing RNase H-mediated knockdown of target gene RNA. In some embodiments, this disclosure demonstrates that oligonucleotide properties (e.g., activity, toxicity, etc.) can be modulated by chemical modifications of sugars, nucleobases, and / or nucleotide inter-linkings. In some embodiments, this disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides having a common base sequence and comprising one or more modified internucleotide links (or "non-natural internucleotide links") that can be used to replace the natural phosphate internucleotide links (-OP(O)(OH)O-) present in natural DNA and RNA, which can be in salt form (-OP(O)(O)) at physiological pH. -The oligonucleotide may contain (O-) bonds, one or more modified sugar moieties, and / or one or more native phosphate ester bonds. In some embodiments, the provided oligonucleotide may contain two or more types of modified nucleotide bonds. In some embodiments, the provided oligonucleotide contains non-negatively charged nucleotide bonds. In some embodiments, the non-negatively charged nucleotide bonds are neutral nucleotide bonds. In some embodiments, the neutral nucleotide bonds include triazole, alkyne, or cyclic guanidine moieties. Such moieties are optionally substituted. In some embodiments, the provided oligonucleotide contains a neutral nucleotide bond and another nucleotide bond that is not a neutral backbone. In some embodiments, the provided oligonucleotide contains a neutral nucleotide bond and a phosphate thioester nucleotide bond. In some embodiments, the provided oligonucleotide composition comprising multiple oligonucleotides is chiral controlled, and the levels of the multiple oligonucleotides in the composition are controlled or predetermined, and the multiple oligonucleotides share a common stereochemical configuration at one or more chiral nucleotide bonds. For example, in some embodiments, multiple oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral nucleotides at their inter-chiral nucleotide linkages, each independently being Rp or Sp; in some embodiments, multiple oligonucleotides share a common stereochemical configuration at each chiral nucleotide linkage. In some embodiments, when the oligonucleotides in the composition share a common stereochemical configuration (independently Rp or Sp configuration), the chiral nucleotide linkage is referred to as a chiral-controlled nucleotide linkage. In some embodiments, the modified nucleotide linkages are non-negatively charged (neutral or cationic) nucleotide linkages because, at certain pH (e.g., human physiological pH (~7.4), pH of the delivery site (e.g., organelle, cell, tissue, organ, organism, etc.), it is mostly (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 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.) present in neutral or cationic form (compared to anionic form (e.g., -OP(O)(O)). - -O- (the anionic form linked by natural phosphate esters), -OP(O)(S -(e.g., anionic form of thiophosphate-linked nucleotides). In some embodiments, the modified internucleotide links are neutral internucleotide links because they are predominantly present in a neutral form at a given pH. In some embodiments, the modified internucleotide links are cationic internucleotide links because they are predominantly present in a cationic form at a given pH. In some embodiments, the pH is human physiological pH (~7.4). In some embodiments, the modified internucleotide links are neutral internucleotide links because at least 90% of the internucleotide links are present in a neutral form in aqueous solution at pH 7.4. In some embodiments, the modified internucleotide links are neutral internucleotide links because at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the internucleotide links are present in a neutral form in 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, when present in its neutral form, the non-negatively charged internucleotide linkages (e.g., neutral internucleotide linkages) do not have a pKa less than 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the pKa of the internucleotide linkages in this disclosure may be represented by the pKa of the CH3-internucleotide linkage-CH3 (i.e., replacing the two nucleoside units linked by the internucleotide linkage with two -CH3 groups). Without wishing to be bound by any particular theory, in at least some cases, neutral internucleotide linkages in oligonucleotides can provide improved properties and / or activities 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 intron escape, and / or improved nuclear uptake, etc.

[0040] In some embodiments, the negatively charged internucleotide linkages have structures such as In-1, In-2, In-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc. In some embodiments, the non-negatively charged internucleotide linkages comprise a triazole or alkyne moiety. In some embodiments, the non-negatively charged internucleotide linkages comprise a cyclic guanidine moiety. In some embodiments, the modified internucleotide linkages comprising a cyclic guanidine moiety moiety have the following structures: In some embodiments, the neutral nucleotide linking comprising the cyclic guanidine moiety is chiral controlled. In some embodiments, this disclosure relates to compositions comprising oligonucleotides, said oligonucleotides comprising at least one neutral nucleotide linking and at least one phosphate thioester nucleotide linking.

[0041] In some embodiments, this disclosure relates to compositions comprising oligonucleotides, said oligonucleotides comprising at least one neutral nucleotide link and at least one phosphate thioester nucleotide link, wherein said phosphate thioester nucleotide link is a chiral-controlled nucleotide link of the Sp configuration.

[0042] In some embodiments, this disclosure relates to compositions comprising oligonucleotides, said oligonucleotides comprising at least one neutral nucleotide linker and at least one phosphate thioester nucleotide linker, wherein said phosphate thioester is an Rp-configuration chiral-controlled nucleotide linker.

[0043] In some embodiments, this disclosure relates to compositions comprising oligonucleotides, said oligonucleotides comprising at least one neutral nucleotide linking selected from: neutral nucleotide linking comprising an optionally substituted triazole group, neutral nucleotide linking comprising an optionally substituted alkynyl group, and containing a Tmg group. Neutral nucleotide linkages and at least one thiophosphate ester.

[0044] In some embodiments, this disclosure relates to compositions comprising oligonucleotides comprising at least one neutral nucleotide linker selected from: neutral nucleotide linkers comprising an optionally substituted triazole group, neutral nucleotide linkers comprising an optionally substituted alkynyl group, and neutral nucleotide linkers comprising a Tmg group, and at least one thiophosphate ester, wherein the thiophosphate ester is a chiral-controlled nucleotide linker with an Sp configuration.

[0045] In some embodiments, this disclosure relates to compositions comprising oligonucleotides comprising at least one neutral nucleotide linker selected from: neutral nucleotide linkers comprising an optionally substituted triazole group, neutral nucleotide linkers comprising an optionally substituted alkynyl group, and neutral nucleotide linkers comprising a Tmg group, and at least one thiophosphate ester, wherein the thiophosphate ester is a chiral-controlled nucleotide linker with an Rp configuration.

[0046] Different types of nucleotide linkages differ in nature. Without wishing to be bound by any theory, this disclosure notes that native phosphate ester linkages (phosphodiester nucleotide linkages) are anionic and may be unstable when used alone in vivo without further chemical modifications; thiophosphate nucleotide linkages are anionic, generally more stable in vivo than native phosphate ester linkages, and generally more hydrophobic; neutral nucleotide linkages, such as the neutral nucleotide linkages containing cyclic guanidine moieties exemplified in this disclosure, are neutral at physiological pH, may be more stable in vivo than native phosphate ester linkages, and are more hydrophobic.

[0047] In some implementations, the chiral-controlled internucleotide linkages are neutral at physiological pH, chiral-controlled, stable in vivo, hydrophobic, and can increase incorpus escape.

[0048] In some embodiments, the provided oligonucleotide comprises one or more regions, such as block regions, wing regions, core regions, 5′-terminal regions, 3′-terminal regions, intermediate regions, seed regions, post-seed regions, etc. In some embodiments, the regions (e.g., block regions, wing regions, core regions, 5′-terminal regions, 3′-terminal regions, intermediate regions, etc.) comprise non-negatively charged internucleotide bonds, for example, having the formula In-1, In-2, In-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, etc. In some embodiments, the regions comprise neutral internucleotide bonds. In some embodiments, the regions comprise internucleotide bonds containing a triazole or alkyne moiety. In some embodiments, the regions comprise internucleotide bonds containing a cyclic guanidine moiety. In some embodiments, the regions comprise internucleotide bonds containing a cyclic guanidine moiety moiety. In some embodiments, the regions comprise internucleotide bonds having the following structure: In some implementations, such nucleotide linkages are chiral controlled.

[0049] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more nucleotide linkages, sugars, and / or base modifications.

[0050] In some embodiments, this disclosure provides an oligonucleotide composition capable of directing single-stranded RNA interference and comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more nucleotide inter-linkings, and / or one or more sugars and / or one or more base modifications. In some embodiments, the oligonucleotides or oligonucleotide compositions are also capable of directing RNase H-mediated knockdown of target gene RNA.

[0051] 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 link is a modified internucleotide link. In some embodiments, the nucleotide comprises a base, a sugar, and an internucleotide link, wherein each of the base, sugar, and internucleotide link is independently and optionally naturally occurring or non-naturally occurring. In some embodiments, the nucleoside comprises a base and a sugar, wherein each of the base and sugar is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2′-deoxy) and RNA (2′-OH) nucleotides; and those nucleotides comprising one or more modifications at the base, sugar, and / or internucleotide link. 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 linking may have a structure of Formula I as described in this disclosure. In some embodiments, the internucleotide linking is a phosphorus-free portion used to link two natural or non-natural sugars.

[0052] In some embodiments, this disclosure provides a chirality-controlled oligonucleotide composition that has improved properties and / or activity compared to reference conditions (e.g., the absence of the composition or the absence of a reference composition (e.g., a stereorandom composition of oligonucleotides having the same base sequence and chemical modifications)).

[0053] In some embodiments, this disclosure provides a chiral-controlled oligonucleotide composition that directs a greater reduction in the expression, activity, and / or level of a gene or its gene product, single-stranded RNA interference, and / or RNase H-mediated knockdown, compared to reference conditions (e.g., the absence of the composition or the absence of a reference composition (e.g., a stereorandom composition of oligonucleotides having the same base sequence and chemical modifications)).

[0054] In some embodiments, the oligonucleotide composition comprising multiple oligonucleotides is stereorandom because the multiple oligonucleotides do not share a common stereochemistry at any chiral nucleotide linking site. In some embodiments, the oligonucleotide composition comprising multiple oligonucleotides is chiral controlled because the multiple oligonucleotides share a common stereochemistry at one or more chiral nucleotide linking sites. In some embodiments, the chiral controlled oligonucleotide composition comprising a first plurality of oligonucleotides exhibits reduced sensitivity to endonucleases and exonucleases compared to a stereorandom oligonucleotide composition comprising a first plurality of oligonucleotides.

[0055] In some embodiments, the oligonucleotide composition can direct the reduction of expression and / or levels of a target gene or its gene product. In some embodiments, the oligonucleotide composition can direct single-stranded RNA interference. In some embodiments, the oligonucleotide composition can direct RNase H-mediated knockdown. In some embodiments, the oligonucleotide composition can direct RNase H-mediated knockdown and RNA interference of target gene RNA. In some embodiments, the oligonucleotide composition can direct RNase H-mediated knockdown of a first RNA target and RNA interference of a second RNA target, wherein the first and second RNA targets may be the same or different.

[0056] In some embodiments, the composition comprises two or more of the following polymers: a first plurality of oligonucleotides and / or a second plurality of oligonucleotides, wherein the first and second plurality of oligonucleotides can independently direct the knockdown of the same or different targets via RNA interference and / or RNase H-mediated knockdown.

[0057] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides sharing:

[0058] 1) Common base sequence;

[0059] 2) Common main chain keying style;

[0060] 3) Common stereochemistry independently at at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 chiral nucleotide links (“chiral-controlled nucleotide links”);

[0061] The composition is chiral controlled because the level of the first plurality of oligonucleotides in the composition is predetermined.

[0062] In some embodiments, the oligonucleotide composition comprising multiple oligonucleotides (e.g., a first plurality of oligonucleotides) is chiral controlled because the multiple oligonucleotides independently share a common stereochemistry at one or more chiral nucleotide linking sites. In some embodiments, the multiple oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral nucleotide linking sites, each independently being Rp or Sp. In some embodiments, the multiple oligonucleotides share a common stereochemical configuration at each chiral nucleotide linking site. In some embodiments, chiral nucleotide linking is referred to as chiral controlled nucleotide linking when the oligonucleotides at a predetermined level in the composition share a common stereochemical configuration (independently Rp or Sp).

[0063] In some embodiments, the provided composition comprises a predetermined level of oligonucleotides, such as the first plurality of oligonucleotides of certain exemplary compositions, comprising 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 nucleotide linkages.

[0064] In some embodiments, at least 5 nucleotides are chirally controlled; in some embodiments, at least 10 nucleotides are chirally controlled; in some embodiments, at least 15 nucleotides are chirally controlled; and in some embodiments, each chiral nucleotide is chirally controlled.

[0065] In some embodiments, 1%–100% of the chiral nucleotide linkages are chiral controlled. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the chiral nucleotide linkages are chiral controlled.

[0066] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides sharing:

[0067] 1) Common base sequence;

[0068] 2) Common main chain keying style; and

[0069] 3) Common backbone chiral center pattern, the composition being substantially pure products of a single oligonucleotide because the oligonucleotides in the composition at predetermined levels have a common base sequence and length, a common backbone bonding pattern and a common backbone chiral center pattern.

[0070] In some implementations, the common main-chain chiral center pattern includes at least one internucleotide bond containing a chiral center with chiral control.

[0071] In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides having or containing a common base sequence in the provided composition. In some embodiments, the oligonucleotides having or comprising a common base sequence in the provided composition are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition that have or contain a common base sequence, base modifications, sugar modifications, and / or modified internucleotide bonds. In some embodiments, the oligonucleotides in the provided composition having or comprising a common base sequence, base modification, sugar modification, and / or modified internucleotide bonds are at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition.In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition having or containing a common base sequence, base modification pattern, sugar modification pattern, and / or modified internucleotide linking pattern. In some embodiments, all oligonucleotides in the provided composition having or comprising a common base sequence, base modification pattern, sugar modification pattern, and / or modified internucleotide linking pattern constitute at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition. In some embodiments, the predetermined level of oligonucleotides is at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the provided composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, and / or a common modified internucleotide linking pattern. In some embodiments, all oligonucleotides in the provided composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, and / or a common modified internucleotide linking pattern constitute at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of all oligonucleotides in the composition.

[0072] In some embodiments, the predetermined level is 1-100%. 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*(1 / 2) g ), where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least 10*(1 / 2) g ), where g is the number of chiral-controlled nucleotide bonds. In some embodiments, the predetermined level is at least 100*(1 / 2) g ), where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.80). g , where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.80). g , where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.80). g , where g is the number of chiral-controlled nucleotide bonds. In some embodiments, the predetermined level is at least (0.85). g , where g is the number of chiral-controlled nucleotide bonds. In some embodiments, the predetermined level is at least (0.90). g , where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.95). g , where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.96). g , where g is the number of chiral-controlled nucleotide bonds. In some embodiments, the predetermined level is at least (0.97). g , where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.98). g , where g is the number of chiral-controlled nucleotide linkages. In some embodiments, the predetermined level is at least (0.99). g, where g is the number of chiral-controlled internucleotide links. In some embodiments, to determine the level of an oligonucleotide having g chiral-controlled internucleotide links in the composition, the product of the diastereomeric purity of each of the g chiral-controlled internucleotide links is used as said level: (diastereomeric purity of chiral-controlled internucleotide link 1) * (diastereomeric purity of chiral-controlled internucleotide link 2) * ... * (diastereomeric purity of chiral-controlled internucleotide link g), wherein the diastereomeric purity of each chiral-controlled internucleotide link is independently represented by the diastereomeric purity of a dimer containing the same internucleotide link and the nucleoside side-linking the internucleotide link and prepared by a method comparable to that of the oligonucleotide (e.g., comparable or preferably the same oligonucleotide preparation cycle, which includes comparable or preferably the same reagents and reaction conditions).

[0073] In some implementations, the level of oligonucleotide and / or diastereomeric purity can be determined by analytical methods (e.g., chromatography, spectrophotometry, spectroscopy, or any combination thereof).

[0074] This disclosure particularly covers the understanding that stereorandom oligonucleotide articles contain multiple distinct chemical entities, for example, differing from each other in terms of stereochemical structure (or stereochemistry) of the individual backbone chiral centers within the oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral centers, stereorandom oligonucleotide articles provide uncontrolled compositions comprising an undefined level of oligonucleotide stereoisomers. Even if these stereoisomers may have the same base sequence and / or chemical modifications, they are distinct chemical entities at least due to their different backbone stereochemistry, and as demonstrated herein, they can possess different properties, such as sensitivity to nucleases, activity, distribution, etc. 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, this disclosure demonstrates that improvements in properties and activity achieved by controlling the stereochemistry within the oligonucleotide can be comparable to or even better than those achieved by using chemical modifications.

[0075] In some embodiments, the provided oligonucleotides, such as oligonucleotides capable of directing the reduction of expression and / or levels of a target gene or its gene product, oligonucleotides capable of directing single-stranded RNA interference (e.g., single-stranded RNAi agents, ssRNA, or ssRNAi), or oligonucleotides capable of directing single-stranded RNA interference and RNase H-mediated knockdown, are represented by the following structures:

[0076] 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′,

[0077] The variables are each independent as described in this disclosure.

[0078] In some embodiments, PX0 is a 5′-terminal structure. In some embodiments, PX1 to PX26 are each independently an intermolecular nucleotide link. In some embodiments, PX27 is an intermolecular nucleotide link or OH. In some embodiments, N1 to N27 independently represent nucleosides. In some embodiments, N1-PX1 to N27-PX27 independently represent nucleotides. Any nucleoside may be the same as or different from any adjacent nucleoside. Any nucleotide may be the same as or different from any adjacent nucleotide. In some embodiments, wherein any one of mz to yz is >1, each base of N18 to N27 may be the same as or different; and / or each nucleoside of N18 to N27 may be the same as or different and may independently contain the same as or different modifications (e.g., 2′-modification). In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz may each be 0 to 10 independently. In some embodiments: if in -(N18-PX18) mz In the given condition, if mz > 1, then each N18 can be the same or different and / or each PX18 can be the same or different; if in -(N19-PX19) nz In the given condition, if nz > 1, then each N19 can be the same or different and / or each PX19 can be the same or different; if in -(N20-PX20) pz In the given condition, if pz > 1, then each N20 can be the same or different and / or each PX20 can be the same or different; if in -(N21-PX21)rz In the given condition, if rz > 1, then each N21 can be the same or different and / or each PX21 can be the same or different; if in -(N22-PX22) sz In the given condition, if sz > 1, then each N22 can be the same or different and / or each PX22 can be the same or different; if in -(N23-PX23) tz In the given condition, if tz > 1, then each N23 can be the same or different and / or each PX23 can be the same or different; if in -(N24-PX24) vz In the given condition, if vz > 1, then each N24 can be the same or different and / or each PX24 can be the same or different; if -(N25 - PX25) wz In the given condition, wz > 1, then each N25 can be the same or different and / or each PX26 can be the same or different; if in (N26-PX26-N27-PX27)... yz - In the case where yz > 1, then each N26 can be the same or different and each N27 can be the same or different, and each N26 and N27 can be the same or different, and / or each PX26 can be the same or different, and / or each PX27 can be the same or different, and / or each PX26 and PX27 can be the same or different; etc.

[0079] In some implementations, N or PX may each independently and optionally also include one or more additional chemical moieties, such as a targeting moieties, carbohydrate moieties, GalNAc moieties, lipid moieties, etc.

[0080] In some embodiments, PX1 to PX27 each independently represent an internucleotide link, wherein PX1 to PX27 may be the same or different. In some embodiments, PX1 to PX27 each independently represent an internucleotide link, which is a phosphodiester, a thiophosphate, a Sp-configured thiophosphate, an Rp-configured thiophosphate, an internucleotide link, a Sp-configured internucleotide link, or an Rp-configured internucleotide link, wherein PX1 to PX27 may be the same or different.

[0081] In some implementations, the 3′-terminal region is represented as: -(N26-PX26-N27-PX27) yz -(CAP) zz , where yz = 1 and zz = 0, or yz = 0 and zz = 1, or yz = 1 and zz = 1; -(N26-PX26-N27-PX27) yz , where yz = 1; -(CAP) zz Where zz = 1; or -(N26-PX26-N27-PX27) yz-(CAP) zz , where yz = 1 and zz = 1.

[0082] In some embodiments, PX27 represents an internucleotide link or OH. In some embodiments, zz = 0, yz = 1, and PX27 is -OH. In some embodiments, zz = 1, yz = 1, and PX27 is an internucleotide link.

[0083] In some embodiments, PX27 represents an internucleotide link, which is a phosphodiester, a thiophosphate, a Sp-configured thiophosphate, an Rp-configured thiophosphate, an internucleotide link, a Sp-configured internucleotide link, or an Rp-configured internucleotide link. In some embodiments, PX27 is -OH.

[0084] In some implementations, where zz = 1 (e.g., in the presence of CAP), PX27 represents an internucleotide link, which is a phosphodiester, a thiophosphate, a Sp-configured thiophosphate, an Rp-configured thiophosphate, an internucleotide link, a Sp-configured internucleotide link, or an Rp-configured internucleotide link.

[0085] In some embodiments, the oligonucleotide comprises a 5′-terminal region, a seed region, a post-seed region, and a 3′-terminal region in a 5′ to 3′ sequence, and optionally also includes additional chemical moieties.

[0086] In some embodiments, the 5′-terminal region is the entire portion of the oligonucleotide located at the 5′ end of the seed region. In some embodiments, the 3′-terminal region is the entire portion of the oligonucleotide located at the 3′ end of the post-seed region.

[0087] In some implementations, the 5′-terminal region is represented as any of the following: PX0-, PX0-N1-, PX0-N1-PX1-, PX0-N1-PX1-N2-, PX0-N1-PX1-N2-PX2-, PX0-N1-PX1-N2-PX2-, or PX0-N1-PX1-N2-PX2-N3-PX3-.

[0088] In some implementations, the 5′-terminal region is represented as any of the following: PX0-, PX0-N1-, or PX0-N1-PX1-.

[0089] In some implementations, the 5′-terminal structure is represented as any of the following: PX0-, PX0-N1-, or PX0-N1-PX1-.

[0090] In some implementations, the 5′-terminal structure is denoted as PX0-.

[0091] In some implementations, the 5′-terminal structure is a 5′-terminal group.

[0092] In some embodiments, the 5′-terminal structure contains a 5′-terminal group.

[0093] In some embodiments, -N1-PX1- represents the 5′ nucleotide moiety. In some embodiments, -N1- represents a 5′ nucleoside.

[0094] In some implementations, the 5′-terminal nucleoside is -N1-.

[0095] In some implementations, the 5′-terminal nucleotide is -N1-PX1-.

[0096] In some embodiments, the provided oligonucleotide may comprise a 5′-terminal region, 5′-terminal structure, 5′-terminal group, 5′-terminal nucleoside, or 5′-terminal nucleotide as described herein or known in the art.

[0097] In some embodiments, -PX0-, -PX0-N1-, and -PX0-N1-PX1- are represented by the following structures described herein: 5′-terminal structure, 5′-terminal region, 5′-nucleotide, modified 5′-nucleotide, 5′-nucleotide analog, or 5′-nucleoside, modified 5′-nucleoside, or 5′-nucleoside analog.

[0098] In some implementations, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents a seed region. In some implementations, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents a seed region. In some implementations, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents a seed region. In some implementations, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents a seed region. In some implementations, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents a seed region. In some implementations, -N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents a seed region. In some implementations, -N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents a seed region.

[0099] In some implementations, -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 (e.g., the region between the seed region and the 3′-terminal region).

[0100] In some implementations, -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 region after the seed.

[0101] In some implementations, -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 region after the seed.

[0102] In some implementations, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents the 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 region after the seed.

[0103] In some implementations, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents the 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 region after the seed.

[0104] In some implementations, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents 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 region after the seed.

[0105] In some implementations, -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents the 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 region after the seed.

[0106] In some implementations, -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents the 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 region after the seed.

[0107] In some implementations, -N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents the 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 region after the seed.

[0108] In some implementations, -N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents the 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 region after the seed.

[0109] In some implementations, 5′-PX0-N1-PX1- represents the 5′-terminal region; -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7- represents the seed region; -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; and -(N26-PX26-N27-PX27) yz -(CAP) zz -3′ indicates the 3′-terminal region.

[0110] In some implementations, 5′-PX0-N1-PX1- represents the 5′-terminal region; -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents the seed region; -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 This represents the region after the seed; and -(N26-PX26-N27-PX27) yz -(CAP) zz -3′ indicates the 3′-terminal region.

[0111] In some implementations, 5′-PX0-N1-PX1-N2-PX2- represents the 5′-terminal region; -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7- represents the seed region; -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 This represents the region after the seed; and -(N26-PX26-N27-PX27) yz -(CAP) zz -3′ indicates the 3′-terminal region.

[0112] In some implementations, 5′-PX0-N1-PX1-N2-PX2- represents the 5′-terminal region; -N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8-PX8- represents the seed region; -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 This represents the region after the seed; and -(N26-PX26-N27-PX27) yz -(CAP) zz -3′ indicates the 3′-terminal region.

[0113] In some implementations, 5′-PX0-N1-PX1- represents the 5′-terminal region; -N2-PX2-N3-PX3-N4-PX4-N5-PX5-N6-PX6-N7-PX7-N8- represents the seed region; -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 This represents the region after the seed; and -(N26-PX26-N27-PX27) yz -(CAP)zz -3′ indicates the 3′-terminal region.

[0114] In some implementations, 5′-PX0-N1-PX1-N2-PX2- represents the 5′-terminal region; -N3-PX3-N4-PX4-N5-PX5-N6-PX6- represents the seed region; -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 This represents the region after the seed; and -(N26-PX26-N27-PX27) yz -(CAP) zz -3′ indicates the 3′-terminal region.

[0115] In some implementations, mz, nz, pz, rz, sz, tz, vz, and wz are each independently 0 to 10.

[0116] In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 49 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 45 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 40 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 35 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 30 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 25 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 23 nucleotides. In some embodiments, mz, nz, pz, rz, sz, tz, vz, and wz can each independently be 0 to 10, and the total length of the oligonucleotide does not exceed about 21 nucleotides.

[0117] In some implementations, -(N26-PX26-N27-PX27) yz - indicates a 3′-terminal dinucleotide, where yz is 1. When yz = 0, a 3′-terminal dinucleotide does not exist. When yz = 1, a 3′-terminal dinucleotide exists.

[0118] In some implementations, -(CAP) zz - indicates an optional 3′-terminal cap, where zz is 0 or 1. When zz = 0, CAP does not exist. When zz = 1, CAP exists.

[0119] In some embodiments, if yz = 1, then zz = 0. In some embodiments, if yz = 0, zz = 1. In some embodiments, yz = 1 and zz = 1, indicating that the molecule contains both a 3′-terminal dinucleotide and CAP.

[0120] In some embodiments, the 5′-terminal structure, 5′-terminal region, 5′-nucleotide moiety, seed region, post-seed region, 3′-terminal dinucleotide, and / or 3′-terminal cap independently have any structure described herein or known in the art. In some embodiments, any structure of the 5′-terminus described herein or known in the art, and / or any structure of the 5′-nucleotide moiety described herein or known in the art, and / or any structure of the seed region described herein or known in the art, and / or any structure of the post-seed region described herein or known in the art, and / or any structure of the 3′-terminal dinucleotide described herein or known in the art, and / or any structure of the 3′-terminal cap described herein or known in the art may be combined.

[0121] In some embodiments, the provided oligonucleotide comprises one or more blocks. In some embodiments, the provided oligonucleotide comprises one or more blocks, wherein the blocks comprise one or more consecutive nucleosides, and / or nucleotides, and / or sugars or bases, and / or internucleotide bonds. In some embodiments, the blocks cover the entire seed region or a portion thereof. In some embodiments, the blocks cover the entire post-seed region or a portion thereof.

[0122] In some implementations, the provided oligonucleotides are block copolymers.

[0123] In some implementations, the provided oligonucleotides are alternating polymers.

[0124] In some embodiments, the provided oligonucleotides are alternating polymers containing alternating blocks. In some embodiments, the block polymers or alternating polymers can be defined by chemical modifications (including their presence or absence) (e.g., base modifications, sugar modifications, internucleotide linking modifications, stereochemistry, etc.) or their patterns.

[0125] In some embodiments, the provided oligonucleotide is capable of directing the reduction of expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotide is capable of directing the reduction of expression and / or level of a target gene or its gene product via RNA interference. In some embodiments, the provided oligonucleotide is capable of directing the reduction of expression and / or level of a target gene or its gene product via biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotide is capable of directing the reduction of expression and / or level of a target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotide comprises one or more blocks containing two or more different nucleotide inter-linkings. In some embodiments, the provided oligonucleotide comprises one or more blocks containing a total of two or more modified nucleotide inter-linkings and native phosphate ester inter-linkings. In some embodiments, the provided oligonucleotide comprises one or more blocks containing two or more different modified nucleotide inter-linkings. In some embodiments, the provided oligonucleotide comprises alternating blocks containing two or more different nucleotide inter-linkings. In some embodiments, the provided oligonucleotide comprises alternating blocks comprising a total of two or more modified internucleotide links and native phosphate ester links. In some embodiments, the provided oligonucleotide comprises alternating blocks comprising two or more different modified internucleotide links. In some embodiments, the blocks comprising modified internucleotide links have a main-chain chiral center pattern as described herein. In some embodiments, each block comprising modified internucleotide links has the same main-chain chiral center pattern. In some embodiments, the blocks comprising modified internucleotide links have different main-chain chiral center patterns. In some embodiments, the blocks comprising modified internucleotide links have different lengths and / or modifications. In some embodiments, the blocks comprising modified internucleotide links have the same length and / or modifications. In some embodiments, the blocks comprising modified internucleotide links have the same length. In some embodiments, the blocks comprising modified internucleotide links have the same internucleotide links.

[0126] In some embodiments, the provided oligonucleotide is capable of directing single-stranded RNA interference and is contained in a first block (seed region-block) in the seed region and a second block (seed region-block) in the post-seed region, each block independently containing one or more modified nucleotide inter-links. In some embodiments, the seed region-block and the post-seed region-block each independently contain 2, 3, 4, 5, 6, 7 or more modified nucleotide inter-links. In some embodiments, the seed region-block contains 4 or more modified nucleotide inter-links. In some embodiments, the seed region-block contains 5 or more modified nucleotide inter-links. In some embodiments, the seed region-block contains 6 or more modified nucleotide inter-links. In some embodiments, the seed region-block contains 7 modified nucleotide inter-links. In some embodiments, the post-seed region-block contains 4 or more modified nucleotide inter-links. In some embodiments, the post-seed region-block contains 5 or more modified nucleotide inter-links. In some embodiments, the post-seed region-block contains six or more modified nucleotide inter-links. In some embodiments, the post-seed region-block contains seven or more modified nucleotide inter-links. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least four modified nucleotide inter-links. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least five modified nucleotide inter-links. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least six modified nucleotide inter-links. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least seven modified nucleotide inter-links. In some embodiments, the modified nucleotide inter-links within the block are sequential. In some embodiments, each link in the seed region-block is independently a phosphate thioester link. In some embodiments, each link in the seed region-block is independently chiral controlled. In some embodiments, each link in the seed region-block is sp. In some embodiments, each link of the post-seed region-block is independently a modified nucleotide link. In some embodiments, each link of the post-seed region-block is independently a phosphate thioester link. In some embodiments, each link of the post-seed region-block is independently chiral controlled. In some embodiments, each link of the post-seed region-block is sp.

[0127] In some embodiments, the provided oligonucleotide comprises one or more sugar modifications. In some embodiments, the sugar modification is at the 2′-position. In some embodiments, the sugar modification is selected from 2′-F, 2′-OMe, and 2′-MOE. 2′-F is also known as 2′-fluoro. 2′-OMe is also known as 2′-O-methyl. 2′-MOE is also known as 2′-methoxyethyl or MOE.

[0128] In some embodiments, the provided oligonucleotide is capable of directing single-stranded RNA interference and is contained in a first block (seed region-block) in the seed region and a second block (seed region-block) in the post-seed region, each block independently containing one or more 2′-F. In some embodiments, the seed region-block and the post-seed region-block each independently contain 2, 3, 4, 5, 6, 7 or more 2′-F. In some embodiments, the seed region-block and the post-seed region-block each independently contain 2, 3, 4, 5, 6, 7 or more consecutive 2′-F. In some embodiments, the seed region-block contains 4 or more 2′-F. In some embodiments, the seed region-block contains 5 or more 2′-F. In some embodiments, the seed region-block contains 6 or more 2′-F. In some embodiments, the seed region-block contains 7 2′-F. In some embodiments, the post-seed region-block contains 4 or more 2′-F. In some embodiments, the post-seed region-block contains five or more 2′-Fs. In some embodiments, the post-seed region-block contains six or more 2′-Fs. In some embodiments, the post-seed region-block contains seven or more 2′-Fs. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least four 2′-Fs. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least five 2′-Fs. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least six 2′-Fs. In some embodiments, the seed region-block and the post-seed region-block each independently contain at least seven 2′-Fs. In some embodiments, the 2′-Fs within the block are consecutive.

[0129] In some embodiments, the oligonucleotide comprises one, two, three, four, five, six, seven, eight, nine, ten or more 2′-F moieties. In some embodiments, the oligonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more continuous sugar moieties containing 2′-F moieties.

[0130] In some embodiments, the oligonucleotide contains only two 2′-F nucleotides, with the two nucleotides located at positions 2 and 14. Non-limiting examples of such oligonucleotides include: Figure 1F The oligonucleotides in format 70, as well as WV-7540 and WV-7543.

[0131] In some embodiments, the oligonucleotide contains only two 2′-F nucleotides, with the two nucleotides at positions 2 and 14, and wherein the first nucleotide (5′-terminal nucleotide or -N1-PX1-) is 2′-deoxy. Non-limiting examples of such oligonucleotides include: Figure 1F Format 70, as well as WV-7540 and WV-7543.

[0132] In some embodiments, the oligonucleotide contains only two 2′-F nucleotides, with the two nucleotides at positions 2 and 14, and wherein the first nucleotide (5′-terminal nucleotide or -N1-PX1-) is a 2′-deoxy-T. Non-limiting examples of such oligonucleotides include: Figure 1F Format 70, as well as WV-7540 and WV-7543.

[0133] In some embodiments, the oligonucleotide contains only two 2′-F nucleotides, with the two nucleotides at positions 2 and 14, and wherein the first nucleotide (5′-terminal nucleotide or -N1-PX1-) is 2′-deoxy, and the 5′-terminal structure (PX0) is -OH. Non-limiting examples of such oligonucleotides include: Figure 1F The oligonucleotides in format 70, as well as WV-7540 and WV-7543.

[0134] In some embodiments, the oligonucleotide contains only two 2′-F nucleotides, with the two nucleotides at positions 2 and 14, and wherein the first nucleotide (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: Figure 1F Format 70, as well as WV-7540 and WV-7543.

[0135] In some embodiments of this document, with respect to oligonucleotides, "first" (e.g., first nucleotide) refers to the 5′ end of the oligonucleotide, and "last" or "terminus" (e.g., last nucleotide or terminal nucleotide) refers to the 3′ end.

[0136] In some embodiments, the provided oligonucleotide comprises a sugar with specific modifications, which alternates with unmodified or differently modified sugars. In some embodiments, the sugar with specific modifications appears in one or more blocks.

[0137] In some embodiments, the provided oligonucleotide comprises one or more blocks containing sugars with a specific 2′ modification, alternating with sugars that are independently unmodified or have different modifications. In some embodiments, the provided oligonucleotide comprises one or more blocks containing sugars with a 2′-F modification, alternating with sugars that are independently unmodified or have different modifications. In some embodiments, the provided oligonucleotide comprises one or more blocks containing sugars with a 2′-OMe modification, alternating with sugars that are independently unmodified or have different modifications. In some embodiments, the provided oligonucleotide comprises one or more blocks containing sugars with a 2′-OMe modification, alternating with sugars with a 2′-F modification.

[0138] In some embodiments, the sugar block has or comprises a 2′-modified pattern of any of the following: ff, fffm, fffmm, fffmmm, fffmmmm, fffmmmmm, fffmmmmmm, fffmmmmmmf, fffmmmmmmff, fffmmmmmmffm, fffmmmmmmffmm, fffmmmmmmffmmf, fffmmmmmmffmmfm, fffmmmmmmffmmfmf, fffmmmmmmffmmfmfm, fffmmmmmmffmmfmfmf, fffmmmmmmffmmfmfmfm, fffmmmmmmffmmfmfmfmm, fffmmmmmmffmmfmfmfmmm, ffmmffmm, ffmmmmmmffmmfmfmfmmm, fmfmfmfmfmfmfm, fmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmfmf, fmfmfmfmfmfmfmfmfmfm, fmfmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmfmm, fmfmfmfmfmfmfmfmm, fmfmfmfmfmfmm, fmfmfmfmfmfmmm, fmmffmm, fmmmmmmffmmfmfmfmmm, mff, mffm, mffmf, mffmff, mffmffm, mffmmffmm, mfmfm, mfmfmfmfmfffmfmfmfmmm, mfmfmfmfmfmfmfm, mfmfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmfmfmmm, mfmfmfmfmfmfmfmfmm, mfmfmfmfmfmfmfmm, mfmfmfmfmfmfmm, mfmfmfmfmfmfmmm, mfmfmfmfmfmmm, mfmfmfmfmfmmmfm, mfmfmfmfmfmmmmm, mfmfmfmfmmm, mfmfmfmfmmmfmfm, mfmfmfmfmmmfmmm, mfmfmfmfmmmmmfm, mfmfmfmmm, mfmfmfmmmfmfmfm, mfmfmfmmmfmfmmm, mfmfmfmmmfmfmmmfm, mfmfmfmmmfmmmfmfm,mfmfmmmmmfmfmfm,mfmmm,mfmmmfmfmfmfmfm,mfmmmfmfmfmfmmm,mfmmmfmfmfmmmfm , mfmmmfmfmmmfmfm, mfmmmfmmmfmfmfm, mfmmmfmmmfmfmfm, mfmmmmmfmfmfmfm, mmffm , mmffmm, mmffmm, mmffmmf, mmffmmff, mmffmmffm, mmffmmffmm, mmffmmfmfmfmmm, mmm, mmmffmmfmfmfmmm, mmmfmfmfmfmfmfm, mmmfmfmfmfmfmfmmm, mmmfmfmfmfmmmfm, mmmf mfmfmmmfmfm, mmmfmfmmmfmfmfm, mmmfmmmfmfmfmfm, mmmmffmmfmfmfmmm, mmm, mmmm, mmmmm, mmmmffmmfmfmfmmm, mmmmmmfmfmfmfmfm, mmmmmm, mmmmmmffmmfmfmfmmm, mfmf ,mfmf,mfmfmf,fmfm,fmfmfm,fmfmfmf,dfdf,dfdfdf,dfdfdfdf,fdfd,fdfdfd,fdfd fdfd, dfdfmfmf, dfmfmf, mfdfmf, or dfmfdf, where m represents 2′-OMe, f represents 2′-F, and d represents no substitution at the 2′-position. In some implementations, the seed region and / or post-seed region may contain sugar-modified blocks.

[0139] In some embodiments, the block is a stereochemical block. In some embodiments, the block is an Rp block because the internucleotide link of each nucleotide in the block is Rp. In some embodiments, the seed region-block is an Rp block. In some embodiments, the post-seed region-block is an Rp block. In some embodiments, the block is an Sp block because the internucleotide link of each nucleotide in the block is Sp. In some embodiments, the seed region-block is an Sp block. In some embodiments, the post-seed region-block is an Sp block. In some embodiments, the provided oligonucleotide comprises both Rp and Sp blocks. In some embodiments, the provided oligonucleotide comprises one or more Rp blocks but does not comprise Sp blocks. In some embodiments, the provided oligonucleotide comprises one or more Sp blocks but does not comprise Rp blocks. In some embodiments, the provided oligonucleotide comprises one or more PO blocks, wherein the internucleotide link of each nucleotide in the block is a native phosphate ester link.

[0140] In some embodiments, the seed region-block is an Sp block, wherein each sugar moiety contains a 2′-F modification. In some embodiments, the seed region-block is an Sp block, wherein each intermolecular link is a modified intermolecular link, and each sugar moiety contains a 2′-F modification. In some embodiments, the seed region-block is an Sp block, wherein each intermolecular link is a phosphate thioester link, and each sugar moiety contains a 2′-F modification. In some embodiments, the seed region-block contains four or more nucleoside units. In some embodiments, the nucleoside unit is a nucleoside. In some embodiments, the seed region-block contains five or more nucleoside units. In some embodiments, the seed region-block contains six or more nucleoside units. In some embodiments, the seed region-block contains seven or more nucleoside units. In some embodiments, the post-seed region-block is an Sp block, wherein each sugar moiety contains a 2′-F modification. In some embodiments, the post-seed region-block is an Sp block, wherein each internucleotide link is a modified internucleotide link, and each sugar moiety contains a 2′-F modification. In some embodiments, the post-seed region-block is an Sp block, wherein each internucleotide link is a phosphate thioester link, and each sugar moiety contains a 2′-F modification. In some embodiments, the post-seed region-block contains four or more nucleoside units. In some embodiments, the post-seed region-block contains five or more nucleoside units. In some embodiments, the post-seed region-block contains six or more nucleoside units. In some embodiments, the post-seed region-block contains seven or more nucleoside units. In some embodiments, the seed region and / or post-seed region may contain blocks. In some embodiments, the seed region and / or post-seed region contains stereochemical blocks.

[0141] In some embodiments, the nucleoside type in a region, block, or oligonucleotide is followed by a specified type of internucleotide link, such as a natural phosphate ester link, a modified internucleotide link, an Rp chiral internucleotide link, a Sp chiral internucleotide link, etc. In some embodiments, A is followed by a Sp nucleotide link. In some embodiments, A is followed by an Rp nucleotide link. In some embodiments, A is followed by a natural phosphate ester link (PO). In some embodiments, U is followed by a Sp nucleotide link. In some embodiments, U is followed by an Rp nucleotide link. In some embodiments, U is followed by a natural phosphate ester link (PO). In some embodiments, C is followed by a Sp nucleotide link. In some embodiments, C is followed by an Rp nucleotide link. In some embodiments, C is followed by a natural phosphate ester link (PO). In some embodiments, G is followed by a Sp nucleotide link. In some embodiments, G is followed by an Rp nucleotide link. In some embodiments, G is followed by a natural phosphate ester link (PO). In some embodiments, C and U are followed by Sp nucleotide links. In some embodiments, C and U are followed by an Rp nucleotide linker. In some embodiments, C and U are followed by a natural phosphate ester linker (PO). In some embodiments, A and G are followed by an Sp nucleotide linker. In some embodiments, A and G are followed by an Rp nucleotide linker. In some embodiments, A and G are followed by a natural phosphate ester linker (PO).

[0142] In some embodiments, the provided oligonucleotide comprises alternating blocks comprising modified sugar moieties and unmodified sugar moieties. In some embodiments, the modified sugar moieties comprise 2′-modification. In some embodiments, the provided oligonucleotide comprises alternating 2′-OMe-modified sugar moieties and unmodified sugar moieties.

[0143] In some embodiments, the provided oligonucleotide comprises one or more 2′-F modified sugar moieties, wherein the 3′-nucleotide inter-linking is a modified nucleotide inter-linking. In some embodiments, the modified nucleotide inter-linking is a phosphate thioester. In some embodiments, the modified nucleotide inter-linking is chiral controlled and is Rp. In some embodiments, the modified nucleotide inter-linking is chiral controlled and is Sp. In some embodiments, the provided oligonucleotide comprises one or more 2′-OR 1 The modified sugar moiety has natural phosphate ester bonds between its 3′-nucleotides.

[0144] In some embodiments, the provided oligonucleotide has a main-chain chiral center pattern comprising (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m (unless otherwise specified, the description of the modified and stereochemical pattern is as commonly used in the art from 5′ to 3′). In some embodiments, the provided main-chain chiral center pattern comprises (Sp)m(Rp)n. In some embodiments, the provided main-chain chiral center pattern comprises (Rp)n(Sp)m. In some embodiments, the provided main-chain chiral center pattern comprises (Np)t(Rp)n(Sp)m. In some embodiments, the provided main-chain chiral center pattern comprises (Np)tRp(Sp)m. In some embodiments, the provided main-chain chiral center pattern comprises (Sp)tRp(Sp)m. In some implementations, the provided mainchain chiral center pattern includes repeating (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m units. In some implementations, the repeating unit is (Sp)m(Rp)n. In some implementations, the repeating unit is SpRp. In some implementations, the repeating unit is SpSpRp. In some implementations, the repeating unit is RpRpSp. In some implementations, the repeating unit is (Rp)n(Sp)m. In some implementations, the repeating unit is (Np)t(Rp)n(Sp)m. In some implementations, the repeating unit is (Sp)t(Rp)n(Sp)m.

[0145] In some implementations, t, n, and m are each independently 1-20. In some implementations, n is 1. In some implementations, m is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some implementations, m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some implementations, t is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some implementations, t is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some implementations, m and t are each independently at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 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, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, m is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, t is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0146] In some implementations, regions containing main-chain chiral centers of patterns or repeating patterns of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m (structurally starting from the first and ending at the last, having internucleotide linkages of patterns or repeating patterns of (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m; "(repeated)(Sp)m(Rp)n region", "(repeated)((Rp)n(Sp)m region", "(repeated)(Np)t(Rp)n(Sp)m region", or "(repeated)(Sp)n(Rp)n(Sp)m region" are included. The (Sp)t(Rp)n(Sp)m region depends on whether it is repeated or not. For example, the (Sp)t(Rp)n(Sp)m region ((Sp)7(Rp)1(Sp)3) in WV-2555: mA*SmGmCmUmU*SC*ST*ST*SG*ST*SC*SC*RA*SG*SC*SmUmUmUmA*SmU) does not contain 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 the unmodified natural 2′-deoxyribose moiety of DNA. In some embodiments, it contains a main-chain chiral center (which contains or (S A 5′-terminal region is flanked by a region of the pattern or repeating pattern of (p)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m), which structurally terminates at the 3′ end of a nucleoside moiety (which is connected at its 3′ end to the first nucleotide of a region of the pattern or repeating pattern of the main chain chiral center (which contains either (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m). For example, the flanked 5′-terminal region in WV-2555: mA*SmGmCmUmU*SC*ST*ST*SG*ST*SC* SC*RA*SG*SC*SmUmUmUmA*SmU). In some embodiments, a region containing a main-chain chiral center (which contains either (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m or (Sp)t(Rp)n(Sp)m)) is side-connected to a 3′-terminal region, which structurally begins at the nucleotide moiety (which is connected at its 5′-terminus to the last nucleotide of the region containing a main-chain chiral center (which contains either (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m or (Sp)t(Rp)n(Sp)m)).For example, the 3′-terminal regions flanked in WV-2555 are: mA*SmGmCmUmU*SC*ST*ST*SG*ST*SC*SC*RA*SG*SC*SmUmUmUmA*SmU). In some embodiments, regions containing a pattern or repeating pattern of main-chain chiral centers are flanked by 5′-terminal and 3′-terminal regions, the main-chain chiral centers comprising (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 flanked 5′-terminal and / or 3′-terminal regions contain modified internucleotide bonds. In some embodiments, the flanked 5′-terminal and / or 3′-terminal regions contain modified internucleotide bonds comprising Sp-linked phosphates. In some embodiments, the flanked 5′-terminal and / or 3′-terminal regions contain Sp-thiophosphate bonds. In some embodiments, the side-attached 5′-terminal region and / or 3′-terminal region comprises one or more natural phosphate ester links. In some embodiments, the side-attached 5′-terminal region and / or 3′-terminal region comprises one or more consecutive natural phosphate ester links. In some embodiments, the side-attached 5′-terminus comprises only one modified internucleotide link (i.e., a 5′-terminal internucleotide link) and one or more consecutive natural phosphate ester links (e.g., in WV-2555: mA*SmGmCmUmU*SC*ST*ST*SG*ST*SC*SC*RA*SG*SC*SmUmUmUmA*SmU(SOOOSSSSSSSRSSSOOOS)). In some embodiments, the side-attached 3′-terminus contains only one modified internucleotide link (i.e., a 3′-terminal internucleotide link) and one or more consecutive natural phosphate ester links (e.g., in WV-2555: mA*SmGmCmUmU*SC*ST*ST*SG*ST*SC*SC*RA*SG*SC*SmUmUmUmA*SmU(SOOOSSSSSSSRSSSOOOS)). In some embodiments, the side-attached 5′-terminal region and / or 3′-terminal region contains a 2′-modified sugar unit. In some embodiments, each sugar unit in the 5′-terminal region and / or 3′-terminal region is modified independently. In some embodiments, each sugar unit in the 5′-terminal region and / or 3′-terminal region independently contains a 2′-modification (e.g., in WV-2555: mA*SmGmCmUmU*SC*ST*ST*SG*ST*SC*SC*RA*SG*SC*SmUmUmUmA*SmU.). In some embodiments, each sugar unit in the 5′-terminal region and / or 3′-terminal region contains the same 2′-modification.In some embodiments, the 2′-modification is 2′-OR, where R is an optionally substituted C1-6 aliphatic compound. 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 (which includes the type of C2-C4 bridge).

[0147] In some implementations, the provided main chain chiral center pattern includes (Rp / Sp)-(all Rp or all Sp)-(Rp / Sp). In some implementations, the provided main chain chiral center pattern includes (Rp)-(all Sp)-(Rp). In some implementations, the provided main chain chiral center pattern includes (Sp)-(all Sp)-(Sp). In some implementations, the provided main chain chiral center pattern includes (Sp)-(all Rp)-(Sp). In some implementations, the provided main chain chiral center pattern includes (Rp / Sp)-(repeating (Sp)m(Rp)n)-(Rp / Sp). In some implementations, the provided main chain chiral center pattern includes (Rp / Sp)-(repeating SpSpRp)-(Rp / Sp).

[0148] In some embodiments, the provided main-chain chiral center pattern is (Rp / Sp)-(all Rp or all Sp)-(Rp / Sp). In some embodiments, the provided main-chain chiral center pattern is (Sp)-(all Sp)-(Sp). In some embodiments, the inter-chiral nucleotide bond is Sp. In some embodiments, the provided main-chain chiral center pattern is (Rp)-(all Sp)-(Rp). In some embodiments, the provided main-chain chiral center pattern is (Sp)-(all Rp)-(Sp). In some embodiments, the provided main-chain chiral center pattern is (Rp / Sp)-(repeating (Sp)m(Rp)n)-(Rp / Sp). In some embodiments, the provided main-chain chiral center pattern is (Rp / Sp)-(repeating SpSpRp)-(Rp / Sp).

[0149] In some implementations, the seed region and / or the post-seed region or any part thereof may contain a pattern of main chain chiral centers.

[0150] In some embodiments, the provided oligonucleotides contain a specific type of internucleotide linking, which alternates with different types of internucleotide links. In some embodiments, the various types of internucleotide links include, but are not limited to, phosphodiester, thiophosphate, stereorandom thiophosphate, stereocontrolled thiophosphate (Rp or Sp), dithiophosphate, or any other type of internucleotide linking described herein or known in the art.

[0151] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides are:

[0152] 1) They share a common base sequence; and

[0153] 2) It contains one or more modified sugar moieties and modified nucleotides linked together.

[0154] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the first plurality of oligonucleotides are:

[0155] 1) It has a common base sequence complementary to the target sequence in the transcript; and

[0156] 2) It contains one or more modified sugar moieties and modified nucleotides linked together.

[0157] In some embodiments, the reference condition is the absence of the composition. In some embodiments, the reference condition is the presence of a reference composition. Exemplary reference compositions comprising a reference plurality of oligonucleotides are fully described in this disclosure. In some embodiments, the reference plurality of oligonucleotides have different structural elements (chemical modifications, stereochemistry, etc.) compared to the first plurality of oligonucleotides in the provided composition. In some embodiments, the provided oligonucleotide composition comprising the first plurality of oligonucleotides is chiral-controlled because the first plurality of oligonucleotides contains one or more chiral-controlled internucleotide links. In some embodiments, the provided oligonucleotide composition comprising the first plurality of oligonucleotides is chiral-controlled because the first plurality of oligonucleotides contains 1-20 chiral-controlled internucleotide links. In some embodiments, the first plurality of oligonucleotides contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 chiral-controlled internucleotide links. In some embodiments, the reference composition is a stereorandom article of oligonucleotides having the same chemical modifications. In some embodiments, the reference composition is a mixture of stereoisomers, while the provided composition is a stereoisomer of a single-stranded RNAi agent. In some embodiments, in the provided composition, the reference oligonucleotides have the same base sequence as the first oligonucleotides. In some embodiments, in the provided composition, the reference oligonucleotides have the same chemical modifications as the first oligonucleotides. In some embodiments, in the provided composition, the reference oligonucleotides have the same sugar modifications as the first oligonucleotides. In some embodiments, in the provided composition, the reference oligonucleotides have the same base modifications as the first oligonucleotides. In some embodiments, in the provided composition, the reference oligonucleotides have the same internucleotide linking modifications as the first oligonucleotides. In some embodiments, in the provided composition, the reference oligonucleotides have the same base sequence and the same chemical modifications as the first oligonucleotides. In some embodiments, in the provided composition, the reference oligonucleotides have the same stereochemistry as the first oligonucleotides but with different chemical modifications, such as base modifications, sugar modifications, internucleotide linking modifications, etc.

[0158] In some embodiments, this disclosure provides compositions comprising oligonucleotides, wherein the oligonucleotides are complementary or substantially complementary to a target RNA sequence and have a total length of about 15 to about 49 nucleotides, wherein the oligonucleotides comprise at least one non-natural base, sugar, and / or internucleotide bond.

[0159] In some embodiments, this 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 total length of about 15 to about 49 nucleotides, and is capable of directing target-specific RNA interference, wherein the single-stranded RNAi agent comprises at least one non-natural base, sugar, and / or nucleotide linker.

[0160] In some implementations, the length is a total of 15 to 49, about 17 to about 49, 17 to 49, about 19 to about 29, 19 to 29, about 19 to about 25, 19 to 25, about 19 to about 23, or 19 to 23 nucleotides.

[0161] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides are:

[0162] 1) It has a common base sequence that is complementary to or substantially complementary to the target sequence in the transcript; and

[0163] 2) Contains one or more modified sugar moieties and modified nucleotides linked together.

[0164] The oligonucleotide composition is characterized in that, when it is contacted with a transcript, the knockdown of the transcript is improved compared to the knockdown observed under reference conditions selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0165] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein the first plurality of oligonucleotides are:

[0166] 1) It has a common base sequence complementary to the target sequence in the transcript; and

[0167] 2) Contains one or more modified sugar moieties and modified nucleotides linked together.

[0168] The oligonucleotide composition is characterized in that, when it comes into contact with transcripts in an RNA interference system, the RNAi-mediated knockdown of the transcripts is improved compared to the knockdown observed under reference conditions selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0169] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, wherein the first plurality of oligonucleotides belongs to a specific oligonucleotide type, which is defined as follows:

[0170] 1) Base sequence;

[0171] 2) Main chain key style;

[0172] 3) Main chain chiral center pattern; and

[0173] 4) Main chain phosphorus modification patterns.

[0174] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference, wherein said first plurality of oligonucleotides belong to a specific oligonucleotide type, defined as follows:

[0175] 1) Base sequence;

[0176] 2) Main chain key style;

[0177] 3) Main chain chiral center pattern; and

[0178] 4) Main chain phosphorus modification patterns.

[0179] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides belonging to a certain oligonucleotide type, wherein the oligonucleotide type is defined by the following:

[0180] 1) Base sequence;

[0181] 2) Main chain key style;

[0182] 3) Main chain chiral center pattern; and

[0183] 4) Main-chain phosphorus modification patterns,

[0184] The composition is chiral controlled because it is rich in oligonucleotides belonging to a specific oligonucleotide type, relative to substantially racemic articles of oligonucleotides having the same base sequence.

[0185] The oligonucleotide composition is characterized in that, when it is contacted with a transcript, the knockdown of the transcript is improved compared to the knockdown observed under reference conditions selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0186] In some embodiments, this disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides, the first plurality of oligonucleotides being capable of directing single-stranded RNA interference and belonging to a certain oligonucleotide type, wherein the oligonucleotide type is defined as follows:

[0187] 1) Base sequence;

[0188] 2) Main chain key style;

[0189] 3) Main chain chiral center pattern; and

[0190] 4) Main-chain phosphorus modification patterns,

[0191] The composition is chiral controlled because it is rich in oligonucleotides belonging to a specific oligonucleotide type, relative to substantially racemic articles of oligonucleotides having the same base sequence.

[0192] The oligonucleotide composition is characterized in that, when it comes into contact with transcripts in an RNA interference system, the RNAi-mediated knockdown of the transcripts is improved compared to the knockdown observed under reference conditions selected from the group consisting of the absence of the composition, the presence of the reference composition, and combinations thereof.

[0193] In some embodiments, the provided oligonucleotide has any format shown in Figure 1, or any structural element of any format shown in Figure 1.

[0194] In some embodiments, the provided single-stranded RNAi agent has any of the formats shown in Figure 1, or any structural elements of any format shown in Figure 1.

[0195] In particular, the data provided in this disclosure demonstrate that various oligonucleotides in the disclosed formats can direct the reduction of expression and / or levels of a target gene or its gene product (when it targets any of several different sequences) in any of several different genes. In some embodiments, the data provided in this disclosure demonstrate that various RNAi agents in the disclosed formats can direct RNA interference in any of many different genes against any of many different sequences.

[0196] In some embodiments, the oligonucleotide has format 1. In some embodiments, the oligonucleotide has format 2. In some embodiments, the oligonucleotide has format 3. In some embodiments, the oligonucleotide has format 4. In some embodiments, the oligonucleotide has format 5. In some embodiments, the oligonucleotide has format 6. In some embodiments, the oligonucleotide has format 7. In some embodiments, the oligonucleotide has format 8. In some embodiments, the oligonucleotide has format 9. In some embodiments, the oligonucleotide has format 10. In some embodiments, the oligonucleotide has format 11. In some embodiments, the oligonucleotide has format 12. In some embodiments, the oligonucleotide has format 13. In some embodiments, the oligonucleotide has format 14. In some embodiments, the oligonucleotide has format 15. In some embodiments, the oligonucleotide has format 16. In some embodiments, the oligonucleotide has format 17. In some embodiments, the oligonucleotide has format 18. In some embodiments, the oligonucleotide has format 19. In some embodiments, the oligonucleotide has format 20. In some embodiments, the oligonucleotide has format 21. In some embodiments, the oligonucleotide has format 22. In some embodiments, the oligonucleotide has format 23. In some embodiments, the oligonucleotide has format 24. In some embodiments, the oligonucleotide has format 25. In some embodiments, the oligonucleotide has format 26. In some embodiments, the oligonucleotide has format 27. In some embodiments, the oligonucleotide has format 28. In some embodiments, the oligonucleotide has format 29. In some embodiments, the oligonucleotide has format 30. In some embodiments, the oligonucleotide has format 31. In some embodiments, the oligonucleotide has format 32. In some embodiments, the oligonucleotide has format 33. In some embodiments, the oligonucleotide has format 34. In some embodiments, the oligonucleotide has format 35. In some embodiments, the oligonucleotide has format 36. In some embodiments, the oligonucleotide has format 37. In some embodiments, the oligonucleotide has format 38. In some embodiments, the oligonucleotide has format 39. In some embodiments, the oligonucleotide has format 40. In some embodiments, the oligonucleotide has format 41. In some embodiments, the oligonucleotide has format 42. In some embodiments, the oligonucleotide has format 43. In some embodiments, the oligonucleotide has format 44. In some embodiments, the oligonucleotide has format 45. In some embodiments, the oligonucleotide has format 46. In some embodiments, the oligonucleotide has format 47. In some embodiments, the oligonucleotide has format 48. In some embodiments, the oligonucleotide has format 49. In some embodiments, the oligonucleotide has format 50.In some embodiments, the oligonucleotide has format 51. In some embodiments, the oligonucleotide has format 52. In some embodiments, the oligonucleotide has format 53. In some embodiments, the oligonucleotide has format 54. In some embodiments, the oligonucleotide has format 55. In some embodiments, the oligonucleotide has format 56. In some embodiments, the oligonucleotide has format 57. In some embodiments, the oligonucleotide has format 58. In some embodiments, the oligonucleotide has format 59. In some embodiments, the oligonucleotide has format 60. In some embodiments, the oligonucleotide has format 61. In some embodiments, the oligonucleotide has format 62. In some embodiments, the oligonucleotide has format 63. In some embodiments, the oligonucleotide has format 64. In some embodiments, the oligonucleotide has format 65. In some embodiments, the oligonucleotide has format 66. In some embodiments, the oligonucleotide has format 67. In some embodiments, the oligonucleotide has format 68. In some embodiments, the oligonucleotide has format 69. In some embodiments, the oligonucleotide has format 70. In some embodiments, the oligonucleotide has format 71. In some embodiments, the oligonucleotide has format 72. In some embodiments, the oligonucleotide has format 73. In some embodiments, the oligonucleotide has format 74. In some embodiments, the oligonucleotide has format 75. In some embodiments, the oligonucleotide has format 76. In some embodiments, the oligonucleotide has format 77. In some embodiments, the oligonucleotide has format 78. In some embodiments, the oligonucleotide has format 79. In some embodiments, the oligonucleotide has format 80. In some embodiments, the oligonucleotide has format 81. In some embodiments, the oligonucleotide has format 82. In some embodiments, the oligonucleotide has format 83. In some embodiments, the oligonucleotide has format 84. In some embodiments, the oligonucleotide has format 85. In some embodiments, the oligonucleotide has format 86. In some embodiments, the oligonucleotide has format 87. In some embodiments, the oligonucleotide has format 88. In some embodiments, the oligonucleotide has format 89. In some embodiments, the oligonucleotide has format 90. In some embodiments, the oligonucleotide has format 91. In some embodiments, the oligonucleotide has format 92. In some embodiments, the oligonucleotide has format 93. In some embodiments, the oligonucleotide has format 94. In some embodiments, the oligonucleotide has format 95. In some embodiments, the oligonucleotide has format 96. In some embodiments, the oligonucleotide has format 97. In some embodiments, the oligonucleotide has format 98. In some embodiments, the oligonucleotide has format 99. In some embodiments, the oligonucleotide has format 100.In some embodiments, the oligonucleotide has format 101. In some embodiments, the oligonucleotide has format 102. In some embodiments, the oligonucleotide has format 103. In some embodiments, the oligonucleotide has format 104. In some embodiments, the oligonucleotide has format 105. In some embodiments, the oligonucleotide has format 106. In some embodiments, the oligonucleotide has format 107. Tables 71A to 71C show various non-limiting examples of stereocontrolled (chiral controlled) oligonucleotide formats. In some embodiments, the oligonucleotide has format S1. In some embodiments, the oligonucleotide has format S2. In some embodiments, the oligonucleotide has format S3. In some embodiments, the oligonucleotide has format S4. In some embodiments, the oligonucleotide has format S5. In some embodiments, the oligonucleotide has format S6. In some embodiments, the oligonucleotide has format S7. In some embodiments, the oligonucleotide has format S8. In some embodiments, the oligonucleotide has format S9. In some embodiments, the oligonucleotide has format S10. In some embodiments, the oligonucleotide has format S11. In some embodiments, the oligonucleotide has format S12. In some embodiments, the oligonucleotide has format S13. In some embodiments, the oligonucleotide has format S14. In some embodiments, the oligonucleotide has format S15. In some embodiments, the oligonucleotide has format S16. In some embodiments, the oligonucleotide has format S17. In some embodiments, the oligonucleotide has format S18. In some embodiments, the oligonucleotide has format S19. In some embodiments, the oligonucleotide has format S20. In some embodiments, the oligonucleotide has format S21. In some embodiments, the oligonucleotide has format S22. In some embodiments, the oligonucleotide has format S23. In some embodiments, the oligonucleotide has format S24. In some embodiments, the oligonucleotide has format S25. In some embodiments, the oligonucleotide has format S26. In some embodiments, the oligonucleotide has format S27. In some embodiments, the oligonucleotide has format S28. In some embodiments, the oligonucleotide has format S29. In some embodiments, the oligonucleotide has format S30. In some embodiments, the oligonucleotide has format S31. In some embodiments, the oligonucleotide has format S32. In some embodiments, the oligonucleotide has format S33. In some embodiments, the oligonucleotide has format S34. In some embodiments, the oligonucleotide has format S35. In some embodiments, the oligonucleotide has format S36. In some embodiments, the oligonucleotide has format S37. In some embodiments, the oligonucleotide has format S38. In some embodiments, the oligonucleotide has format S39.In some embodiments, the oligonucleotide has format S40. In some embodiments, the oligonucleotide has format S41. In some embodiments, the oligonucleotide has format S42. In some embodiments, the oligonucleotide has format S43. In some embodiments, the oligonucleotide has format S44.

[0197] In some embodiments, an oligonucleotide having any of the structures described and / or shown herein is capable of directing RNA interference. In some embodiments, an oligonucleotide having any of the structures described and / or shown herein is capable of directing RNase H-mediated knockdown. In some embodiments, an oligonucleotide having any of the structures described and / or shown herein is capable of directing RNA interference and / or RNase H-mediated knockdown. In some embodiments, the oligonucleotide comprises any oligonucleotide described herein or any structural element of any format described herein or shown in Figure 1. In some embodiments, the oligonucleotide comprises any oligonucleotide described herein or any structural element of any format described herein or shown in Figure 1 and is capable of directing RNA interference. In some embodiments, the oligonucleotide comprises any oligonucleotide described herein or any structural element of any format described herein or shown in Figure 1 and is capable of directing RNase H-mediated knockdown. In some embodiments, the oligonucleotide comprises any oligonucleotide described herein or any structural element of any format described herein or shown in Figure 1 and is capable of directing RNA interference and / or RNase H-mediated knockdown.

[0198] In some embodiments, the RNAi agent comprises one or more of the following: a 5′-terminal structure, a 5′-terminal region, a seed region, a post-seed region, and a 3′-terminal region, and optional additional chemical moieties. In some embodiments, the seed region is any seed region described herein or known in the art. In some embodiments, the post-seed region can be any region between the seed region described herein or known in the art and the 3′-terminal region. In some embodiments, the 3′-terminal region can be any 3′-terminal region described herein or known in the art. In some embodiments, any optional additional chemical moieties can be any optional additional chemical moieties described herein or known in the art. Any individual 5′-terminal structure, 5′-terminal region, seed region, post-seed region, 3′-terminal region, and optional additional chemical moieties described herein or known in the art can be independently combined with any other 5′-terminal structure, 5′-terminal region, seed region, post-seed region, 3′-terminal region, and optional additional chemical moieties described herein or known in the art. In some implementations, as a non-limiting example, the region of a single-stranded RNAi agent is a 5′-terminal structure, a 5′-terminal region, a seed region, a post-seed region, a portion of a seed region, a portion of a post-seed region, or a 3′-terminal dinucleotide.

[0199] 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 includes the base sequence of any oligonucleotide disclosed herein. In some embodiments, the base sequence of the provided oligonucleotide comprises a sequence of 15 consecutive bases of any oligonucleotide base sequence disclosed herein. In some embodiments, the base sequence of the provided oligonucleotide comprises a sequence of 20 consecutive bases (with up to 5 mismatches) of any oligonucleotide base sequence disclosed herein.

[0200] In some embodiments, the provided oligonucleotide is capable of directing the reduction of expression and / or levels of a target gene or its gene product. In some embodiments, the provided oligonucleotide is capable of directing single-stranded RNAi interference. In some embodiments, the provided oligonucleotide is capable of directing RNase H-mediated knockdown. In some embodiments, the provided oligonucleotide is capable of directing both single-stranded RNAi interference and RNase H-mediated knockdown. In some embodiments, the oligonucleotide comprises a sequence targeting any transcript or gene targeted by the oligonucleotides disclosed herein.

[0201] In some embodiments, the provided oligonucleotide targets ACVR2B or MSTN-R. In some embodiments, the provided oligonucleotide targets APOB. In some embodiments, the provided oligonucleotide targets APOC3. In some embodiments, the provided oligonucleotide targets FXI (factor XI). In some embodiments, the provided oligonucleotide targets KRT14. In some embodiments, the provided oligonucleotide targets MSTN. In some embodiments, the provided oligonucleotide targets PCSK9.

[0202] In some implementations, the provided oligonucleotide targets PNPLA3.

[0203] In some embodiments, the 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, the provided oligonucleotides can be used to reduce or inhibit the activity, level, and / or expression of a gene or its gene product, wherein abnormal or excessive activity, level, or / or expression of the gene or its gene product, a harmful mutation in the gene or its gene product, or the tissue or intercellular or intracellular distribution of the gene or its gene product is associated with, causes, and / or is related to a disease. In some embodiments, the provided oligonucleotides can be used to treat a disease and / or prepare medicaments for treating a disease, said disease being associated with, caused by, or related to abnormal or excessive activity, level, and / or expression, or abnormal distribution of a gene or its gene product.

[0204] In some embodiments, the provided oligonucleotides can be used to treat conditions related to a specific gene or gene product, or to prepare medicaments for treating conditions related to a specific gene or gene product. In some embodiments, this disclosure relates to methods using the oligonucleotides disclosed herein, said oligonucleotides being able to target ACVR2B and being used to treat ACVR2B-related conditions and / or to prepare treatments for ACVR2B-related conditions. In some embodiments, this disclosure relates to methods using the oligonucleotides disclosed herein, said oligonucleotides being able to target APOB and being used to treat APOB-related conditions and / or to prepare treatments for APOB-related conditions.

[0205] In some embodiments, this disclosure relates to methods using oligonucleotides disclosed herein that are capable of targeting APOC3 and can be used to treat APOC3-related conditions and / or to prepare treatments for APOC3-related conditions.

[0206] In some embodiments, this disclosure relates to methods using the oligonucleotides disclosed herein, said oligonucleotides being capable of targeting FXI (factor XI) and used to treat FXI (factor XI)-related conditions and / or to prepare treatments for FXI (factor XI)-related conditions. In some embodiments, this disclosure relates to methods using the oligonucleotides disclosed herein, said oligonucleotides being capable of targeting KRT or KRT14 and used to treat KRT or KRT14-related conditions and / or to prepare treatments for KRT or KRT14-related conditions. In some embodiments, this disclosure relates to methods using the oligonucleotides disclosed herein, said oligonucleotides being capable of targeting myostatin (MSTN) and used to treat MSTN-related conditions and / or to prepare treatments for MSTN-related conditions. In some embodiments, this disclosure relates to methods using the oligonucleotides disclosed herein, said oligonucleotides being capable of targeting PCSK9 and used to treat PCSK9-related conditions and / or to prepare treatments for PCSK9-related conditions.

[0207] In some embodiments, this disclosure relates to methods using oligonucleotides disclosed herein, said oligonucleotides being able to target PNPLA3 and being used to treat PNPLA3-related conditions and / or to prepare treatments for PNPLA3-related conditions.

[0208] In some embodiments, the oligonucleotide capable of targeting a gene comprises a base sequence that is a portion of the target gene's base sequence or is complementary to or substantially complementary to a portion of the target gene's base sequence. In some embodiments, the portion is at least 15 bases in length. In some embodiments, the base sequence of the single-stranded RNAi agent may comprise or consist of a base sequence having a specified maximum number of mismatches with a specified base sequence.

[0209] In some implementations, a mismatch is a difference in the base sequence or length when two sequences are aligned and compared to the maximum extent. As a non-limiting example, a mismatch is defined as a difference between a base at a specific position in one sequence and a corresponding base at a position in another sequence. Thus, for example, a mismatch is defined as a position in one sequence having a specific base (e.g., A) and a corresponding position in another sequence having a different base (e.g., G, C, or U). Similarly, a mismatch is defined as a position in one sequence having a base (e.g., A) and a corresponding position in another sequence lacking a base (e.g., the position being a baseless nucleotide containing a phosphate sugar backbone but without a base) or skipping the position. Single-stranded cuts in either sequence (or sense or antisense strand) may not be considered mismatches; for example, if one sequence contains the sequence 5′-AG-3′ and another sequence contains the sequence 5′-AG-3′ with a single-stranded gap between A and G, it will not be considered a mismatch. Base modifications are generally not considered mismatches. For example, if one sequence contains a C and another sequence contains a modified C at the same position (e.g., 5mC), it is not considered a mismatch. In some embodiments, replacing U with T or vice versa is not considered a mismatch for the purpose of counting mismatches.

[0210] In some embodiments, the oligonucleotide is complementary or fully or 100% complementary to the target sequence (e.g., RNA, such as mRNA), meaning that the base sequence of the oligonucleotide has no mismatch with a sequence that is fully complementary to the target gene (e.g., base pairs paired via Watson-Crick base pairing). Not wishing to be bound by any particular theory, this disclosure states that for single-stranded RNAi agents, the 5′-terminal nucleotide portion or the 3′-terminal dinucleotide does not necessarily need to pair with the target bases. These can be mismatches. Furthermore, antisense oligonucleotides or single-stranded RNAi agents may have a small number of internal mismatches and still direct the expression and / or reduction of the level of the target gene or its gene product, and / or direct knockdown and / or RNA interference mediated by RNase H. If the first base sequence of the oligonucleotide (e.g., an antisense oligonucleotide or single-stranded RNAi agent) has a small number of mismatches with a reference base sequence that is 100% complementary to the target sequence, then the first base sequence is substantially complementary to the target sequence. In some embodiments, the oligonucleotide (e.g., antisense oligonucleotide or single-stranded RNAi agent) may have a base sequence that is complementary to or substantially complementary to the target sequence. In some embodiments, complementarity is determined based on Watson-Crick base pairings (guanine-cytosine and adenine-thymine / uracil), wherein guanine, cytosine, adenine, thymine, and uracil may be optionally and independently modified, but retain their unmodified pairing hydrogen bond pattern. In some embodiments, the sequence complementary to another sequence contains at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases.

[0211] In some embodiments, the oligonucleotides, oligonucleotide compositions, or oligonucleotide types have a common backbone linking pattern. In some embodiments, the common backbone linking pattern comprises at least 10 modified nucleotide links.

[0212] In some embodiments, the common main-chain linking pattern comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises at least 15 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises at least 19 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises no more than 19 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises no more than 15 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises 11 to 21 modified nucleotide links. In some embodiments, the common main-chain linking pattern comprises 0 phosphodiester. In some embodiments, the common backbone linking pattern comprises one phosphodiester. In some embodiments, the common backbone linking pattern comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 phosphodiesters. In some embodiments, the common backbone linking pattern comprises at least 2 to 19 phosphodiesters. In some embodiments, the phosphodiesters are optionally continuous or discontinuous. In some embodiments, the internucleotide linkages are optionally continuous or discontinuous.

[0213] In some embodiments, the common main-chain bonding pattern comprises at least 10 thiophosphate bonds. In some embodiments, the common main-chain bonding pattern comprises at least 11 thiophosphate bonds. In some embodiments, the common main-chain bonding pattern comprises at least 12 to 19 thiophosphate bonds. In some embodiments, the common main-chain bonding pattern comprises at least 12, 13, 14, 15, 16, 17, 18, 19, or 20 thiophosphate bonds. In some embodiments, the common main-chain bonding pattern comprises 0 phosphodiester bonds. In some embodiments, the common main-chain bonding pattern comprises 1 to 6 phosphodiester bonds and 13 to 19 thiophosphate bonds. In some embodiments, the phosphodiester bonds are optionally continuous or discontinuous. In some embodiments, the thiophosphate bonds are optionally continuous or discontinuous.

[0214] In some embodiments, the oligonucleotide, oligonucleotide composition, or oligonucleotide type has a common backbone chiral center pattern. In some embodiments, the common backbone chiral center pattern comprises at least one sp-configured intermolecular nucleotide link. In some embodiments, the common backbone chiral center pattern comprises at least one sp-configured phosphate thioester intermolecular nucleotide link.

[0215] In some embodiments, the common main-chain chiral center pattern comprises at least 5 internucleotide links with Sp configurations. In some embodiments, the common main-chain chiral center pattern comprises at least 6 to 19 internucleotide links with Sp configurations. In some embodiments, the common main-chain chiral center pattern comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 internucleotide links with Sp configurations. In some embodiments, the common main-chain chiral center pattern comprises no more than 8 internucleotide links with Rp configurations. In some embodiments, the common main-chain chiral center pattern comprises no more than 1 to 7 internucleotide links with Rp configurations. In some embodiments, the common main-chain chiral center pattern comprises no more than 8 internucleotide links that are not chiral (as a non-limiting example, phosphodiester). In some embodiments, the common main-chain chiral center pattern comprises no more than 1, 2, 3, 4, 5, 6, or 7 internucleotide links that are not chiral. In some embodiments, the common main-chain chiral center pattern comprises at least 10 Sp-configured nucleotide links and no more than 8 non-chiral nucleotide links. In some embodiments, the common main-chain chiral center pattern comprises at least 11 Sp-configured nucleotide links and no more than 7 non-chiral nucleotide links. In some embodiments, the common main-chain chiral center pattern comprises at least 12 Sp-configured nucleotide links and no more than 6 non-chiral nucleotide links. In some embodiments, the common main-chain chiral center pattern comprises at least 13 Sp-configured nucleotide links and no more than 6 non-chiral nucleotide links. In some embodiments, the common main-chain chiral center pattern comprises at least 14 Sp-configured nucleotide links and no more than 5 non-chiral nucleotide links. In some embodiments, the common main-chain chiral center pattern comprises at least 15 Sp-configured nucleotide links and no more than 4 non-chiral nucleotide links. In some embodiments, the Sp-configured nucleotide links are optionally continuous or discontinuous. In some embodiments, the internucleotide linkages of the Rp configuration are optionally continuous or discontinuous. In some embodiments, the internucleotide linkages of non-chiral nucleotides are optionally continuous or discontinuous.

[0216] In some embodiments, the oligonucleotides in the provided compositions have a common main-chain phosphorus modification pattern. In some embodiments, the provided compositions are chiral controlled oligonucleotide compositions because the compositions contain a predetermined level of a single oligonucleotide type, wherein the oligonucleotide type is defined by:

[0217] 1) Base sequence;

[0218] 2) Main chain key style;

[0219] 3) Main chain chiral center pattern; and

[0220] 4) Main chain phosphorus modification patterns.

[0221] As noted above and as understood in the art, in some embodiments, the base sequence of an oligonucleotide may refer to the identity and / or modification state of nucleoside residues in the oligonucleotide (e.g., sugar and / or base composition, relative to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize with specific complementary residues).

[0222] In some implementations, a specific oligonucleotide type can be defined as follows:

[0223] 1A) Base identity;

[0224] 1B) Base modification patterns;

[0225] 1C) Sugar-modified patterns;

[0226] 2) Main chain key style;

[0227] 3) Main chain chiral center pattern; and

[0228] 4) Main chain phosphorus modification patterns.

[0229] Therefore, in some embodiments, specific types of oligonucleotides may share the same bases, but have different patterns of base modifications and / or sugar modifications. In some embodiments, specific types of oligonucleotides may share the same base and base modification patterns (including, for example, the absence of base modifications), but have different patterns of sugar modifications.

[0230] In some embodiments, specific types of oligonucleotides are chemically identical because they have the same base sequence (including length), the same chemical modification patterns of sugar and base moieties, the same backbone bonding patterns (e.g., native phosphate ester bonding, non-negatively charged bonding, thiophosphate ester bonding, thiophosphate triester bonding, and combinations thereof), the same backbone chiral center pattern (e.g., stereochemical pattern of chiral nucleotide inter-linking (Rp / Sp)), and the same backbone phosphorus modification pattern (e.g., modification patterns of internucleotide phosphorus atoms (such as -S)). - ) and Equation I's -LR 1 ).

[0231] In some embodiments, this disclosure provides a chiral-controlled oligonucleotide composition comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) internucleotide bonds (particularly oligonucleotides comprising multiple (e.g., more than 5, 6, 7, 8, 9, or 10) chiral nucleotide bonds). In some embodiments, in the stereoselective or racemic articles of oligonucleotides, at least one chiral nucleotide bond is formed to have a diastereoselectivity of less than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral-controlled articles of oligonucleotides, each chiral nucleotide bond is formed to have a diastereoselectivity greater than 90:10, 95:5, 96:4, 97:3, or 98:2. In some embodiments, for stereoselective or chiral-controlled articles of oligonucleotides, each chiral nucleotide bond is formed to have a diastereoselectivity greater than 95:5. In some embodiments, for articles with stereoselectivity or chirality controlled by oligonucleotides, each chiral nucleotide link is formed with a diastereoselectivity greater than 96:4. In some embodiments, for articles with stereoselectivity or chirality controlled by oligonucleotides, each chiral nucleotide link is formed with a diastereoselectivity greater than 97:3. In some embodiments, for articles with stereoselectivity or chirality controlled by oligonucleotides, each chiral nucleotide link is formed with a diastereoselectivity greater than 98:2. In some embodiments, for articles with stereoselectivity or chirality controlled by oligonucleotides, each chiral nucleotide link is formed with a diastereoselectivity greater than 99:1. In some embodiments, the diastereoselectivity of chiral nucleotide interlinking in oligonucleotides can be measured by a model reaction (e.g., formation of a dimer under substantially identical or comparable conditions), wherein the dimer has the same internucleotide interlinking as the chiral nucleotide interlinking, the 5′-nucleotide of the dimer is identical to the 5′-terminal nucleoside of the chiral nucleotide interlinking, and the 3′-nucleotide of the dimer is identical to the 3′-terminal nucleoside of the chiral nucleotide interlinking. In some embodiments, the chiral-controlled internucleotide interlinking has a diastereomeric purity of 90%–100% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) at the linking phosphorus. In some embodiments, the chiral-controlled internucleotide linking has a diastereomeric purity of 95%-100% (e.g., 95%, 96%, 97%, 98%, 99%, or 99.5%) at the linking phosphorus. In some embodiments, the chiral-controlled internucleotide linking has a diastereomeric purity of 97%-100% (e.g., 97%, 98%, 99%, or 99.5%) at the linking phosphorus. In some embodiments, the chiral-controlled internucleotide linking has at least 97% diastereomeric purity.In some embodiments, the chiral-controlled internucleotide linkages have at least 98% diastereomeric purity. In some embodiments, the chiral-controlled internucleotide linkages have at least 99% diastereomeric purity. In some embodiments, the achiral-controlled (racemic / stereorandom) internucleotide linkages have less than 90% diastereomeric purity.

[0232] This disclosure provides, in particular, oligonucleotide compositions and techniques for optimizing properties.

[0233] This disclosure particularly provides oligonucleotide compositions and techniques for optimizing properties (e.g., improved single-stranded RNA interference, RNase H-mediated knockdown, etc.). In some embodiments, this disclosure provides methods for reducing immune responses associated with the administration of oligonucleotides and compositions thereof (i.e., methods for administering oligonucleotide compositions such that adverse immune responses to the oligonucleotides in the composition are reduced, for example, relative to adverse immune responses observed with a reference composition of nucleotides having comparable or identical nucleotide sequences). In some embodiments, this disclosure provides methods for increasing binding to certain proteins by oligonucleotides and compositions thereof. In some embodiments, this disclosure provides methods for increasing binding to certain proteins by oligonucleotides and compositions thereof. In some embodiments, this disclosure provides methods for enhancing the delivery of oligonucleotides and compositions thereof. This disclosure particularly covers the understanding that, in some embodiments, optimal delivery of oligonucleotides to their targets involves a balance of binding of the oligonucleotide to certain proteins such that the oligonucleotide can be transported to the desired location, and involves the release of the oligonucleotide such that the oligonucleotide can be appropriately released from certain proteins to perform its desired function, such as hybridizing with its target, cleaving its target, inhibiting translation, regulating transcript processing, etc. As exemplified in this disclosure, this disclosure particularly recognizes that the properties of oligonucleotides can be improved through chemical modification and / or stereochemistry.

[0234] In some embodiments, this disclosure provides a method for treating or preventing a disease, which includes administering the oligonucleotide composition described herein to a subject.

[0235] In some implementations, the disease is one in which, after administration of the provided composition, knocking down the target nucleic acid by single-stranded RNA interference can repair, restore, or introduce new beneficial functions.

[0236] In some implementations, the disease is cancer.

[0237] In some embodiments, the common sequence includes sequences selected from Table 1A. In some embodiments, the common sequence is a sequence selected from Table 1A. In some embodiments, the main chain chiral center pattern is selected from those patterns described in Table 1A.

[0238] In some embodiments, this disclosure provides a method comprising administering a composition comprising a first plurality of oligonucleotides, the composition exhibiting improved delivery compared to a reference composition comprising a plurality of oligonucleotides, each of the plurality of oligonucleotides also having a common base sequence but structurally different from the first plurality of oligonucleotides because:

[0239] The individual oligonucleotides in the reference oligonucleotides are stereochemically distinct from each other; and / or

[0240] At least some of the oligonucleotides in the plurality of oligonucleotides have a structure different from that represented by the plurality of oligonucleotides in the composition.

[0241] In some embodiments, this disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides having a common nucleotide sequence and capable of directing the reduction of expression and / or level of a target gene or its gene product, the improvement comprising:

[0242] The application of an oligonucleotide comprising a first plurality of oligonucleotides is characterized by improved delivery relative to a reference oligonucleotide composition having the same common nucleotide sequence.

[0243] In some embodiments, this disclosure provides a method of administering an oligonucleotide composition comprising a first plurality of oligonucleotides capable of directing single-stranded RNA interference and having a common nucleotide sequence, the improvement comprising:

[0244] The application of an oligonucleotide comprising a first plurality of oligonucleotides is characterized by improved delivery relative to a reference oligonucleotide composition having the same common nucleotide sequence.

[0245] In some embodiments, this disclosure provides single-stranded RNAi agents of oligonucleotides selected from any table disclosed herein (including, but not limited to, Table 1A) or elsewhere. In some embodiments, this disclosure provides single-stranded RNAi agents of oligonucleotides selected from any table disclosed herein (including, but not limited to, Table 1A) or elsewhere, wherein said oligonucleotides are conjugated to a lipid moiety.

[0246] In some embodiments, the oligonucleotide is no more than 25 bases in length. In some embodiments, the oligonucleotide is no more than 30 bases in length. In some embodiments, the oligonucleotide is no more than 35 bases in length. In some embodiments, the oligonucleotide is no more than 40 bases in length. In some embodiments, the oligonucleotide is no more than 45 bases in length. In some embodiments, the oligonucleotide is no more than 50 bases in length. In some embodiments, the oligonucleotide is no more than 55 bases in length. In some embodiments, the oligonucleotide is no more than 60 bases in length.

[0247] In some embodiments, the provided oligonucleotide comprises a lipid moiety. In some embodiments, the lipid moiety is incorporated by conjugation to a lipid. In some embodiments, the lipid moiety is a fatty acid. In some embodiments, the oligonucleotide is conjugated to a fatty acid. In some embodiments, the provided single-stranded RNAi agent also comprises a lipid. In some embodiments, the provided single-stranded RNAi agent comprises a lipid moiety conjugated at the 9th or 11th nucleotide (counting from the 5′ end). In some embodiments, the oligonucleotide is conjugated to a fatty acid at a base. In some embodiments, the provided single-stranded RNAi agent comprises a lipid moiety. In some embodiments, the provided single-stranded RNAi agent comprises a lipid moiety conjugated at the base of the 9th or 11th nucleotide (counting from the 5′ end).

[0248] In some embodiments, this disclosure provides compounds (e.g., oligonucleotides) having the structure of formula OI or a salt thereof:

[0249]

[0250] Or its salts, wherein the variables are each independent as described in this disclosure.

[0251] In some implementations, this disclosure provides a structure having the following:

[0252] 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 -(RD ) b

[0253] or compounds of its salts, wherein the variables are each independent as described in this disclosure.

[0254] In some implementations, each A c Independently, it is the oligonucleotide moiety of an oligonucleotide having the formula OI or a salt thereof (e.g., [H]). a -A c Or [H] b -A c (It is an oligonucleotide having the formula OI or a salt thereof). In some embodiments, this disclosure provides oligonucleotides having the following structures: 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 its salts. In some embodiments, this disclosure provides oligonucleotide compositions comprising an oligonucleotide having the following structure: 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 its salts. In some embodiments, this disclosure provides oligonucleotide compositions comprising predetermined levels (as described in this disclosure) of oligonucleotides having the following structure: A c -[-L M -(RD ) 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 its salts. In some embodiments, this disclosure provides chiral-controlled oligonucleotide compositions comprising an oligonucleotide having the following structure: 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 its salts. In some embodiments, multiple (e.g., a first multiple) oligonucleotides or oligonucleotide types are oligonucleotides having the following structure: 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 its salts. In some embodiments, this disclosure provides oligonucleotides having the following structure: Ac -[-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 its salt. In some embodiments, the structure is A. c -[-L M -(R D ) a ] b Or its salt. In some embodiments, the structure is [(A c ) a -L M ] b -R D Or its salt. In some embodiments, the structure is (A c ) a -L M -(A c ) b Or its salt. In some embodiments, the structure is A. c -[-L M -(R D ) a ] b Or its salt.

[0255] In some implementation schemes, A c Each oligonucleotide moiety is independently an oligonucleotide having the following structure: 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 -3′ or its salt.

[0256] In some implementations, the conjugate has A c -[-L LD -(R LD ) a ] b The structure is such that each variable is independent as described in this disclosure.

[0257] In some embodiments, this disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides having the following structures:

[0258] A c -[-L LD -(R LD ) a ] b or [(A c ) a -L LD ] b -R LD ,

[0259] in:

[0260] A c Each is an oligonucleotide moiety independently (e.g., [H]). a -A c Or [H] b -A c (It is an oligonucleotide);

[0261] a is 1-1000;

[0262] b is 1-1000;

[0263] L LD Each is an independent joint part; and

[0264] R LD Each is either a lipid component or a targeting component.

[0265] In some embodiments, this disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides having the following structures:

[0266] A c -[-L LD -(RLD ) a ] b or [(A c ) a -L LD ] b -R LD ,

[0267] in:

[0268] A c Each is an oligonucleotide moiety independently (e.g., [H]). a -A c Or [H] b -A c (It is an oligonucleotide);

[0269] a is 1-1000;

[0270] b is 1-1000;

[0271] L LD Each is independently a covalent bond or an optional substituted C1-C bond. 80 Saturated or partially unsaturated aliphatic groups, wherein one or more methylene units are optionally and independently T LD Alternatively, a group selected from the following optional substituted groups may be used: 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)O-, -OC(O)N(R′)-, -S(O)-, -S(O)2-, -S(O)2N(R′)-, -N(R′)S(O)2-, -SC(O)-, -C(O)S-, -OC(O)- and -C(O)O-;

[0272] R LD Each is independently hydrogen or an optional substituted C1-C 80A saturated or partially unsaturated aliphatic group, wherein one or more methylene units are optionally and independently replaced by optional substituted groups selected from: 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-, -SC(O)-, -C(O)S-, -OC(O)- and -C(O)O-;

[0273] T LD It has the structure of formula I′:

[0274]

[0275] W is O, S, or Se;

[0276] X, Y, and Z are each independently -O-, -S-, -N(-LR) 1 )- or L;

[0277] L is a covalent bond or an optionally substituted straight or branched chain C1-C 10 Aliphatic, wherein one or more methylene units of L are optionally and independently substituted by a group selected from the following optional substituted groups: C1-C6 aliphatic moiety, C1-C6 imenyl group, -C≡C-C1-C6 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-, -SC(O)-, -C(O)S-, -OC(O)- and -C(O)O-;

[0278] R 1 It is a halogen, R, or optionally substituted C1-C 50Aliphatic, wherein one or more methylene units are optionally and independently substituted by a group selected from the following optional substituted groups: 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-, -SC(O)-, -C(O)S-, -OC(O)-, and -C(O)O-

[0279] R′ can be independently -R, -C(O)R, -CO2R, or -SO2R, or:

[0280] Two or more R′ and their intermediary atoms together form a C3-C group with optional substitutions selected from aryl, carbocyclic, heterocyclic and heteroaryl groups. 14 Group;

[0281] -Cy- is selected from phenylene, C3-C 14 subcarbocyclic group, C 10 -C 14 Aspartic, C5-C 14 heteroaryl and C3-C 14 The optional substitution of the divalent ring of the heterocyclic group; and

[0282] R is independently hydrogen or selected from C1-C. 20 Aliphatic, C3-C 20 carbon cyclo group, C6-C 20 Aryl, C5-C 20 heteroaryl and C3-C 20 The optional substituent group of the heterocyclic group;

[0283] In some implementation schemes, [H] a -A c Or [H] b -A c It is an oligonucleotide having the structure of formula OI or a salt thereof. In some embodiments, [H] a -A c Or [H] b -A cIt is an oligonucleotide with the following structure: 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 -3′ or its salt.

[0284] In some implementations, T LD P in P is P * In some implementations, the conjugate has [(A c ) a -L LD ] b -R LD The structure. In some embodiments, the conjugate has (A c ) a -L LD -R LD The structure.

[0285] In some implementations, a is 1-100. In some implementations, a is 1-50. In some implementations, a is 1-40. In some implementations, a is 1-30. In some implementations, a is 1-20. In some implementations, a is 1-15. In some implementations, a is 1-10. In some implementations, a is 1-9. In some implementations, a is 1-8. In some implementations, a is 1-7. In some implementations, a is 1-6. In some implementations, a is 1-5. In some implementations, a is 1-4. In some implementations, a is 1-3. In some implementations, a is 1-2. In some implementations, a is 1. In some implementations, a is 2. In some implementations, a is 3. In some implementations, a is 4. In some implementations, a is 5. In some implementations, a is 6. In some implementations, a is 7. In some implementations, a is 8. In some implementations, a is 9. In some implementations, a is 10. In some implementations, a is greater than 10. In some implementations, b is 1-100. In some implementations, b is 1-50. In some implementations, b is 1-40. In some implementations, b is 1-30. In some implementations, b is 1-20. In some implementations, b is 1-15. In some implementations, b is 1-10. In some implementations, b is 1-9. In some implementations, b is 1-8. In some implementations, b is 1-7. In some implementations, b is 1-6. In some implementations, b is 1-5. In some implementations, b is 1-4. In some implementations, b is 1-3. In some implementations, b is 1-2. In some implementations, b is 1. In some implementations, b is 2. In some implementations, b is 3. In some implementations, b is 4. In some implementations, b is 5. In some implementations, b is 6. In some implementations, b is 7. In some implementations, b is 8. In some implementations, b is 9. In some embodiments, b is 10. In some embodiments, b is greater than 10. In some embodiments, the conjugate has A. c -L LD -R LD The structure. In some implementations, A c Conjugation is achieved through one or more of its sugar, base, and / or nucleotide inter-linking moieties. In some embodiments, A c Conjugation occurs via its 5′-OH (5′-O-). In some embodiments, A c Enzyme combination occurs via its 3′-OH (3′-O-). In some embodiments, A cConjugation occurs via internucleotide bonds. In some implementations, A c Conjugation is achieved through nucleobase pairing. In some implementations, A c Conjugation is achieved via sugars. In some implementations, A... c -(H) b (b is an integer from 1 to 1000, depending on A) c The valence of L is that of the oligonucleotide described herein, for example, one of the oligonucleotides listed in any of the tables. In some embodiments, L LD It is -L-. In some implementations, L LD It contains a thiophosphate group. In some embodiments, L LD It is -C(O)NH-(CH2)6-OP(=O)(S)-O-. In some embodiments, the -C(O)NH terminus is connected to RL. D And the -O-terminus is linked to the oligonucleotide, for example, via a 5′- or 3′-terminus. In some embodiments, R LD C is an optional substitute 10 C 15 C 16 C 17 C 18 C 19 C 20 C 21 C 22 C 23 C 24 or C 25 To 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 Aliphatic. In some implementations, R LD C is an optional substitute 10-80 Aliphatic. In some implementations, R LD C is an optional substitute 20-80 Aliphatic. In some implementations, R LD C is an optional substitute 10-70 Aliphatic. In some implementations, RLD C is an optional substitute 20-70 Aliphatic. In some implementations, R LD C is an optional substitute 10-60 Aliphatic. In some implementations, R LD C is an optional substitute 20-60 Aliphatic. In some implementations, R LD C is an optional substitute 10-50 Aliphatic. In some implementations, R LD C is an optional substitute 20-50 Aliphatic. In some implementations, R LD C is an optional substitute 10-40 Aliphatic. In some implementations, R LD C is an optional substitute 20-40 Aliphatic. In some implementations, R LD C is an optional substitute 10-30 Aliphatic. In some implementations, R LD C is an optional substitute 20-30 Aliphatic. In some implementations, R LD It is unreplaced 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 To 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 Aliphatic. In some implementations, R LD It is unreplaced C 10-80 Aliphatic. In some implementations, R LD It is unreplaced C 20-80 Aliphatic. In some implementations, R LDIt is unreplaced C 10-70 Aliphatic. In some implementations, R LD It is unreplaced C 20-70 Aliphatic. In some implementations, R LD It is unreplaced C 10-60 Aliphatic. In some implementations, R LD It is unreplaced C 20-60 Aliphatic. In some implementations, R LD It is unreplaced C 10-50 Aliphatic. In some implementations, R LD It is unreplaced C 20-50 Aliphatic. In some implementations, R LD It is unreplaced C 10-40 Aliphatic. In some implementations, R LD It is unreplaced C 20-40 Aliphatic. In some implementations, R LD It is unreplaced C 10-30 Aliphatic. In some implementations, R LD It is unreplaced C 20-30 Lipids.

[0286] In some implementation schemes, R LD It is not hydrogen. In some implementations, R LD It is the lipid portion. In some implementations, R LD It is the targeted portion. In some implementations, R LD It is a targeted portion containing the carbohydrate portion. In some implementations, R LD It's the GalNAc part.

[0287] In some embodiments, the single-stranded RNAi agent is any of the aforementioned compositions, which also contains one or more additional components.

[0288] In some implementations, the provided oligonucleotides are capable of degrading target transcripts, such as RNA, via RNase H and RNAi mechanisms.

[0289] In some embodiments, the conjugation of the lipid moiety to the oligonucleotide improves at least one property of the oligonucleotide. In some embodiments, the improved properties include increased activity (e.g., increased ability to direct the expression and / or reduction of levels of target genes or their gene products, and / or to direct single-stranded RNA interference, and / or to direct 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 properties include reduced hTLR9 agonist activity. In some embodiments, the improved properties include hTLR9 antagonist activity. In some embodiments, the improved properties include increased hTLR9 antagonist activity.

[0290] Generally speaking, the properties of oligonucleotide compositions as described herein can be evaluated using any appropriate assay.

[0291] Those skilled in the art will recognize and / or be able to readily develop appropriate assays for a particular oligonucleotide composition.

[0292] definition

[0293] As used herein, unless otherwise specified, the following definitions apply. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version) and the Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th edition, edited by Smith, MB and March, J., John Wiley & Sons, New York: 2001.

[0294] Aliphatic: As used herein, "aliphatic" means a straight-chain (i.e., unbranched) or branched substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more unsaturated units (excluding aromatic units), or a combination thereof. In some embodiments, the aliphatic group contains 1-50 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-20 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-10 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-9 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-8 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-7 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms, and in yet another embodiment, the aliphatic group contains 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic groups include, but are not limited to, straight-chain or branched substituted or unsubstituted alkyl, alkenyl, alkynyl and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0295] Alkenyl: As used herein, the term “alkenyl” refers to an alkyl group having one or more double bonds as defined herein.

[0296] Alkyl: As used herein, the term "alkyl" has its usual meaning in the art and may include saturated aliphatic groups, including straight-chain alkyl, branched alkyl, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl, and cycloalkyl-substituted alkyl. In some embodiments, the alkyl group has 1-100 carbon atoms. In some embodiments, the straight-chain or branched alkyl group has about 1-20 carbon atoms in its main chain (e.g., for a straight chain it is C1-C1). 20 For sidechains, it's C2-C. 20 The alkyl group has about 1 to 10 carbon atoms. In some embodiments, the cycloalkyl ring has about 3 to 10 carbon atoms in its ring structure, wherein such ring is monocyclic, bicyclic, or polycyclic, and alternatively has about 5, 6, or 7 carbon atoms in the ring structure. In some embodiments, the alkyl group may be a lower alkyl group, wherein the lower alkyl group contains 1 to 4 carbon atoms (e.g., C1-C4 for straight-chain lower alkyl groups).

[0297] Alkynyl group: As used herein, the term "alkynyl group" refers to an alkyl group as defined herein that has one or more triple bonds.

[0298] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0299] Approximately: As used herein, unless otherwise stated or apparent from the context, the term "approximately" or "about" with respect to a numerical value is generally considered to include values ​​that fall within a range of 5%, 10%, 15%, or 20% of the value in either direction (greater or less) (the difference being that such values ​​would be less than 0% of a possible value or more than 100% of a possible value). In some embodiments, the use of the term "about" with respect to dosage means ±5 mg / kg / day.

[0300] Aryl: As used herein, the term "aryl" alone or as part of a larger portion such as "aralkyl," "aralkyloxy," or "aryloxyalkyl" refers to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in said system is aromatic. In some embodiments, an aryl is a monocyclic, bicyclic, or polycyclic ring system having a total of five to fourteen ring members, wherein at least one ring in said system is aromatic and wherein each ring in said system contains 3 to 7 ring members. In some embodiments, an aryl is a biaryl. The term "aryl" may be used interchangeably with the term "aryl ring." In certain embodiments of this disclosure, "aryl" refers to an aromatic ring system that may carry one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, binaphthalene, anthracene, etc. The scope of the term "aryl" as used herein also includes groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimide, naphthimide, phenanthridine, or tetrahydronaphthyl.

[0301] Characteristic portion: As used herein, the “characteristic portion” of a phrase protein or polypeptide is a portion containing a single continuous amino acid or a collection of multiple continuous amino acid segments that collectively characterize the protein or polypeptide. Each such continuous segment will generally contain at least two amino acids. Furthermore, those skilled in the art will understand that the characterization of a protein typically requires at least 5, 10, 15, 20, or more amino acids. Generally, a characteristic portion is a portion that, in addition to the sequence identity specified above, also shares at least one functional characteristic with the associated intact protein.

[0302] Characteristic structural element: The term "characteristic structural element" or "structural element" refers to a unique structural element present in all members of a polypeptide, small molecule, or nucleic acid family and thus usable by those skilled in the art to define members of said family. In some embodiments, structural elements of a single-stranded RNAi agent include, but are not limited to: 5′-terminal structure, 5′-terminal region, 5′ nucleotide moiety, seed region, post-seed region, 3′-terminal region, 3′-terminal dinucleotide, 3′ cap, modification pattern, stereochemical pattern in the backbone, additional chemical moieties, etc.

[0303] Comparable: The term "comparable" is used herein to describe two (or more) sets of conditions or situations that are sufficiently similar to each other to allow for comparison of obtained results or observed phenomena. In some embodiments, multiple sets of comparable conditions or situations are characterized by a number of substantially identical features and one or a few different features. Those skilled in the art will appreciate that when multiple sets of conditions are characterized by a sufficient number and type of substantially identical features, the multiple sets of conditions are comparable to each other to ensure that reasonable conclusions can be drawn that differences in results or observed phenomena obtained in different sets of conditions or situations are caused by or indicate changes in those different features.

[0304] Alicyclic: The terms “cycloaliphatic,” “carbocycle,” “carbocyclyl,” “carbocyclic radical,” and “carbocyclic ring” are used interchangeably and, as used herein, refer to saturated or partially unsaturated, but non-aromatic, cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems having 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, the alicyclic group has 3-6 carbons. In some embodiments, the alicyclic group is saturated and is cycloalkyl. The term “alicyclic” may also include aliphatic rings fused to one or more aromatic or non-aromatic rings (such as decahydronaphthyl or tetrahydronaphthyl). In some embodiments, the alicyclic group is bicyclic. In some embodiments, the alicyclic group is tricyclic. In some embodiments, the alicyclic group is polycyclic. In some embodiments, "alicyclic" refers to C3-C6 monocyclic hydrocarbons, or C8-C6 monocyclic hydrocarbons. 10 Bicyclic or polycyclic hydrocarbons that are fully saturated or contain one or more unsaturated but not aromatic units, having a single connection point to the rest of the molecule, or C9-C. 16Polycyclic hydrocarbons are those that are fully saturated or contain one or more unsaturated but not aromatic units, and have a single connection point to the rest of the molecule.

[0305] Dosing regimen: As used herein, a “dosing regimen” or “treatment regimen” refers to a set of unit doses (usually more than one) administered individually to a subject at multiple time intervals. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each of which is separated from the others by time intervals of equal length; in some embodiments, a dosing regimen includes multiple doses and at least two distinct time intervals separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dosage. In some embodiments, the different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen includes a first dose administered at a first dosage, followed by one or more additional doses at a second dosage different from the first dosage. In some embodiments, a dosing regimen includes a first dose at a first dosage, followed by one or more additional doses at a second dosage identical to the first dosage.

[0306] Equivalent Agents: Those skilled in the art will understand upon reading this disclosure that the scope of agents available in the context of this disclosure is not limited to those explicitly mentioned or exemplified herein. Specifically, those skilled in the art will recognize that active agents generally have a structure consisting of a core portion and connected side portions, and will further understand that simple variations of such core and / or side portions may not significantly alter the activity of the agent. For example, in some embodiments, substituting one or more side portions with groups having comparable three-dimensional structural and / or chemical reactivity characteristics may produce substituted compounds or portions equivalent to or partially equivalent to the parent reference compound. In some embodiments, adding or removing one or more side portions may produce substituted compounds equivalent to the parent reference compound. In some embodiments, altering the core structure, for example, by adding or removing a few bonds (typically no more than 5, 4, 3, 2, or 1 bond, and often only a single bond), may produce substituted compounds equivalent to the parent reference compound. In many embodiments, equivalent compounds may be prepared using readily available starting materials, reagents, and conventional or provided synthetic procedures, by methods shown, for example, in the general reaction schemes described below, or by modifications thereof. These reactions may also utilize known variants that are not mentioned in this paper.

[0307] Equivalent dose: The term "equivalent dose" is used herein to compare doses of different pharmaceutically active agents that achieve the same biological outcome. Two different agents are considered "equivalent" to each other if doses yield comparable levels or extents of biological outcome. In some embodiments, the equivalent dose of different pharmaceutical formulations used according to this disclosure is determined using in vitro and / or in vivo assays as described herein. In some embodiments, one or more lysosomal activators for use according to this disclosure are utilized at doses equivalent to a reference lysosomal activator; in some such embodiments, the reference lysosomal activator for such purposes is selected from the group consisting of: small molecule allosteric activators (e.g., pyrazolpyrimidine), iminosaccharides (e.g., isofagomine), antioxidants (e.g., N-acetylcysteine), and cell transport regulators (e.g., Rabla peptides).

[0308] Heteroaliphatic: As used herein, the term "heteroaliphatic" has its usual meaning in the art and refers to an aliphatic group in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). In some embodiments, one or more units selected from C, CH, CH2, and CH3 are independently replaced by one or more heteroatoms (including their oxidized and / or substituted forms). In some embodiments, the heteroaliphatic group is a heteroalkyl group. In some embodiments, the heteroaliphatic group is a heteroalkenyl group.

[0309] Heteroalkyl: As used herein, the term “heteroalkyl” has its usual meaning in the art and refers to an alkyl group in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, nitrogen, sulfur, silicon, phosphorus, etc.). Examples of heteroalkyl groups include, but are not limited to, alkoxy, poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl, piperidinyl, morpholinyl, etc.

[0310] Heteroaryl: As used herein, the terms "heteroaryl" and "heteroaryl-" used alone or as part of a larger portion (e.g., "heteroarylalkyl" or "heteroarylalkoxy") refer to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic and at least one aromatic ring atom is a heteroatom. In some embodiments, a heteroaryl is a group having 5 to 10 ring atoms (i.e., monocyclic, bicyclic, or polycyclic), and in some embodiments, a group having 5, 6, 9, or 10 ring atoms. In some embodiments, a heteroaryl has 6, 10, or 14 π electrons shared in the cyclic array; and in addition to a carbon atom, it has one to five heteroatoms. Heteroaryl groups include, but are not limited to, thiophene, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, indazinyl, purinyl, naphridinyl, and pteridinyl. In some embodiments, the heteroaryl group is a heterobiaryl group, such as bipyridinyl. As used herein, the terms “heteroaryl” and “heteroaryl-” also include groups in which the heteroaryl ring is fused to one or more aryl, alicyclic, or heterocyclic rings, wherein the linking group or linking point is on the heteroaryl ring. Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, indazoleyl, benzimidazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cenolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinazinyl, carbazoleyl, acridineyl, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or polycyclic. The term "heteroaryl" is used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaryl family," any of which includes an optionally substituted ring. The term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group, wherein the alkyl and heteroaryl portions are independently optionally substituted.

[0311] Heteroatom: As used herein, the term "heteroatom" means an atom that is not carbon or hydrogen. In some embodiments, the heteroatom is oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any quaternized form of basic nitrogen; or a substituted nitrogen of a heterocycle (e.g., N in a 3,4-dihydro-2H-pyrrole group), NH (as in a pyrroleyl group), or NR. + (e.g., in N-substituted pyrroleyl groups, etc.).

[0312] Heterocycle: As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic moiety (e.g., 3-30 members) having one or more heteroatom ring atoms. In some embodiments, the heterocyclyl group is a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more, preferably one to four, heteroatoms as defined above, in addition to a carbon atom. When referring to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, and nitrogen, nitrogen can be N (e.g., in 3,4-dihydro-2H-pyrroleyl), NH (e.g., in pyrroleyl), or + NR (as in N-substituted pyrrolidinyl groups). Heterocycles can be attached to their side groups at any heteroatom or carbon atom that produces a stable structure, and any ring atom can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolyl, piperazine, dioxyl, dioxapentylyl, diazapyridine, oxonitrilepyridine, thiopyridine, morpholinyl, and quininecycloyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical” are used interchangeably herein and also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or alicyclic rings, such as indololinyl, 3H-indolyl, benzodihydropyranyl, phenanthridineyl, or tetrahydroquinolinyl. Heterocyclic groups can be monocyclic, bicyclic, or polycyclic. The term “heterocyclic alkyl” refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl group and the heterocyclic moiety are independently optional substitutions.

[0313] Intraperitoneal: As used herein, the phrases “intraperitoneal administration” and “administered intraperitoneal” have the meaning as understood in the art and refer to the administration of a compound or composition into the peritoneum of a subject.

[0314] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, etc., rather than in an organism (e.g., an animal, plant, and / or microorganism).

[0315] In vivo: As used in this article, the term “in vivo” refers to events that occur within an organism (e.g., an animal, plant, or microorganism).

[0316] Lower alkyl groups: The term "lower alkyl group" refers to C14-C ... 1-4 Straight-chain or branched alkyl groups. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0317] Lower haloalkyl: The term "lower haloalkyl" refers to a C14 alkyl group that has been substituted with one or more halogen atoms. 1-4 Straight-chain or branched alkyl groups.

[0318] Optionally Substituted: As described herein, the compounds of this disclosure (e.g., oligonucleotides) may contain optionally substituted and / or substituted moieties. Generally, the term "substituted," regardless of whether the term "optionally" precedes it, means that one or more hydrogens of the specified moieties are replaced by suitable substituents. Unless otherwise specified, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and when more than one position in any given structure can be substituted by more than one substituent selected from the specified group, the substituents at each position may be the same or different. In some embodiments, the optionally substituted group is unsubstituted. Combinations of substituents contemplated by this disclosure are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" means a compound that is substantially unchanged when subjected to conditions that allow it to be produced, detected, and in some embodiments, recovered, purified, and used for one or more purposes disclosed herein.

[0319] A suitable monovalent substituent on a substituted atom (e.g., a suitable carbon atom) is independently a halogen; -(CH2) 0-4 R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o -O-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 CH(OR o )2; by R o Substituted -(CH2) 0-4 Ph; by R o Substituted -(CH2) 0-4O(CH2) 0-1 Ph; by R o The substituted -CH=CHPh can be replaced by R o Substituted -(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 o ;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NR o 2; -C(S)NR o 2;-C(S)SR o ;-SC(S)SR o ;-(CH2) 0-4OC(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)2NR o 2;-N(R o )S(O)2R o ;-N(OR o )R o ;-C(NH)NR o 2;-Si(R o )3;-OSi(R o )3;-B(R o )2;-OB(R o )2;-OB(OR o )2;-P(R o )2;-P(OR o )2;-OP(R o )2;-OP(OR o )2;-P(O)(R o )2;-P(O)(OR o )2;-OP(O)(R o )2;-OP(O)(OR o )2;-OP(O)(OR o )(SR o );-SP(O)(R o )2;-SP(O)(OR o )2;-N(R o )P(O)(R o )2;-N(R o )P(O)(OR o )2;-P(R o )2[B(R o )3];-P(ORo )2[B(R o )3];-OP(R o )2[B(R o )3];-OP(OR o )2[B(R o )3];-(C 1-4 (linear or branched alkylene)ON(R) o )2; or -(C 1-4 (straight-chain or branched alkylene)C(O)ON(R) o )2, where each R o It can be substituted as defined below and is independently hydrogen, C 1-20 Aliphatic, having 1-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus. 1-20 Aliphatic group, -CH2-(C 6-14 Aryl), -O(CH2) 0-1 (C 6-14 Aryl), -CH2-(5-14 membered heteroaryl ring), 5-20 membered monocyclic, bicyclic or polycyclic, having 0-5 independently selected heteroatoms selected from nitrogen, oxygen, sulfur, silicon and phosphorus, saturated, partially unsaturated or aryl rings, or, despite the above definition, two independently occurring R... o Together with its intercalary atoms, it forms a 5-20 membered monocyclic, bicyclic, or polycyclic, saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus, which may be substituted as defined below.

[0320] R o (or two independent Rs) o Suitable monovalent substituents on the ring formed by the ring and its intercalating atoms are independently halogens, -(CH2). 0-2 R · -(halogenated R) · -(CH2) 0-2 OH, -(CH2) 0-2 OR · -(CH2) 0-2 CH(OR · )2、-O(halogenated R · -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- 2SH、-(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-chain or branched alkylene)C(O)OR · or -SSR · , where each R · It is either unsubstituted or, when previously "halogenated," substituted by only one or more halogens, and independently selected from C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, and 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur. R o Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0321] For example, suitable divalent substituents on suitable carbon atoms are independently the following: =O, =S, =NNR. * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * -O(C(R) * 2)) 2-3 O- or -S(C(R) * 2)) 2-3 S-, where each independently occurring R * Selected from hydrogen, C can be substituted as defined below. 1-6 Aliphatic, and having 0-4 unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings independently selected from nitrogen, oxygen, and sulfur. Suitable divalent substituents attached to the ortho-substituted carbon of the "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independently occurring R * Selected from hydrogen, C can be substituted as defined below. 1-6 Aliphatic, and unsubstituted 5-6 member saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0322] R * Suitable substituents on the aliphatic group are independently halogens, -R ·-(halogenated R) · -OH, -OR · -O (halogenated R) · -CN, -C(O)OH, -C(O)OR · -NH2, -NHR · -NR · 2 or -NO2, where each R · It is either unsubstituted or, in the case of prior "halogenation," substituted only by one or more halogens, and is independently C. 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 saturated, partially unsaturated and / or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0323] Oral administration: As used herein, the phrases “oral administration” and “administered orally” have the meaning as understood in the art as referring to the administration of a compound or composition through the oral cavity.

[0324] Parenteral administration: As used herein, the phrases “parenteral administration” and “administered parenterally” have the meaning as understood in the art, referring to a mode of administration other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intravertebral, and intrasternal injections and infusions.

[0325] Partially unsaturated: As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to cover rings having multiple unsaturated sites, and not to include aryl or heteroaryl moiety as defined herein.

[0326] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together 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, when administered to a relevant population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for: oral administration, such as enemas (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), pills, powders, granules, or pastes for application to the tongue; parenteral administration, as, for example, sterile solutions or suspensions, or sustained-release formulations such as those administered via subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, such as creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or mouth; intravaginal or rectal administration, such as as pessaries, creams, or foams; sublingual administration; ocular administration; transdermal administration; or nasal administration, pulmonary administration, and administration to other mucosal surfaces.

[0327] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” means compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications and in proportion to a reasonable benefit / risk ratio.

[0328] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, relating to the carrying or transport of a subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with other components of the formulation and does not harm the patient. Examples of materials that can serve 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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; maltose; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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 glycerol, 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; ethanol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0329] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salt" means a salt suitable for use in the context of a pharmaceutical product, i.e., a salt suitable for contact with human and lower animal tissues without excessive toxicity, irritation, anaphylactic reactions, etc., and in proportion to a reasonable benefit / risk ratio, to the extent of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to: non-toxic acid addition salts, which are salts of an amino group formed with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or an organic acid (such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods used in the art (such as ion exchange). In some implementations, pharmaceutically acceptable salts include, but are not limited to, adipic acid salts, alginates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, hydrogen sulfates, borates, butyrates, camphorates, camphor sulfonates, citrates, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-p-ethylheptanate, glycerophosphates, gluconate, hemisulfates, heptanate, hexanoates, hydroiodides, 2-hydroxyethanesulfonate, and lacturonic acid. Salts, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, papoate, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. In some embodiments, the provided compounds contain one or more acidic groups, such as oligonucleotides, and pharmaceutically acceptable salts are alkali metal, alkaline earth metal, or ammonium salts (e.g., ammonium salts of N(R)3, where each R is independently defined and described in this disclosure). Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. 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 implementations, pharmaceutically acceptable salts, where appropriate, include non-toxic ammonium, quaternary ammonium, and amine cations formed using balancing ions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl, sulfonate, and arylsulfonate having 1 to 6 carbon atoms.In some embodiments, the provided compound comprises more than one acidic group; for example, the provided oligonucleotide may comprise two or more acidic groups (e.g., in natural phosphate ester-linked and / or modified nucleotide-linked structures). In some embodiments, a pharmaceutically acceptable salt or general salt of such compound comprises two or more cations, which may be the same or different. In some embodiments, in the pharmaceutically acceptable salt (or general salt), all ionizable hydrogen atoms in the acidic groups are replaced by cations. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of the provided oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of the provided oligonucleotide, wherein each acidic phosphate ester group is present in salt form (all sodium salts); for example, containing 19 Na atoms. + The sodium salt of WV-2555, or containing 23 Na atoms. + Sodium salt of WV-2555.

[0330] Prodrug: Generally speaking, as used herein and as understood in the art, a "prodrug" is an entity that, upon administration to an organism, is metabolized in vivo to deliver a target active agent (e.g., a therapeutic or diagnostic agent). Typically, such metabolism involves the removal of 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:

[0331] a) Design of Prodrugs, H. Bundgaard, ed. (Elsevier, 1985) and Methods in Enzymology, 42: 309-396, K. Widder et al., eds. (Academic Press, 1985);

[0332] b) Prodrugs and Targeted Delivery, edited by J.Rautio (Wiley, 2011);

[0333] c)A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen;

[0334] d) Bundgaard, Chapter 5 "Design and Application of Prodrugs", H. Bundgaard, pp. 113-191 (1991);

[0335] e) Bundgaard, Advanced Drug Delivery Reviews, 8: 1-38 (1992);

[0336] f) Bundgaard et al., Journal of Pharmaceutical Sciences, 77: 285 (1988); and

[0337] g) Kakeya et al., Chem. Pharm. Bull., 32: 692 (1984).

[0338] Like the other compounds described herein, the prodrug can be provided in any of a variety of forms, such as crystalline form, salt form, etc. In some embodiments, the prodrug is provided as its pharmaceutically acceptable salt.

[0339] Protecting Groups: As used herein, the term “protecting group” is well known in the art and includes those detailed in *Protecting Groups in Organic Synthesis*, TW Greene and PGM Watts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference in their entirety. It also includes those protecting groups described in *Current Protocols in Nucleic Acid Chemistry*, ed., Serge L. Beaucage et al., 06 / 2012, which are particularly suitable for nucleoside and nucleotide chemistry, Chapter 2, which are incorporated herein by reference in their entirety. Suitable amino protecting groups include methyl carbamate, ethyl carbamate, 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfonyl)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothiopheneyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzoyl methyl carbamate (Phenoc), and amino... 2,2,2-Trichloroethyl carbamate (Troc), 2-trimethylsilyl ethyl carbamate (Teoc), 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-biphenyl)ethyl carbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′-pyridyl and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylformamido)ethyl carbamate, tert-butyl carbamate (BOC), 1-adamantane carbamate (Adoc), vinyl carbamate (Voc), ammonia Allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamonyl carbamate (Coc), 4-nitrocinnamonyl carbamate (Noc), 8-quinoline carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2-carbamate4-Dichlorobenzoate, 4-methylsulfinylbenzene carbamate (Msz), 9-anthraylmethyl carbamate, diphenyl methyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithionecyclohexyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonium ethyl carbamate (Peoc), 2-triphenylphosphonium isopropyl carbamate (Ppoc), 1,1-dimethyl-2-carbamate - Cyanoethyl ester, m-chloro-p-acyloxybenzyl ester of carbamate, p-(dihydroxyboryl)benzyl ester of carbamate, 5-benzisoxazolyl methyl ester of carbamate, 2-(trifluoromethyl)-6-chromone methyl ester of carbamate (Tcroc), m-nitrophenyl ester of carbamate, 3,5-dimethoxybenzyl ester of carbamate, o-nitrophenyl ester of carbamate, 3,4-dimethoxy-6-nitrophenyl ester of carbamate, phenyl(o-nitrophenyl) methyl ester of carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, tert-amyl ester of carbamate, thioamino S-benzoic acid, p-cyanobenzoic acid, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl carbamate, p-decyloxybenzoic acid, 2,2-dimethoxycarbonyl vinyl carbamate, o-(N,N-dimethylformamide)benzoic acid, 1,1-dimethyl-3-(N,N-dimethylformamide)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinic acid ester 1,4,6-Tri-tert-butylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, 2,4,6-tri-tert-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, 2,4,6-carbamate6-Trimethylbenzyl ester, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridinecarboxamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetylacetamide, (N′-dithiobenzylmethyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4 - Chloropreneamide, 3-methyl-3-nitrobutamide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazoline-2-one, N-benzodicarboximide, N-dithiosuccinimide (Dts), N-2,3-diphenylcis-butene diimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilazylazine adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1 3-Diphenylmethyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridinone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptanamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (P hF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferroceneylmethylamino (Fcm), N-2-pyridinemethylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-phenylmethyleneamine, N-p-methoxyphenylmethyleneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)trimethylmethyl]methyleneamine, N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylenediamine, N-p-nitrophenylmethyleneamine, N-salicylamine, N-5-chlorosalicylamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-Dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylalkylboronic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkyl aminophosphate, diphenylaminophosphate, diphenylaminophosphate, benzenesulfinamide, o-nitrobenzenesulfinamide (Nps), 2,4-dinitrobenzenesulfinamide, pentachlorobenzenesulfinamide, 2-nitro-4-methoxybenzenesulfinamide, triphenylmethylsulfinamide, 3-nitropyridinesulfinamide (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-pentamethylsomn-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracitesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylmethylsulfonamide, trifluoromethylsulfonamide, and benzoylmethylsulfonamide.

[0340] Suitable protected carboxylic acids also include, but are not limited to, silyl, alkyl, alkenyl, aryl, and arylalkyl-protected carboxylic acids. Examples of suitable silyl groups include trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, etc. Examples of suitable alkyl groups include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, triphenylmethyl, tert-butyl, tetrahydropyran-2-yl. Examples of suitable alkenyl groups include allyl. Examples of suitable aryl groups include optionally substituted phenyl, biphenyl, or naphthyl groups. Examples of suitable arylalkyl groups include optionally substituted benzyl groups (e.g., p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl) as well as 2-pyridinemethyl and 4-pyridinemethyl.

[0341] Suitable hydroxyl protecting groups include methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silanoxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), and tetrahydropyranyl (THP). ), 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methylbridged benzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzylmethyloxyethyl, 1-methyl -1-Benzylmethyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylhydroselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxyphenyl, 3,4-dimethoxyphenyl, o-nitrophenyl, p-nitrophenyl, p-halophenylmethyl, 2,6-dichlorophenylmethyl, p-cyanophenylmethyl, p-phenylphenylmethyl, 2-pyridinemethyl, 4-pyridinemethyl, 3-methyl-2-pyridinemethyl N-oxobridge, diphenylmethyl, p,p′-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl , di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromobenzoylmethyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorobenzoiminophenyl)methyl, 4,4′,4″-tris(acetylpropionyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)oxanthracene, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiopentane-2-yl, benzisothiazolyl S,S-dioxane bridge, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triphenylmethylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxy Phthalate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantane, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (trimethylbenzoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-( Triphenylphosphonium (PP) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzenemethyl carbonate, 3,4-dimethoxybenzenemethyl carbonate, o-nitrobenzenemethyl carbonate, p-nitrobenzenemethyl carbonate, S-benzenemethylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthio) Methoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, dichlorophenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthyl ester, nitrate ester, alkyl N,N,N′,N-tetramethylphosphonic acid diamine, N-phenylcarbamate, borate ester, dimethylphosphinosulfonyl, 2,4-dinitrophenyl sulfenic acid ester, sulfate ester, methanesulfonate (methanesulfonate), benzyl methylsulfonate and toluenesulfonate (Ts). For the protection of 1,2-diol or 1,3-diol.Protecting groups include methylene acetal, ethylene acetal, 1-tert-butyl ethylene ketal, 1-phenyl ethylene ketal, (4-methoxyphenyl) ethylene acetal, 2,2,2-trichloroethylene acetal, acetone compounds, cyclopentyl ketal, cyclohexyl ketal, cycloheptyl ketal, benzene methylene acetal, p-methoxybenzene methylene acetal, 2,4-dimethoxybenzene methylene ketal, 3,4-dimethoxybenzene methylene acetal, 2-nitrobenzene methylene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene orthoester, 1-methoxyethylene orthoester, 1 -Ethoxyethylene orthoester, 1,2-dimethoxyethylene orthoester, α-methoxybenzyl orthoester, 1-(N,N-dimethylamino)ethylene derivative, α-(N,N′-dimethylamino)benzyl derivative, 2-oxacyclopentyl orthoester, di-tert-butylsilane (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxane) derivative (TIPDS), tetra-tert-butoxydisiloxane-1,3-diendyl derivative (TBDS), cyclic carbonates, cyclic borates, ethyl borates, and phenyl borates.

[0342] In some embodiments, the hydroxyl protecting group is acetyl, tert-butyl, tert-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 (triphenylmethyl), 4,4′-dimethoxytriphenylmethyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, benzoylcarbamate, chloroacetyl, trichloroacetyl, trifluoroacetyl, neopentanoyl, 9-fluorenylmethyl carbonate, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, triphenylmethyl, monomethoxytriphenylmethyl (MMTr), 4,4′-dimethoxytriphenylmethyl (DMTr) and 4,4′,4″-Trimethoxytriphenylmethyl (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)triphenylmethyl, diphenylcarbamoyl, acetylpropionyl, 2-(dibromomethyl)benzoyl (Dbmb), 2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenyloxanthracene-9-yl (pixyl), or 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In some embodiments, the hydroxyl protecting group is independently selected from acetyl, benzyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and 4,4′-dimethoxytriphenylmethyl. In some embodiments, the hydroxyl protecting group is selected from the group consisting of triphenylmethyl, monomethoxytriphenylmethyl, and 4,4′-dimethoxytriphenylmethyl.

[0343] In some embodiments, the phosphorus-linked protecting group is a group attached to a phosphorus-linked (e.g., internucleotide link) connection throughout the oligonucleotide synthesis. In some embodiments, the protecting group is attached to the sulfur atom of a thiophosphate group. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide thiophosphate link. In some embodiments, the protecting group is attached to the oxygen atom of an internucleotide phosphate link. 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-butylformamido)-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.

[0344] 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 comprises only naturally occurring amino acids. In some embodiments, a protein comprises one or more non-naturally occurring amino acids (e.g., portions that form one or more peptide bonds with neighboring amino acids). In some embodiments, one or more residues in the protein chain contain non-amino acid portions (e.g., glycans, etc.). In some embodiments, a protein comprises more than one polypeptide chain linked, for example, by one or more disulfide bonds or otherwise associated. 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. Available modifications include, for example, terminal acetylation, amidation, methylation, etc. The term "peptide" is generally used to refer to polypeptides of length less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, a protein is an antibody, an antibody fragment, its biologically active portion, and / or its characteristic portion.

[0345] RNA Interference: As used herein, the term “RNA interference” or “RNAi” refers to a posttranscriptional targeted gene silencing process involving RISC (RNA-induced silencing complex). RNAi is reported to occur naturally when ribonuclease III (Dicer) cleaves longer dsRNAs into shorter fragments (called siRNAs). Naturally occurring siRNAs (small interfering RNAs) are typically about 21 to 23 nucleotides in length, have a double strand of about 19 base pairs and two single-stranded overhangs, and are usually RNA. These RNA fragments are then reported to direct the degradation of target nucleic acids, such as mRNA or pre-mRNA. Dicer has also reportedly involved the excision of 21 and 22 nucleotides of short-length RNA (stRNA) involving translation control from a precursor RNA with a conserved structure. (Hutvagner et al. 2001, Science, 293, 834). Those skilled in the art will recognize that RNAi can be mediated by single-stranded or double-stranded oligonucleotides comprising sequences complementary to or substantially complementary to a target sequence (e.g., in target mRNA). Therefore, in some embodiments of this disclosure, single-stranded oligonucleotides as described herein can act as RNAi agents; in some embodiments, double-stranded oligonucleotides as described herein can act as RNAi agents. In some embodiments, the RNAi reaction involves a nuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which directs the cleavage of a single-stranded mRNA complementary to the antisense strand of the siRNA. In some embodiments, the RISC directs the cleavage of a target RNA complementary to the provided oligonucleotide, which can be used as a single-stranded RNAi agent. In some embodiments, the cleavage of the target RNA occurs in the middle of a region complementary to the antisense strand of the siRNA duplex or the single-stranded RNAi agent. In some embodiments, RNA interference is directed by a single-stranded oligonucleotide that acts as a single-stranded RNAi agent, which can direct RNA interference in mechanisms involving the RISC pathway.

[0346] RNAi agents: As used herein, the terms "RNAi agent," "iRNA agent," etc., refer to oligonucleotides that, when administered to a system in which a target gene product is being expressed or has been expressed (e.g., a transcript of the target gene, such as pre-mRNA or mRNA), reduces the level and / or activity (e.g., translation) of said target gene product. In some embodiments, the RNAi agent may be or comprise a single-stranded or double-stranded oligonucleotide. In some embodiments, the RNAi agent may have a structure recognized in the art, such as siRNA (short repressor RNA), shRNA (short or small hairpin RNA), dsRNA (double-stranded RNA), microRNA, etc. In some embodiments, the RNAi agent may specifically bind to an RNA target (e.g., a transcript of the target gene). In some embodiments, after binding to its target, the RNAi agent is loaded into a RISC (RNA-induced silencing complex). In some embodiments, the RNAi agent directs the degradation of its target and / or the inhibition of its target translation through a mechanism involving the RISC (RNA-induced silencing complex) pathway. In some embodiments, the RNAi agent is an oligonucleotide that activates the RISC complex / pathway. In some embodiments, the RNAi agent contains an antisense strand sequence. In some embodiments, the RNAi agent contains only one oligonucleotide chain (e.g., a single-stranded oligonucleotide). In some embodiments, the single-stranded RNAi agent oligonucleotide may be or contains a sense or antisense strand sequence, as described in Sioud 2005 J.Mol.Biol.348:1079-1090. In some embodiments, the RNAi agent is a compound capable of directing RNA interference. In some embodiments, the RNAi agent may have a structure or format as present in a "canonical" siRNA structure. In some embodiments, the RNAi agent may have a structure different from a "canonical" siRNA structure. To name just a few examples, in some embodiments, the RNAi agent may be longer or shorter than the canonical form, may be blunt-ended, and / or may contain one or more modifications, mismatches, vacancies, and / or nucleotide substitutions. In some embodiments, the RNAi agent contains a 3′-terminal cap as described in this disclosure. Not wishing to be bound by any particular theory, the applicant proposes that, in some embodiments, the 3′-terminal cap may allow two functions: (1) to allow RNA interference; and (2) to increase the duration of activity and / or biological half-life of the RNAi agent (which may be achieved, for example, by increasing binding to the PAZ domain of Dicer and / or one or more Ago proteins) and / or to reduce or prevent the degradation of the RNAi agent (e.g., by nucleases, such as those in serum or intestinal fluid). In some embodiments, the RNAi agent of this disclosure targets (e.g., binds, anneals, etc.) target mRNA.In some embodiments, exposure of an RNAi agent to its target results in a reduction in activity, level, and / or expression, such as “knockdown” or “knockout” of the target. Specifically, in some embodiments, in cases of diseases, symptoms, and / or conditions characterized by overexpression and / or hyperactivity of a target gene, administration of an RNAi agent to cells, tissues, or subjects sufficiently knocks down the target gene to restore normal activity levels or reduces activity to a level that can alleviate, improve, mitigate, suppress, prevent, delay the onset of, reduce the severity of, and / or decrease the occurrence of one or more symptoms or features of the disease, symptoms, and / or condition. In some embodiments, the RNAi agent is double-stranded and contains an antisense strand, which is a single-stranded RNAi agent as described herein, and its combination with the sense strand can guide RNA interference.

[0347] Sample: As used herein, a “sample” is a specific organism or material obtained therefrom. In some embodiments, a sample is a biological sample obtained or derived from a target source as described herein. In some embodiments, the target source includes organisms such as animals or humans. In some embodiments, a biological sample includes biological tissues or fluids. In some embodiments, a biological sample is or includes bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; body fluids containing cells; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washes or lavages, such as catheter lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces; other body fluids, secretions and / or excretions; and / or cells obtained therefrom, etc. In some embodiments, a biological sample is or includes cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a target source by any appropriate means. For example, in some embodiments, primary biological samples are obtained by methods selected from the group consisting of: biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, body fluid collection (e.g., blood, lymph, feces, etc.). In some embodiments, as will be apparent from the context, the term "sample" refers to an article obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). For example, filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by techniques such as amplification or mRNA reverse transcription, separation, and / or purification of certain components of the primary sample. 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 an organism other than a human.

[0348] Single-stranded RNA interference: As used herein, the phrases “single-stranded RNAi” or “single-stranded RNA interference” refer at least in part to a process or method of gene silencing directed by the administration of a single-stranded RNAi agent to a system (e.g., cells, tissues, organs, subjects, etc.), wherein RNAi will be directed by an RNAi agent and requires a RISC pathway. The term may be used in certain contexts herein to distinguish it from “double-stranded RNAi” or “double-stranded RNA interference,” wherein a double-stranded RNAi agent is administered to a system and can be further processed, for example, to load one of its two strands into a RISC pathway to, for example, inhibit translation, cleave target RNA, etc.

[0349] Single-stranded RNAi agents: As used herein, the phrase "single-stranded RNAi agent" refers to a single-stranded oligonucleotide that can direct single-stranded RNA interference (RNAi or iRNA) gene silencing via a RISC pathway. Single-stranded RNAi agents may include polymers of one or more single-stranded nucleotides.

[0350] Subject: As used herein, the term "subject" or "test subject" means any organism to which the provided compound or composition is administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes, in accordance with this disclosure. 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 have and / or be susceptible to diseases, conditions, and / or symptoms.

[0351] Essentially: As used herein, the term “essentially” refers to a qualitative situation that represents the overall or near-overall range or degree of a target characteristic or property. A base sequence substantially complementary to the second sequence is not identical to the second sequence, but is largely or nearly identical to it. Furthermore, those skilled in the art of biology will understand that biological and chemical phenomena rarely (if they have ever occurred) reach completeness and / or proceed to completeness, or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to capture the inherent lack of potential completeness in many biological and / or chemical phenomena.

[0352] Suffering from: An individual who has been diagnosed with and / or exhibits one or more symptoms of a disease, condition and / or illness.

[0353] Susceptible: An individual "susceptible" to a disease, condition, and / or symptom is an individual at higher risk of developing said disease, condition, and / or symptom than the general population. In some embodiments, an individual susceptible to a disease, condition, and / or symptom may not have been diagnosed with said disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, and / or symptom may exhibit symptoms of said disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, and / or symptom may not exhibit symptoms of said disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, and / or symptom will develop said disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, and / or symptom will not develop said disease, condition, and / or symptom.

[0354] Systemic: As used herein, the phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” have their meanings as understood in the art and refer to the administration of a compound or composition to bring it into the recipient’s system.

[0355] Tautomerism: As used herein, the phrase "tautomerism" describes different isomers of an organic compound that can readily interconvert. Tautomers are characterized by the migration of hydrogen atoms or protons, accompanied by the conversion of single bonds and adjacent double bonds. In some embodiments, tautomers may arise from proton shift tautomerism (i.e., proton repositioning). In some embodiments, tautomers may arise from valence tautomerism (i.e., rapid reconfiguration of bond electrons). All such tautomerisms are intended to be included within the scope of this disclosure. In some embodiments, the tautomerisms of the compound exist in dynamic equilibrium with each other, so attempting to prepare individual substances would result in the formation of mixtures. In some embodiments, the tautomerisms of the compound are separable and separable compounds. In some embodiments of this disclosure, chemical compositions may be provided as pure formulations comprising or including a single tautomerism of the compound. In some embodiments of this disclosure, chemical compositions may be provided as mixtures of two or more tautomerisms of the compound. In some embodiments, such mixtures contain equal amounts of different tautomer forms; in other embodiments, such mixtures contain different amounts of at least two different tautomer forms of the compound. In some embodiments of this disclosure, the chemical composition may contain all tautomer forms of the compound. In some embodiments of this disclosure, the chemical composition may contain less than all tautomer forms of the compound. In some embodiments of this disclosure, the chemical composition may contain one or more tautomer forms of the compound, the amounts of which vary over time due to interconversion. In some embodiments of this disclosure, the tautomer is a keto-enol tautomer. Those skilled in the art will recognize that keto-enol tautomers can be “trapped” (i.e., chemically modified to retain it in the “enol” form) using any suitable reagent known in the chemical field to provide an enol derivative that can subsequently be isolated using one or more suitable techniques known in the art. Unless otherwise specified, this disclosure covers all tautomer forms of the relevant compound, whether in pure form or as blends of each other.

[0356] Therapeutic agent: As used herein, the phrase “therapeutic agent” means any agent that, when administered to a subject, has a therapeutic effect and / or induces the desired biological and / or pharmacological action. In some embodiments, a therapeutic agent is any substance that can be used to reduce, improve, alleviate, inhibit, prevent, delay the onset of a disease, condition and / or symptom, reduce its severity, and / or decrease the occurrence of one or more of its symptoms or features.

[0357] Therapeutic Effective Amount: As used herein, the term "therapeutic effective amount" refers to the 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, condition, and / or symptom when administered to a subject suffering from or susceptible to such a disease, condition, and / or symptom. As will be appreciated 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 to be delivered, and the target cells or tissues. For example, an effective amount of a compound in a formulation for treating a disease, condition, and / or symptom is an amount that reduces, improves, alleviates, inhibits, prevents, delays, reduces, and / or decreases the occurrence of one or more of its symptoms or features. In some embodiments, the therapeutically effective amount is administered in a single dose; in other embodiments, multiple unit doses are required to deliver the therapeutically effective amount.

[0358] Treatment: As used herein, the term "treatment" means any method used to partially or completely alleviate, improve, mitigate, suppress, prevent, delay the onset of a disease, condition, and / or symptom, reduce its severity, and / or decrease the occurrence of one or more of its symptoms or features. Treatment may be administered to a subject who does not exhibit symptoms of a disease, condition, and / or symptom. In some embodiments, treatment may be administered to a subject who exhibits only early symptoms of a disease, condition, and / or symptom, for example, to achieve the purpose of reducing the risk of developing lesions associated with said disease, condition, and / or symptom.

[0359] Unsaturated: As used in this article, the term “unsaturated” means that a part has one or more unsaturated units.

[0360] Unit dose: As used herein, the term "unit dose" refers to the amount administered as a single dose and / or as a physically discrete unit of a pharmaceutical composition. In many embodiments, a unit dose contains a predetermined amount of active agent. In some embodiments, a unit dose contains the entire single dose of the pharmaceutical agent. In some embodiments, more than one unit dose is administered to achieve an overall single dose. In some embodiments, multiple unit doses are required or anticipated to achieve the desired effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained-release formulation containing a predetermined amount of one or more therapeutic agents, or a drug delivery device, etc. It will be understood that a unit dose can be present in a formulation that includes any of a variety of components in addition to the therapeutic agent. For example, an acceptable carrier (e.g., a pharmaceutically acceptable carrier), diluent, stabilizer, buffer, preservative, etc., may be included as described below. Those skilled in the art will understand that in many embodiments, the overall appropriate daily dose of a particular therapeutic agent may include a portion of a unit dose or multiple unit doses, and may be determined, for example, by an attending physician within the bounds of reasonable medical judgment. In some implementations, the specific effective dose level for any particular subject or organism may depend on a variety of factors, including the condition being treated and its severity; the activity of the specific active compound used; the specific composition used; the subject's age, weight, general health status, sex, and diet; the timing of administration and excretion rate of the specific active compound used; the duration of treatment; drugs and / or other therapies used in combination with or concurrently with the specific compound used; and similar factors well known in the medical field.

[0361] Wild-type: As used herein, the term "wild-type" has its meaning as understood in the art, referring to an entity that has the structure and / or activity found in nature in a "normal" state or context (as opposed to mutation, disease, alteration, etc.). Those skilled in the art will understand that wild-type genes and peptides often exist in many different forms (e.g., alleles).

[0362] Nucleic Acids: As used herein, the term “nucleic acid” includes any nucleotide and its polymers. As used herein, the term “polynucleotide” refers to a polymeric form of nucleotide (ribonucleotide (RNA) or deoxyribonucleotide (DNA)) of any length. These terms refer to the primary structure of a molecule and therefore include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. These terms include analogues of RNA or DNA derived from modified nucleotides and / or modified polynucleotides (such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides) as equivalents. The terms cover polynucleotides or oligonucleotides (RNA) and polydeoxynucleotides or oligodeoxynucleotides (DNA); RNA or DNA derived from nucleotides and / or modified nucleotides of N-glycosides or C-glycosides; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate ester bridges and / or modified nucleotide inter-linking. The terminology covers nucleic acids containing any combination of nucleosides, modified nucleosides, sugars, modified sugars, phosphate ester bridges, or modified internucleotide bonds. Examples include, but are not limited to, nucleic acids containing a ribose moiety, nucleic acids containing a deoxyribose moiety, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing both ribose and modified ribose moieties. Unless otherwise specified, the prefix "most" generally refers to nucleic acids containing 2 to approximately 10,000 nucleotide monomer units, and the prefix "oligo" generally refers to nucleic acids containing 2 to approximately 200 nucleotide monomer units.

[0363] 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 nucleotides linked together. Naturally occurring bases (guanine (G), adenine (A), cytosine (C), thymine (T), and uracil (U)) are derivatives of purines or pyrimidines, but it should be understood that naturally occurring and unnaturally occurring base analogues are also included. Naturally occurring sugars are pentose (five-carbon sugars) deoxyribose (which forms DNA) or ribose (which forms RNA), but it should be understood that naturally occurring and unnaturally occurring sugar analogues are also included. Nucleotides are linked together by nucleotide linkages to form nucleic acids or polynucleotides. Many nucleotide linkages are known in the art (such as, but not limited to, phosphate esters, thiophosphate esters, borophosphate esters, etc.). Artificial nucleic acids include PNA (peptide nucleic acid), phosphate triesters, thiophosphates, H-phosphonates, aminophosphates, borophosphates, methylphosphonates, phosphonoacetates, thiophosphonoacetates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, natural nucleotides comprise naturally occurring bases, sugars, and intermolecular bonds. As used herein, the term "nucleotide" also encompasses structural analogs used in place of natural or naturally occurring nucleotides, such as modified nucleotides and nucleotide analogs.

[0364] Modified nucleotides: The term "modified nucleotide" includes any chemical portion that differs structurally from a natural nucleotide but is capable of performing at least one function of a natural nucleotide. In some embodiments, the modified nucleotide contains modifications at sugar, base, and / or nucleotide inter-linking sites. In some embodiments, the modified nucleotide comprises modified sugars, modified nucleobases, and / or modified nucleotide inter-linking sites. In some embodiments, the modified nucleotide is capable of having at least one function of a nucleotide, such as forming a subunit in a polymer capable of base pairing with a nucleic acid containing at least a complementary base sequence.

[0365] Analog: The term "analog" includes any chemical part that is structurally different from a reference chemical part or class of parts but is capable of performing at least one function of such reference chemical part or class of parts. As non-limiting examples, nucleotide analogs are structurally different from nucleotides but perform at least one function of nucleotides; nucleobase analogs are structurally different from nucleosides but perform at least one function of nucleosides; etc.

[0366] Nucleoside: The term "nucleoside" refers to the portion of a substance in which a nucleobase or modified nucleobase is covalently bonded to a sugar or modified sugar.

[0367] Modified nucleosides: The term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside, but containing chemical modifications that distinguish it from a natural nucleoside. Non-limiting examples of modified nucleosides include those containing modifications at the bases and / or sugars. Non-limiting examples of modified nucleosides include those having a 2′ modification at the sugar. Non-limiting examples of modified nucleosides also include baseless nucleosides (which lack a nucleobase). In some embodiments, the modified nucleoside is capable of having at least one function of a nucleoside, for example, forming a moiety in a polymer capable of base pairing with a nucleic acid containing at least a complementary base sequence.

[0368] Nucleoside analogs: The term "nucleoside analog" refers to a chemical moiety that is chemically distinct from a natural nucleoside but capable of performing at least one function of a nucleoside. In some embodiments, nucleoside analogs comprise sugar analogs and / or nucleobase analogs. In some embodiments, the modified nucleoside is capable of having at least one function of a nucleoside, for example, by forming a portion in a polymer that is capable of base pairing with a nucleic acid containing a complementary base sequence.

[0369] Sugar: The term "sugar" refers to a monosaccharide or polysaccharide in a closed and / or open form. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, furanopentose, pyranopentose, and pyranohexose moieties. As used herein, the term "sugar" also encompasses structural analogs used to replace conventional sugar molecules, such as diols, polymers forming the backbone of nucleic acid analogs, diol nucleic acids ("GNAs"), etc. As used herein, the term "sugar" also encompasses structural analogs used to replace naturally occurring or naturally occurring nucleotides, such as modified sugars and nucleotide sugars.

[0370] Modified sugars: The term "modified sugar" refers to a portion of sugar that can be substituted. Modified sugars mimic the spatial arrangement, electronic properties, or some other physicochemical property of sugars.

[0371] Nucleotides: The term "nucleotide" refers to a portion of a nucleic acid that involves hydrogen bonds that enable one nucleic acid strand to bind to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleotides are adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the naturally occurring nucleotides are modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleotides are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleotide is a "modified nucleotide," such as a nucleotide other than adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleotides are methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobases mimic the spatial arrangement, electronic properties, or other physicochemical properties of nucleobases and retain the hydrogen bonds that enable one nucleic acid strand to bind to another in a sequence-specific manner. In some embodiments, the modified nucleobases can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting unwinding behavior, recognition by intracellular enzymes, or activity of oligonucleotide duplexes. As used herein, the term "nucleobase" also encompasses structural analogs used to replace naturally occurring or naturally occurring nucleotides, such as modified nucleobases and nucleobase analogs.

[0372] Modified nucleobase: The terms "modified nucleobase," "modified base," etc., refer to a chemical portion that is chemically different from a nucleobase but capable of performing at least one function of a nucleobase. In some embodiments, the modified nucleobase is a nucleobase comprising a modification. In some embodiments, the modified nucleobase can have at least one function of a nucleobase, for example, forming a portion in a polymer capable of base pairing with a nucleic acid containing at least a complementary base sequence.

[0373] 3′-Terminal Cap: The term “3′-terminal cap” refers to a non-nucleotide chemical portion that binds to the 3′ end of an oligonucleotide (e.g., an RNAi agent). In some embodiments, the 3′-terminal cap replaces a 3′-terminal dinucleotide. In some embodiments, the 3′-terminal cap of an oligonucleotide performs at least one of the following functions: allows oligonucleotide-guided RNA interference, protects the oligonucleotide from degradation or reduces the amount or rate of oligonucleotide degradation (e.g., by nucleases), reduces off-target effects of the sense strand, or increases the activity, duration, or efficacy of oligonucleotide-guided RNA interference. By describing the 3′-terminal cap as “non-nucleotide,” it means that the 3′-terminal cap is not a nucleotide portion or oligonucleotide portion linked to the sugar portion of the remainder of the oligonucleotide, as if it were part of the oligonucleotide chain. Certain exemplary 3′-terminal caps are described herein. It will be understood by those skilled in the art that other 3′-terminal caps known in the art may be used in accordance with this disclosure.

[0374] Blocking group: The term "blocking group" refers to a group that masks the reactivity of a functional group. The functional group can then be demasked by removing the blocking group. In some embodiments, the blocking group is a protecting group.

[0375] Part: The term "part" refers to a specific segment or functional group of a molecule. A chemical part is often considered to be a chemical entity embedded in or attached to a molecule.

[0376] Solid support: The term "solid support" refers to any support that enables the synthesis of nucleic acids. In some embodiments, the term refers to a glass or polymer that is insoluble in the medium used to perform the reaction steps to synthesize nucleic acids and is derived to include reactive groups. In some embodiments, the solid support is highly cross-linked polystyrene (HCP) or controlled-porosity glass (CPG). In some embodiments, the solid support is controlled-porosity glass (CPG). In some embodiments, the solid support is a hybrid support of controlled-porosity glass (CPG) and highly cross-linked polystyrene (HCP).

[0377] Linker or connecting portion: The terms "linker," "connecting portion," etc., refer to any chemical portion that links one chemical portion to another. In some embodiments, a linker is a portion that links one oligonucleotide to another oligonucleotide in a polymer. In some embodiments, a linker is optionally a portion located between the terminal nucleoside and a solid support or between the terminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

[0378] Gene: As used herein, the terms “gene,” “recombinant gene,” and “gene construct” refer to a DNA molecule or a portion thereof that encodes a protein or a portion thereof. A DNA molecule may contain open reading frames (such as exon sequences) encoding a protein and may also include intron sequences. As used herein, the term “intron” refers to a DNA sequence present in a given gene that is not translated into a protein and is present between exons in some, but not all, cases. It may be desirable for the gene to be operatively linked with (or may contain) one or more promoters, enhancers, repressors, and / or other regulatory sequences to regulate gene activity or expression, as is well known in the art.

[0379] Complementary DNA: As used herein, “complementary DNA” or “cDNA” includes recombinant polynucleotides synthesized by reverse transcription of mRNA from which intercalation sequences (introns) have been removed.

[0380] Homology: “Homology,” “identity,” or “similarity” refers to the sequence similarity between two nucleic acid molecules. Homology and identity can each be determined by comparing positions in sequences that can be aligned for comparison purposes. Molecules are identical at that position when equivalent positions in the compared sequences are occupied by the same bases; molecules are said to be homologous (similar) at that position when equivalent sites are occupied by the same or similar nucleic acid residues (e.g., similar in spatial and / or electronic properties). Homology / similarity or identity percentages are expressed as a function of the number of identical or similar nucleic acids at positions shared by the compared sequences. “Irrelevant” or “non-homologous” sequences share less than 40%, less than 35%, less than 30%, or less than 25% identity with the sequences described herein. The absence of residues (amino acids or nucleic acids) or the presence of additional residues also reduces identity and homology / similarity when comparing two sequences.

[0381] In some embodiments, the term "homology" describes a mathematically based sequence similarity comparison used to identify genes with similar functions or motifs. The nucleic acid sequences described herein can be used as "query sequences" for searching public databases, for example, to identify 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 using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of this disclosure. In some embodiments, for obtaining vacancy alignments for comparative purposes, vacancy BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and vacancy BLAST procedures, the default parameters of the corresponding procedures (e.g., XBLAST and BLAST) can be used (see www.ncbi.nlm.nih.gov).

[0382] Identity: As used in this article, “identity” means the percentage of identical nucleotide residues at corresponding positions in two or more sequences when sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but 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, ed., 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., ed., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied). Math., 48:1073 (1988). The method used to determine identity is designed to give the maximum match between the sequences being tested. Furthermore, the method used to determine identity is encoded in publicly available computer programs. Computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF 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 available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM). NIHBethesda, Md. 20894; Altschul, S. et al., J.Mol.Biol. 215: 403-410 (1990). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0383] Oligonucleotides: The term “oligonucleotide” refers to a polymer or oligomer of nucleotides and may contain any combination of natural and non-natural nucleobases, sugars, and internucleotide bonds.

[0384] Oligonucleotides can be single-stranded or double-stranded. As used herein, the term "oligonucleotide chain" encompasses single-stranded oligonucleotides. Single-stranded oligonucleotides may have double-stranded regions (formed by the two parts of a single-stranded oligonucleotide), and double-stranded oligonucleotides comprising two oligonucleotide chains may, for example, have single-stranded regions at regions where the two oligonucleotide chains are not complementary to each other. In some embodiments, oligonucleotides are capable of directing the reduction of expression and / or levels of a target gene or its gene product. In some embodiments, oligonucleotides are capable of directing the reduction of expression and / or levels of a target gene or its gene product via RNA interference. In some embodiments, oligonucleotides are capable of directing the reduction of expression and / or levels of a target gene or its gene product via biochemical mechanisms that do not involve RNA interference or RISC (including, but not limited to, RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, oligonucleotides are capable of directing the reduction of expression and / or levels of a target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. Exemplary oligonucleotides include, but are not limited to, structural genes, genes including control and termination regions, self-replicating systems (such as viral or plasmid DNA), single-stranded and double-stranded RNAi agents and other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribonucleases, microRNAs, microRNA mimics, supermicroRNAs, aptamers, antisense microRNAs, microRNA antagonists, Ul connectives, triple-stranded oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

[0385] Double-stranded and single-stranded oligonucleotides that effectively induce RNA interference are also referred to herein as RNAi agents or iRNA agents. In some embodiments, these RNA interference-inducing oligonucleotides associate with a cytoplasmic multiprotein complex called an RNAi-induced silencing complex (RISC). In many embodiments, double-stranded RNAi agents are long enough that they can be cleaved by endogenous molecules (e.g., by Dicer) to produce smaller oligonucleotides that can enter the RISC mechanism and participate in RISC-mediated cleavage and / or translational repression of target sequences (e.g., target mRNA sequences).

[0386] The oligonucleotides disclosed herein can have various lengths. In certain embodiments, the length of the oligonucleotide can range from about 2 to about 200 nucleotides. In various related embodiments, the lengths of single-stranded, double-stranded, and triple-stranded oligonucleotides can range from about 4 to about 10 nucleotides, about 10 to about 50 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides. In some embodiments, the length of the oligonucleotide is about 10 to about 40 nucleotides. In some embodiments, the length of the oligonucleotide is about 9 to about 39 nucleotides. In some embodiments, the length of the oligonucleotide is at least 4 nucleotides. In some embodiments, the length of the oligonucleotide is at least 5 nucleotides. In some embodiments, the length of the oligonucleotide is at least 6 nucleotides. In some embodiments, the length of the oligonucleotide is at least 7 nucleotides. In some embodiments, the length of the oligonucleotide is at least 8 nucleotides. In some embodiments, the length of the oligonucleotide is at least 9 nucleotides. In some embodiments, the length of the oligonucleotide is at least 10 nucleotides. In some embodiments, the length of the oligonucleotide is at least 11 nucleotides. In some embodiments, the length of the oligonucleotide is at least 12 nucleotides. In some embodiments, the oligonucleotide is at least 15 nucleotides long. In some embodiments, the oligonucleotide is at least 20 nucleotides long. In some embodiments, the oligonucleotide is at least 25 nucleotides long. In some embodiments, the oligonucleotide is at least 30 nucleotides long. In some embodiments, the oligonucleotide is a duplex of complementary strands at least 18 nucleotides long. In some embodiments, the oligonucleotide is a duplex of complementary strands at least 21 nucleotides long. In some embodiments, each nucleotide, counted by length, independently contains a nucleobase selected from the optionally substituted nucleotides of adenine, cytosine, guanosine, thymine, and uracil.

[0387] Nucleotide-to-nucleotide linkage: As used herein, the phrase “nucleotide-to-nucleotide linkage” generally refers to the linkage of nucleoside units connecting oligonucleotides or nucleic acids. In some embodiments, the nucleotide-to-nucleotide linkage is a phosphodiester linkage (natural phosphodiester linkage) as present in naturally occurring DNA and RNA molecules. In some embodiments, the term “nucleotide-to-nucleotide linkage” includes modified nucleotide-to-nucleotide linkages. In some embodiments, the nucleotide-to-nucleotide linkage is a “modified nucleotide-to-nucleotide linkage” in which the oxygen atoms of the phosphodiester linkage are each optionally and independently replaced by an organic or inorganic portion. In some embodiments, such organic or inorganic portions are selected from, but not limited to, =S, =Se, =NR′, -SR′, -SeR′, -N(R′)2, B(R′)3, -S-, -Se-, and -N(R′)-, wherein each R′ is independently defined and described as in this disclosure. In some embodiments, the nucleotide-to-nucleotide linkage is a phosphotriester linkage or a phosphothiodiester linkage. Or linked to modified trithiophosphates.

[0388] In some implementations, the internucleotide linkage is, for example, a PNA (peptide nucleic acid) or PMO (diaminophosphate morpholino oligomer) linkage.

[0389] Those skilled in the art will understand that, due to the presence of acidic or basic components in the bonding, nucleotide bonds can exist as anions or cations at a given pH.

[0390] Unless otherwise specified, when used with oligonucleotide sequences, s, s1, s2, s3, s4, s5, s6, and s7 each independently represent internucleotide linkages with the following modifications as shown below:

[0391]

[0392]

[0393] For example, (Rp,Sp)-ATsCs1GA has 1) a phosphate thioester nucleotide linking between T and C. 2) There is a structure between C and G. The internucleotide linkages of thiophosphate triesters. Unless otherwise specified, the Rp / Sp name preceding the oligonucleotide sequence describes the configuration of the chiral phosphorus atom in the internucleotide linkages arranged in a 5′ to 3′ sequence of the oligonucleotide sequence. For example, in (Rp,Sp)-ATsCs1GA, the phosphorus in the “s” linkage between T and C has the Rp configuration, while the phosphorus in the “s1” linkage between C and G has the Sp configuration.

[0394] In some implementations, “all (Rp)” or “all (Sp)” is used to indicate that all chiral phosphorus atoms in an oligonucleotide have the same Rp or Sp configuration, respectively.

[0395] Oligonucleotide type: As used herein, the phrase “oligonucleotide type” is used to define a specific base sequence, main chain bonding pattern (i.e., internucleotide bonding pattern, such as phosphate esters, thiophosphate esters, etc.), main chain chiral center pattern (i.e., phosphorus-linked stereochemistry pattern (Rp / Sp)), and main chain phosphorus modification pattern (e.g., “-XLR” in Formula I). 1 Oligonucleotides with a common designated "type" are structurally identical to each other.

[0396] Those skilled in the art will appreciate that the synthetic methods of this disclosure provide a degree of control during the synthesis of oligonucleotide chains, allowing each nucleotide unit of the oligonucleotide chain to be designed and / or selected in advance to have a specific stereochemistry at the phosphate link and / or a specific modification and / or a specific base and / or a specific sugar at the phosphate link. In some embodiments, oligonucleotide chains are designed and / or selected in advance to have a specific combination of stereocenters at the phosphate link. In some embodiments, oligonucleotide chains are designed and / or determined to have a specific combination of modifications at the phosphate link. In some embodiments, oligonucleotide chains are designed and / or selected to have a specific combination of bases. In some embodiments, oligonucleotide chains are designed and / or selected to have a specific combination of having one or more structural features. In some embodiments, this disclosure provides a composition comprising a plurality of oligonucleotide molecules or a composition composed of a plurality of oligonucleotide molecules (e.g., a chiral-controlled oligonucleotide composition). In some embodiments, all such molecules belong to the same type (i.e., are structurally identical to each other). However, in many embodiments, the provided compositions typically contain a plurality of different types of oligonucleotides in predetermined relative amounts.

[0397] Chiral control: As used herein, "chiral control" refers to the control of the stereochemical design of the chiral linking phosphorus in the chiral internucleotide link within an oligonucleotide. In some embodiments, control is achieved by chiral elements absent in the sugar and base moieties of the oligonucleotide, for example, by using one or more chiral auxiliaries in the oligonucleotide preparation process as shown in this disclosure, said chiral auxiliaries typically being part of a chiral phosphoramide used in the oligonucleotide preparation process. In contrast to chiral control, those skilled in the art will understand that if conventional oligonucleotide synthesis without chiral auxiliaries is used to form the chiral internucleotide link, such conventional oligonucleotide synthesis cannot control the stereochemistry at the chiral internucleotide link. In some embodiments, the stereochemical design of each chiral linking phosphorus in the chiral internucleotide link within the oligonucleotide is controlled.

[0398] Chiral-controlled oligonucleotide compositions: As used herein, the terms “chiral-controlled oligonucleotide composition,” “chiral-controlled nucleic acid composition,” etc., refer to compositions comprising multiple oligonucleotides (or nucleic acids) that share 1) a common base sequence, 2) a common backbone linking pattern, and 3) a common backbone phosphorus modification pattern, wherein the multiple oligonucleotides (or nucleic acids) have the same stereochemistry at one or more chiral nucleotide links (chiral-controlled nucleotide links), and the levels of the multiple oligonucleotides (or nucleic acids) in the composition are predetermined (e.g., by chiral-controlled oligonucleotide articles to form one or more chiral nucleotide links). In some embodiments, the chiral-controlled oligonucleotide composition contains about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 3%) of all oligonucleotides. 0%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are the plurality of oligonucleotides. In some embodiments, the chiral-controlled oligonucleotide composition comprises about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%) of all oligonucleotides sharing a common base sequence. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are the plurality of oligonucleotides.In some embodiments, approximately 1%–100% (e.g., approximately 5%–100%, 10%–100%, 20%–100%, 30%–100%, 40%–100%, 50%–100%, 60%–100%, 70%–100%, 80%–100%, 90%–100%, 95%–100%, 50%–90%) of all oligonucleotides sharing a common base sequence, a common backbone bonding pattern, and a common backbone phosphorus modification pattern in the chiral-controlled oligonucleotide composition. (or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are the plurality of oligonucleotides. In some embodiments, the predetermined level is about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%) of all oligonucleotides in the composition, or all oligonucleotides in the composition that share a common base sequence (e.g., multiple oligonucleotides or oligonucleotide types), or all oligonucleotides in the composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linking type pattern, and / or a common internucleotide linking modification pattern. 0%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).In some embodiments, multiple oligonucleotides share the same stereochemistry at the bonding sites between about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) chiral nucleotides. In some embodiments, multiple oligonucleotides are present in amounts ranging from about 1% to 100% (e.g., about 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%). The chiral nucleotides at 100%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) share the same stereochemistry. In some embodiments, each chiral nucleotide link is a chiral-controlled nucleotide link, and the composition is a fully chiral oligonucleotide composition. In some embodiments, not all chiral nucleotide links are chiral-controlled nucleotide links, and the composition is a partially chiral oligonucleotide composition. In some embodiments, the chiral oligonucleotide composition comprises a predetermined level of a single oligonucleotide 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 more than one oligonucleotide type. 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 oligonucleotide types, said composition comprising predetermined levels of oligonucleotides of multiple oligonucleotide types.

[0399] Chiral homogeneity: As used herein, the phrase “chiral homogeneity” describes an oligonucleotide molecule or type in which all nucleotide units have the same stereochemistry at the phosphorus-linked junction. For example, an oligonucleotide in which all nucleotide units have Rp stereochemistry at the phosphorus-linked junction is chiral homogeneous. Similarly, an oligonucleotide in which all nucleotide units have Sp stereochemistry at the phosphorus-linked junction is chiral homogeneous.

[0400] Predetermined: Predetermined means intentionally selected, for example, the opposite of random occurrence or uncontrolled achievement. Those skilled in the art who read this specification will understand that this disclosure provides techniques that allow selection of specific chemical and / or stereochemical features to be incorporated into oligonucleotide compositions, and also allow controlled preparation of oligonucleotide compositions having such chemical and / or stereochemical features. Such compositions provided are “predetermined” as described herein. Compositions that may contain certain oligonucleotides have happened to be produced by processes that intentionally produce specific chemical and / or stereochemical features that cannot be controlled, so they are not “predetermined” compositions. In some embodiments, a predetermined composition is a composition that can be intentionally reproduced (e.g., by repeating a controlled process). In some embodiments, a predetermined level of multiple oligonucleotides in a composition means controlling the absolute and / or relative amounts (ratios, percentages, etc.) of multiple oligonucleotides in the composition. In some embodiments, a predetermined level of multiple oligonucleotides in a composition is achieved by chiral-controlled oligonucleotide preparation.

[0401] Linked phosphorus: As defined herein, the phrase “linked phosphorus” is used to indicate that the specific phosphorus atom mentioned is a phosphorus atom present in an internucleotide link, said phosphorus atom corresponding to the phosphorus atom of a phosphodiester, such as those present in naturally occurring DNA and RNA, in internucleotide links. In some embodiments, the linked phosphorus atom is in a modified internucleotide link, wherein the oxygen atoms of the phosphodiester link are each optionally and independently replaced by an organic or inorganic portion. In some embodiments, the linked phosphorus atom is P of Formula I. L In some implementations, the bonded phosphorus atom is chiral.

[0402] P-modification: As used herein, the term "P-modification" refers to any modification at the phosphorus bond other than stereochemical modification. In some embodiments, P-modification includes adding, substituting, or removing the covalently attached side portion of the phosphorus bond. In some embodiments, "P-modification" is -XLR 1 X, L and R 1 Each is independent as defined and described in this disclosure.

[0403] Block polymer: As used herein, the term "block polymer" refers to an oligonucleotide chain characterized by the presence of at least two consecutive nucleotide units sharing a common structural feature at an interphosphonucleotide link. A common structural feature means a common stereochemistry at the phosphorus link or a common modification at the phosphorus link. In some embodiments, the consecutive nucleotide units sharing a common structural feature at at least two interphosphonucleotide links are referred to as a "block." In some embodiments, the provided oligonucleotide is a block polymer.

[0404] In some implementations, the block polymer is a "stereoblock polymer," for example, at least two consecutive nucleotide units having the same stereochemistry at the phosphorus linker. Such at least two consecutive nucleotide units form a "stereoblock."

[0405] In some implementations, the block polymer is a "P-modified block polymer," for example, at least two consecutive nucleotide units have the same modification at the phosphate linker. Such at least two consecutive nucleotide units form a "P-modified block." For example, (Rp,Sp)-ATsCsGA is a P-modified block polymer because at least two consecutive nucleotide units, Ts and Cs, have the same P modification (i.e., both are phosphothioesters). In the same oligonucleotide (Rp,Sp)-ATsCsGA, TsCs forms the block, and it is a P-modified block.

[0406] In some embodiments, the block polymer is a “linked block polymer,” for example, at least two consecutive nucleotide units having the same stereochemistry and the same modification at the linking phosphorus. At least two consecutive nucleotide units form a “linked block.” For example, (Rp,Rp)-ATsCsGA is a linked block polymer because at least two consecutive nucleotide units, Ts and Cs, have the same stereochemistry (both Rp) and P modification (both phosphate thioesters). In the same oligonucleotide (Rp,Rp)-ATsCsGA, TsCs forms the block, and it is a linked block.

[0407] In some embodiments, the block aggregate comprises one or more blocks independently selected from stereoblocks, P-modified blocks, and linked blocks. In some embodiments, the block aggregate is a stereoblock aggregate with respect to one block and / or a P-modified block aggregate with respect to another block and / or a linked block aggregate with respect to yet another block.

[0408] Alternating polymers: As used herein, the term "alternating polymer" refers to an oligonucleotide chain characterized by a structural feature pattern for each individual nucleotide unit, wherein two consecutive nucleotide units of the oligonucleotide chain do not share a specific structural feature at the phosphate linkage between the nucleotides. In some embodiments, the alternating polymer is designed such that it contains repeating patterns. In some embodiments, the alternating polymer is designed such that it does not contain repeating patterns. In some embodiments, the provided oligonucleotide is an alternating polymer.

[0409] In some implementations, the alternating polymer is a “stereo-alternating polymer”, for example, two consecutive nucleotide units do not have the same stereochemistry at the phosphorus linker.

[0410] In some implementations, the alternating polymer is a "P-modified alternating polymer," where two consecutive nucleotide units do not have the same modification at the linking phosphorus. For example, all(Sp)-CAs1GsT, where each linking phosphorus has a different P modification than the others.

[0411] In some implementations, the alternating polymer is a “linked alternating polymer”, for example, two consecutive nucleotide units that do not have the same stereochemistry or the same modification at the linking phosphorus.

[0412] Monomer: As used herein, the term "monomer" refers to an oligonucleotide chain whose structural characterization of each individual nucleotide unit is such that all nucleotide units within the chain share at least one common structural feature at the internucleotide phosphorus bond. A common structural feature means a common stereochemistry at the phosphorus bond or a common modification at the phosphorus bond. In some embodiments, the provided oligonucleotide is a monomer.

[0413] In some implementations, the monomer is a "stereomonomer," for example, where all nucleotide units have the same stereochemistry at the phosphorus linker.

[0414] In some implementations, the monomer is a "P-modified monomer," where all nucleotide units have the same modification at the phosphate linker.

[0415] In some implementations, the monomer is a “linked monomer”, where all nucleotide units have the same stereochemistry and the same modification at the linking phosphorus.

[0416] Spacer: As used herein, the term "spacer" refers to an oligonucleotide chain characterized in that at least one nucleotide in the oligonucleotide chain has an internucleotide phosphophosphodiester bond, such as those found in naturally occurring DNA or RNA. In some embodiments, more than one nucleotide in the oligonucleotide chain has an internucleotide phosphophosphodiester bond, such as those found in naturally occurring DNA or RNA. In some embodiments, the provided oligonucleotide is a spacer.

[0417] Skipmer: As used herein, the term "skipmer" refers to a type of spacer polymer in which the phosphorus bonds between every other nucleotide of the oligonucleotide chain are phosphodiester bonds, such as those present in naturally occurring DNA or RNA, and the phosphorus bonds between every other nucleotide of the oligonucleotide chain are modified internucleotide bonds. In some embodiments, the provided oligonucleotide is a skipmer.

[0418] For the purposes of this disclosure, chemical elements are identified according to the CAS version of the periodic table of elements on the inner cover of Handbook of Chemistry and Physics, 67th edition, 1986-87.

[0419] The methods and structures described herein with respect to the compounds and compositions of this disclosure are also applicable to pharmaceutically acceptable acid or base addition salts and all stereoisomer forms of these compounds and compositions. Attached Figure Description

[0420] Figure 1. Figure 1 includes Figures 1A to 1L The cartoon illustrations show various ssRNAi and hybridization formats.

[0421] Figure 2 . Figure 2 Cartoon illustrations of various antisense oligonucleotide formats are shown.

[0422] Figure 3. Figure 3A An exemplary multimer format is shown. Oligonucleotides can be conjugated directly and / or via linkers. As shown, the multimer can contain oligonucleotide monomers of the same or different structures / types. In some embodiments, the monomer of the multimer is an ssRNAi agent. In some embodiments, the monomer of the multimer is an RNase H-dependent antisense oligonucleotide (ASO). Monomers can be conjugated at various positions, such as the 5′-terminus, 3′-terminus, or middle position. Figure 3B An exemplary chemical method for conjugating monomers is shown, which can perform their functions to form polymers through various pathways. Detailed Implementation

[0423] Synthetic oligonucleotides provide molecular tools available for a wide range of applications. For example, oligonucleotides can be used in therapeutics, diagnostics, research, and novel nanomaterials applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. Therefore, various synthetic counterparts have been developed to circumvent these drawbacks. These include, in particular, synthetic oligonucleotides containing chemical modifications (e.g., base modifications, sugar modifications, backbone modifications, etc.) that make these molecules less susceptible to degradation and improve other properties of the oligonucleotides. From a structural perspective, modifications to the phosphate ester bonds between nucleotides can introduce chirality, and certain properties of oligonucleotides can be influenced by the configuration of the phosphorus atom forming the oligonucleotide backbone. For example, in vitro studies have shown that the properties of antisense oligonucleotides (such as binding affinity, sequence specificity for binding complementary RNA, and stability to nucleases) are particularly affected by the chirality of the backbone phosphorus atom.

[0424] This disclosure particularly covers the understanding that structural elements of oligonucleotides, such as chemical modifications (e.g., modifications to sugar, base, and / or nucleotide linkages) or their patterns, conjugations to lipids or other moieties, and / or stereochemistry [e.g., stereochemistry of the chiral center of the backbone (chiral nucleotide linkages) and / or their patterns], can have a significant impact on properties and activities (e.g., stability, specificity, selectivity, activity that reduces the level of target gene products (transcriptions and / or proteins), etc.). In some embodiments, the properties of oligonucleotides can be modulated by optimizing chemical modifications (modifications to base, sugar, and / or nucleotide linkages), chemical modification patterns, stereochemistry, and / or stereochemical patterns.

[0425] In some embodiments, this disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides having controlled structural elements (e.g., controlled chemical modifications and / or controlled backbone stereochemistry patterns) provide unexpected properties and activities, including but not limited to those described herein. In some embodiments, the provided compositions comprising oligonucleotides having chemical modifications (e.g., base modifications, sugar modifications, internucleotide linking modifications, etc.) or their patterns have improved properties and activities. Non-limiting examples of such improved properties include: directing the expression and / or reduction of levels of target genes or their gene products; and / or directing RNA interference; and / or directing RNase H-mediated knockdown. In some embodiments, this disclosure provides techniques (e.g., oligonucleotides, compositions, methods, etc.) for single-stranded RNAi. In some embodiments, the provided oligonucleotides are ssRNAi agents.

[0426] In some implementations, RNA interference is reportedly a post-transcriptional targeted gene silencing technique that uses an RNAi agent to target RNA (e.g., gene transcripts such as messenger RNA (mRNA)) containing a sequence complementary to the RNAi agent for cleavage mediated by the RISC (RNA-induced silencing complex) pathway. Essentially, a type of RNAi is reportedly observed when ribonuclease III (Dicer) cleaves long dsRNA (double-stranded RNA) (e.g., exogenous dsRNA introduced into mammalian cells) into shorter fragments called siRNA. siRNA (small interfering RNA or short repressive RNA) is typically about 21 to 23 nucleotides long and contains a duplex of about 19 base pairs. The smaller RNA fragment then reportedly mediates the degradation of the target mRNA. The RNAi reaction is also reportedly characterized by an endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which directs the cleavage of a single-stranded mRNA complementary to the antisense strand of the siRNA. The cleavage of the target RNA is reportedly carried out in the middle of a region complementary to the antisense strand of the siRNA duplex. It has been reported that the application of RNAi agents to target transcripts leads to a decrease in gene activity, level, and / or expression, such as the "knockdown" or "removal" of target genes or sequences. Artificial siRNAs can be used both as therapeutic agents and for experimental purposes.

[0427] On the one hand, RNA interference agents include single-stranded RNAs that interact with target RNA sequences to guide target RNA cleavage. Not wanting to be bound by theory, a type III endonuclease called Dicer has been reported to break down long double-stranded RNAs introduced into plant and invertebrate cells into siRNAs (Sharp et al., Genes Dev. 2001, 15:485). A ribonuclease III-like enzyme, Dicer, has been reported to process dsRNAs into short interfering RNAs of 19–23 base pairs, characterized by two 3′ overhangs (Bernstein et al., (2001) Nature 409:363). The siRNAs are then reportedly incorporated into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107:309). Upon binding to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15: 188). Therefore, in one respect, this disclosure relates to single-stranded RNAs that promote the formation of RISC complexes to achieve target gene silencing.

[0428] In some implementations, according to this disclosure, a suitable RNAi agent can be selected by any method known in the art or any method that can be conceived by a person skilled in the art. For example, selection criteria may include one or more of the following steps: preliminary analysis of the target gene sequence and design of the RNAi agent; such design may take into account sequence similarity between species (human, cynomolgus monkey, mouse, etc.) and differences from other (non-target) genes; in vitro screening of the RNAi agent (e.g., 10 nM in cells expressing the target transcript); determination of EC50 or IC50 in cells; determination of the viability of cells treated with the RNAi agent, wherein in some embodiments, the target RNAi agent is required not to inhibit the viability of these cells; testing with human PBMCs (peripheral blood mononuclear cells), for example, testing TNF-α levels to estimate immunogenicity, wherein an immune stimulation sequence is generally not required; testing in human whole blood assays, wherein fresh human blood is treated with the RNAi agent and cytokine / chemokine levels [e.g., TNF-α (tumor necrosis factor-α) and / or MCP1 (monocyte chemoattractant protein 1)], wherein an immune stimulation sequence is generally not required; determination of in vivo gene knockdown using cells or tumors in test animals; and optimization of the specificity of the RNAi agent.

[0429] The so-called canonical siRNA structure is reportedly a double-stranded RNA molecule, with each strand approximately 21 nucleotides long. These two strands are reportedly an antisense (or "leading") 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, typically forming two 3' overhangs of 2 nucleotides at each end.

[0430] While the standard siRNA structure is reported to be double-stranded, RNAi agents can also be single-stranded. In some implementations, single-stranded RNAi agents correspond to the antisense strand of double-stranded siRNA, and single-stranded RNAi agents lack the corresponding transit strand.

[0431] However, it has been reported that not all structural elements of the tested single-stranded RNAi agents are effective; it has been reported that introducing some structural elements into oligonucleotides can interfere with single-stranded RNA interference activity.

[0432] In some embodiments, this disclosure provides oligonucleotides and compositions that can be used as RNAi agents. In some embodiments, this disclosure provides oligonucleotides and compositions that can be used as single-stranded RNAi agents. This disclosure particularly provides novel structures of single-stranded oligonucleotides capable of directing RNA interference. Without wishing to be bound by any particular theory, this disclosure points out that single-stranded RNAi agents have advantages over double-stranded RNAi agents. For example, single-stranded RNAi agents are commercially less expensive because only one strand needs to be constructed. Alternatively or additionally, only one strand (the antisense strand) is applied to target the transcript. The off-target effects directed by dsRNA arise from loading the sense strand into a RISC and binding and knocking down unwanted targets (Jackson et al. 2003 Nat. Biotech. 21: 635-637), and single-stranded RNAi agents cause fewer off-target effects compared to their corresponding double-stranded RNAi agents. In addition, some single-stranded RNAi agents, including some disclosed herein, can target specific sequences that have not been successfully targeted by double-stranded RNAi agents (e.g., they can significantly reduce the levels of the sequence and / or sequence products (transcriptions and / or proteins) compared to double-stranded RNAi agents). This disclosure particularly provides novel formats (modifications, stereochemistry, combinations thereof, etc.) of oligonucleotides that can guide single-stranded RNA interference.

[0433] Oligonucleotides

[0434] In some embodiments, the provided oligonucleotide can direct the reduction of expression and / or level of a target gene or its gene product. In some embodiments, the provided oligonucleotide can direct the reduction of target product level. In some embodiments, the provided oligonucleotide can reduce the transcript level of a target gene. In some embodiments, the provided oligonucleotide can reduce the mRNA level of a target gene. In some embodiments, the provided oligonucleotide can reduce the protein level encoded by the target gene. In some embodiments, the provided oligonucleotide can direct the reduction of expression and / or level of a target gene or its gene product via RNA interference. In some embodiments, the provided oligonucleotide can direct the reduction of expression and / or level of a target gene or its gene product via biochemical mechanisms that do not involve RNA interference or RISC (including but not limited to RNase H-mediated knockdown or steric hindrance of gene expression). In some embodiments, the provided oligonucleotide can direct the reduction of expression and / or level of a target gene or its gene product via RNA interference and / or RNase H-mediated knockdown. In some embodiments, the provided oligonucleotides can guide the reduction of expression and / or levels of a target gene or its gene product by spatially blocking translation upon binding to the target gene mRNA, and / or by altering or interfering with mRNA splicing and / or exon inclusion or exclusion. In some embodiments, the provided oligonucleotides comprise one or more structural elements described herein or known in the art according to this disclosure, such as: base sequences; modifications; stereochemistry; internucleotide linking patterns; GC content; long GC extensions; backbone linking patterns; backbone chiral center patterns; backbone phosphorus modification patterns; additional chemical moieties, including but not limited to one or more targeting moieties, lipid moieties, and / or carbohydrate moieties; seed regions; post-seed regions; 5′-terminal structures; 5′-terminal regions; 5′ nucleotide moieties; 3′-terminal regions; 3′-terminal dinucleotides; 3′-terminal caps; etc. In some embodiments, the seed region of the oligonucleotide is or contains the second to eighth, second to seventh, second to sixth, third to eighth, third to seventh, third to seventh, or fourth to eighth, or fourth to seventh nucleotides counted from the 5′ end; and the post-seed region of the oligonucleotide is the region immediately inoculated with the 3′ seed region and located between the seed region and the 3′ end region.

[0435] In some embodiments, the provided composition comprises oligonucleotides. In some embodiments, the provided composition comprises one or more lipid moieties, one or more carbohydrate moieties (unless otherwise specified, in addition to sugar moieties that form nucleoside units having internucleotide bonds), and / or one or more targeting components.

[0436] In some embodiments, this disclosure provides compounds having the structure of formula OI (e.g., oligonucleotides of the provided compositions):

[0437]

[0438] Or its salt, wherein:

[0439] R E It is a 5′-terminal group;

[0440] Each of BA is independently a group selected from the following optionally substituted groups: C 1-30 Alicyclic group, C 6-30 Aryl group, having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 5-30 Heteroaryl groups having 1-10 C atoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon. 3-30 Heterocyclic group, native nucleobase moiety, and modified nucleobase moiety;

[0441] R s Each of them independently is -F, -Cl, -Br, -I, -CN, -N3, -NO, -NO2, -LR′, -L-OR′, -L-SR′, -LN(R′)2, -OL-OR′, -OL-SR′ or -OLN(R′)2;

[0442] s is 0-20;

[0443] L are each independently a covalent bond, or selected from the following divalent, optionally substituted, straight-chain or branched groups: C 1-30 Aliphatic groups and C atoms having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon. 1-30 Heteroaliphatic groups, wherein one or more methylene units are optionally and independently replaced by: 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)(S R′)-、-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-; and one or more carbon atoms are optionally and independently controlled by Cy L Substitute;

[0444] Cy L Each is independently a tetravalent group selected from the following optionally substituted groups: C 3-20 Alicyclic ring, C 6-20 Aryl rings, 5-20 membered heteroaryl rings having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20 membered heterocyclic rings having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon;

[0445] Each ring A is independently a 3-20 member monocyclic, bicyclic, or polycyclic ring with 0-10 independently substituted heteroatoms selected from oxygen, nitrogen, sulfur, phosphorus, and silicon;

[0446] L P Each is an independent nucleotide bond;

[0447] z is 1-1000;

[0448] L 3E It is L or -LL-;

[0449] R 3E It is -R′, -LR′, -OR′ or a solid support;

[0450] R′ can be independently -R, -C(O)R, -C(O)OR, or -S(O)2R;

[0451] R is independently -H or a group selected from the following optional substitutions: C 1-30 Aliphatic, having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 1-30 Hybrid lipids, C 6-30 Aryl, C 6-30 Aryl aliphatic, having 1-10 C atoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 6-30 Aryl heteroaliphatic compounds, comprising 5-30 membered heteroaryl groups having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, and 3-30 membered heterocyclic groups having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon, or

[0452] The two R groups can be chosen to form a covalent bond together independently, or:

[0453] Two or more R groups on the same atom optionally and independently form, together with the atom, an optionally substituted 3-30 member monocyclic, bicyclic, or polycyclic ring, which, in addition to the atom, has 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; or

[0454] Two or more R groups on two or more atoms optionally and independently form, together with their intervening atoms, optionally substituted 3-30 member monocyclic, bicyclic or polycyclic rings, which, in addition to the intervening atoms, have 0-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon.

[0455] In some implementations, L P Each has its own independent structure, which is based on Formula I.

[0456]

[0457] Or its salt form, wherein:

[0458] P L It is P(=W), P or P→B(R′)3;

[0459] W is O, S, or Se;

[0460] R 1 It is -LR, halogen, -CN, -NO2, -Si(R)3, -OR, -SR or -N(R)2;

[0461] X, Y, and Z are each independently -O-, -S-, -N(-LR) 1 )- or L;

[0462] The variables are each independent as described in this disclosure.

[0463] In some implementations, L PEach has its own independent structure of Equation I, and R E It is -C(R) 5s )3、-LP DB -C(R) 5s )2OH、-LR 5s or -LP 5s -LR 5s Or its salt form, wherein each variable is independent as described in this disclosure.

[0464] In some implementations, RE is -C(R) 5s )3、-LP DB -C(R) 5s )2OH、-LR 5s or -LP 5s -LR 5s or its salt form;

[0465] Each BA is independently a group selected from the following optionally substituted groups: C having 1 to 10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus and silicon. 5-30 Heteroaryl group, and C having 1-10 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron and silicon. 3-30 Heterocyclic groups;

[0466] Each ring A is independently a 3-20 membered monocyclic, bicyclic, or polycyclic ring with 0-10 independently substituted heteroatoms selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; and

[0467] L P Each has its own independent structure as described in Formula I, wherein the variables are each independent as described in this disclosure.

[0468] In some implementation schemes, R E It is -C(R) 5s )3、-LP DB -C(R) 5s )2OH、-LR 5s or -LP 5s -LR 5s or its salt form;

[0469] Each BA is independently a C-type carbon atom with 1 to 10 independently selected heteroatoms from oxygen, nitrogen, sulfur, phosphorus, and silicon, with optional substitutions. 5-30 A heteroaryl group, wherein the heteroaryl group comprises one or more heteroatoms selected from oxygen and nitrogen;

[0470] Each ring A is independently a 5-10 member monocyclic or bicyclic saturated ring having 0-5 independently substituted heteroatoms selected from oxygen, nitrogen, sulfur, phosphorus, and silicon; wherein said ring contains at least one oxygen atom; and

[0471] L P Each has its own independent structure as described in Formula I, wherein the variables are each independent as described in this disclosure.

[0472] In some implementation schemes, R E It is -C(R) 5s )3、-LP DB -C(R) 5s )2OH、-LR 5s or -LP 5s -LR 5s or its salt form;

[0473] Each of BA is independently a nucleobase selected from adenine, cytosine, guanosine, thymine, and uracil, either substituted or protected.

[0474] Each ring A is independently a 5-7 membered monocyclic or bicyclic saturated ring with optional substitution of one or more oxygen atoms; and

[0475] L P Each has its own independent structure as described in Formula I, wherein the variables are each independent as described in this disclosure.

[0476] In some implementation schemes, R E It is the 5′-terminal group as described herein. In some embodiments, R E It is -C(R) 5s )3、-LP DB -C(R) 5s )2OH、-LR 5s or -LP 5s -LR 5s Or its salt form, wherein the variables are each independent as described in this disclosure. In some embodiments, R E It is -CH2OH. In some implementations, R E It is -CH2OP(O)(OR)2 or a salt thereof, wherein each R is independent as described in this disclosure. In some embodiments, R E It is -CH2OP(O)(OH)2 or its salt form. In some embodiments, R E It is -CH2OP(O)(OR)(SR) or a salt thereof, wherein each R is independent as described in this disclosure. In some embodiments, R E It is -CH2OP(O)(SH)(OH) or its salt form. In some embodiments, R E It is (E)-CH=CHP(O)(OR)2 or its salt form, wherein each R is independent as described in this disclosure. In some embodiments, R E It is (E)-CH=CHP(O)(OH)2.

[0477] In some embodiments, this disclosure provides oligonucleotide multimers. In some embodiments, the multimers are multimers of the same oligonucleotide. In some embodiments, the multimers are multimers of structurally different oligonucleotides. In some embodiments, each oligonucleotide of the multimer independently performs its function through its own pathway (e.g., RNAi, RNase-H dependent, etc.). In some embodiments, the provided oligonucleotides are in oligomeric or polymeric form, wherein one or more oligonucleotide moieties are connected via linkers (e.g., L, L...). M (etc.) are linked together by nucleobases, sugars, and / or internucleotide bonds of the oligonucleotide moiety. For example, in some embodiments, the provided polymeric compound has (A c ) a -L M -(A c ) b The structure, wherein the variables are each independent as described in this disclosure. Regarding exemplary multimer techniques, oligonucleotides can each function independently through different pathways (e.g., RNAi and / or RNase-H dependent).

[0478] In some embodiments, the provided compound, such as the oligonucleotide of the provided composition, has the following structure:

[0479] 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 ,

[0480] Or its salt, wherein:

[0481] A c Each is an independent oligonucleotide moiety (e.g., [H]). a -A c Or [H] b -A c (It is an oligonucleotide);

[0482] a is 1-1000;

[0483] b is 1-1000;

[0484] L M It is a multi-valent connector; and

[0485] R D Each can be a lipid component, a carbohydrate component, or a target component independently.

[0486] In some embodiments, the provided compound, such as the oligonucleotide of the provided composition, has the following structure:

[0487] 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 ,

[0488] Or its salt, wherein:

[0489] A c Each is an independent oligonucleotide moiety (e.g., [H]). a -A c Or [H] b -A c (It is an oligonucleotide);

[0490] a is 1-1000;

[0491] b is 1-1000;

[0492] R D Each is R independently LD R CD or R TD ;

[0493] R CD It is a straight-chain or branched group selected from the following optional substitutions: C 1-100 Aliphatic groups and C atoms having 1-30 heteroatoms independently selected from oxygen, nitrogen, sulfur, phosphorus, boron, and silicon. 1-100Heteroaliphatic groups, wherein one or more methylene units are optionally and independently replaced by: C 1-6 Alkylene, C 1-6 Alkenylene, -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)(S R′)-、-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-; and one or more carbon atoms are optionally and independently controlled by Cy L Substitute;

[0494] R LD It is selected from C 1-100 The optional substitution of an aliphatic group in a straight-chain or branched group, wherein one or more methylene units are optionally and independently substituted with: 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)(S R′)-、-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-; and one or more carbon atoms are optionally and independently controlled by Cy L Substitute;

[0495] R TD It is the target part;

[0496] L M Each is independently a covalent bond, or selected from the...

Claims

1. A composition comprising oligonucleotides that are single-stranded RNAi agents, wherein the single-stranded RNAi agents are complementary or substantially complementary to a target RNA sequence, have a length of about 15 to about 49 nucleotides, and are capable of directing target-specific RNA interference, wherein the single-stranded RNAi agents comprise at least one non-natural base, sugar, and / or internucleotide linkage.

2. The composition of claim 1, wherein the composition is a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type characterized by: a) a common base sequence and length; b) a common pattern of backbone linkages; c) a common pattern of backbone chiral centers; the composition is chirally controlled in that the composition is enriched in oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence and length; and wherein the oligonucleotides are complementary or substantially complementary to a target RNA sequence; have a length of about 15 to about 49 nucleotides; and are capable of directing target-specific RNA interference.

3. The composition of claim 1, wherein the composition is a composition comprising oligonucleotides that are single-stranded RNAi agents, wherein the single-stranded RNAi agents are complementary or substantially complementary to a target RNA sequence, have a length of about 15 to about 49 nucleotides, wherein the length includes a span of at least about 10 contiguous nucleotides having a 2'-modification pattern of ababababab, wherein a represents a first type of 2'-modification and b represents a second type of 2'-modification, wherein each first type can be the same or different and each second type can be the same or different and each first type is different from each second type, and wherein the single-stranded RNAi agents are capable of directing target-specific RNA interference.

4. The composition of any one of the preceding claims, wherein the oligonucleotides comprise at least one phosphorothioate in the Sp configuration.

5. The composition of any one of the preceding claims, wherein the oligonucleotides comprise at least one phosphorothioate in the Rp configuration.

6. The composition of any one of the preceding claims, wherein a first nucleotide comprising an optionally substituted pyrimidine nucleobase from the 5'-terminus of the oligonucleotide comprises a chiral internucleotide linkage connecting the first nucleotide to a next nucleotide.

7. The composition of any one of the preceding claims, wherein the pattern of backbone chiral centers includes SpSpSpSpSp, wherein each Sp independently represents a chiral internucleotide linkage comprising a Sp-linked phosphorus.

8. The composition of any one of the preceding claims, wherein the pattern of backbone chiral centers includes SpOSpO, wherein each Sp independently represents a chiral internucleotide linkage comprising a Sp-linked phosphorus and O represents a natural phosphodiester linkage.

9. A composition that is an oligonucleotide composition comprising a first plurality of oligonucleotides that share: 1) a common base sequence; 2) a common pattern of backbone linkages; 3) common stereochemistry at one or more chiral internucleotide linkages (stereocontrolled internucleotide linkages) independently; The composition is stereocontrolled in that the level of the first plurality of oligonucleotides in the composition is predetermined.

10. The composition of claim 1, wherein the composition is the composition of claim 9.

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