CN118265789A

CN118265789A - HSD17B 13-related double-stranded oligonucleotide compositions and related methods

Info

Publication number: CN118265789A
Application number: CN202280076577.3A
Authority: CN
Inventors: 钱德拉·瓦尔格赛; 岩本直树; 刘玮; 穆格达·贝德卡; 布雷特·施兰德; 普里扬卡·希瓦·波拉卡莎; 安东尼·拉马蒂纳; 卢西亚诺·H·阿波尼
Original assignee: Wave Life Sciences Pte Ltd
Current assignee: Wave Life Sciences Pte Ltd
Priority date: 2021-11-19
Filing date: 2022-11-18
Publication date: 2024-06-28

Abstract

The present disclosure provides double-stranded oligonucleotides, compositions related to HSD17B13 and methods of using such double-stranded oligonucleotides and compositions to prevent and/or treat a variety of conditions, disorders or diseases associated with HSD17B13 expression. In some embodiments, provided double-stranded oligonucleotides and compositions comprise nucleobase modifications, sugar modifications, internucleotide linkage modifications, and/or patterns thereof, and have improved properties, activity, and/or selectivity. In some embodiments, the provided double stranded oligonucleotides and compositions target HSD17B13.

Description

HSD17B 13-related double-stranded oligonucleotide compositions and related methods

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application number 63/264,360, U.S. provisional application number 63/268,775, and U.S. provisional application number 63/377,482, U.S. provisional application number 63/775, and U.S. provisional application number 28, 9, 2022, filed 11, 19, 2021, each of which is incorporated by reference in its entirety.

Technical Field

The present disclosure provides, among other things, double-stranded (ds) oligonucleotides, compositions, and methods (e.g., methods of making, methods of using, etc.) thereof. In some embodiments, the provided techniques may be used to prevent and/or treat a variety of conditions, disorders, or diseases associated with the expression of hydroxysteroid 17-beta dehydrogenase 13 (HSD 17B 13).

Background

Double stranded (ds) oligonucleotides are useful in a variety of applications, such as therapeutic, diagnostic, and/or research applications. For example, ds oligonucleotides that target HSD17B13 may be used to treat a condition, disorder, or disease associated with HSD17B13 expression, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic Steatohepatitis (ASH), alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis.

Disclosure of Invention

In some embodiments, the disclosure provides ds oligonucleotides and compositions thereof that target HSD17B13 with significantly improved properties and/or high activity. Among other things, the present disclosure provides techniques for designing, manufacturing, and utilizing such ds oligonucleotides and compositions. In particular, in some embodiments, the present disclosure provides ds oligonucleotides comprising useful patterns of internucleotide linkages and/or patterns of sugar modification, which when combined with one or more other structural elements, such as base sequences (or portions thereof), nucleobase modifications (and patterns thereof), additional chemical moieties, and the like, can provide HSD17B 13-targeting ds oligonucleotides and compositions thereof, including but not limited to, that are effective and sufficiently reducing expression, levels, and/or activity of HSD17B13 transcripts and products encoded thereby, with high activity and/or desirable properties. In some embodiments, ds oligonucleotides and compositions that target HSD17B13 reduce the level of HSD17B13 transcripts and are useful for treating and/or preventing HSD17B 13-associated conditions, disorders, or diseases, such as NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis.

In some embodiments, the ds oligonucleotide that targets HSD17B13 is capable of mediating knockdown of HSD17B13, wherein the level, expression, and/or activity of HSD17B13 or a product thereof is reduced. In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating a pan-specific knockdown of HSD17B13, wherein the level, expression, and/or activity of multiple or all of the HSD17B13 alleles is reduced. In some embodiments, the ds oligonucleotide targeting HSD17B13 has a base sequence complementary to a sequence common to multiple or all HSD17B13 alleles.

In certain embodiments, such structural elements include one or more of the following: (1) Chemical modifications (e.g., modification of sugar, base, and/or internucleotide linkages) and patterns thereof; and (2) stereochemistry (e.g., stereochemistry of backbone chiral internucleotide linkages) and changes in the pattern thereof. In certain embodiments, one or more such structural elements may be independently present in one or both of the ds oligonucleotides. In certain embodiments, the properties and/or activities affected by such structural elements include, but are not limited to, participation in, guidance of, or reduction in expression, activity, or level of a gene or gene product thereof, e.g., mediated by RNA interference (RNAi interference).

In certain embodiments, the disclosure demonstrates that compositions comprising ds oligonucleotides (e.g., dsRNAi oligonucleotides, also referred to as dsRNAi agents) with controlled structural elements provide unexpected properties and/or activity.

In certain embodiments, the disclosure includes the recognition that stereochemistry, e.g., stereochemistry of backbone chiral centers, can unexpectedly maintain or improve the properties of ds oligonucleotides. For example, but not limited to, the present disclosure relates in part to ds oligonucleotides comprising one or more of the following:

(1) A guide strand comprising a backbone phosphorothioate chiral center in the Sp configuration between the 3 'terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream, i.e., 5' oriented (N-2) nucleotide;

(2) A guide strand comprising a backbone phosphorothioate chiral center in Rp, sp or alternating configuration between the 5 'end (+1) nucleotide and immediately downstream, i.e., 3' direction (+2) nucleotide and between the +2 nucleotide and immediately downstream (+3) nucleotide;

(3) A guide strand comprising one or more backbone phosphorothioate chiral centers upstream, i.e., in the 5 'direction, of the backbone phosphorothioate chiral center in Sp configuration relative to the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide;

(4) A guide strand comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide and in one or both of the following: between nucleotide (a) +3 and nucleotide +4; and (b) +5 nucleotides and +6 nucleotides;

(5) A passenger chain coupled to one or more of the guide chains, the passenger chain comprising one or more backbone chiral centers in Rp or Sp configuration; and

(6) A passenger strand associated with one or more of the above-described guide strands, the passenger strand comprising a backbone phosphorothioate chiral center in the Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (i.e., 3' direction) (+2) nucleotide and between the 3' terminal nucleotide and the penultimate (N-1) nucleotide;

wherein the ds oligonucleotide further comprises one or more of the following:

(1) A guide strand, wherein one or both of the 5 'and 3' terminal dinucleotides are not joined by Rp, sp, or a sterically random non-negatively charged internucleotide linkage, i.e. the guide strand comprises one or more Rp, sp, or a sterically random non-negatively charged internucleotide linkage downstream (i.e. in the 3 'direction) with respect to the linkage between the 5' terminal dinucleotides and/or upstream (i.e. in the 5 'direction) with respect to the linkage between the 3' terminal dinucleotides;

(2) A guide strand, wherein one or more Rp, sp or stereotactically non-negatively charged internucleotide linkages occur between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

(3) A guide strand, wherein Rp, sp, or a stereotactic nonnegatively charged internucleotide linkage occurs between the third (+3) and fourth (+4) nucleotides relative to the 5 'terminal nucleotide and/or between the tenth (+10) and eleventh (+11) nucleotides relative to the 5' terminal nucleotide of the guide strand;

(4) A passenger strand, wherein one or more Rp, sp or stereotactically nonnegatively charged internucleotide linkages occur upstream, i.e. 5' with respect to the central nucleotide of the passenger strand; and

(5) A passenger strand in which one or more Rp, sp or stereotactic nonnegatively charged internucleotide linkages occur downstream, i.e.in the 3' direction relative to the central nucleotide of the passenger strand, and

Wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactically nonnegatively charged internucleotide linkage. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the disclosure includes recognition that stereochemistry, e.g., stereochemistry at the chiral center at the 5' -end modification of the guide strand, can unexpectedly maintain or improve the properties of the ds oligonucleotides described herein. For example, but not limited to, the present disclosure relates in part to a ds oligonucleotide comprising a guide strand comprising: (1) a phosphorothioate chiral centre in Rp or Sp configuration; (2) Rp, sp or a stereotactically non-negatively charged internucleotide linkage, wherein the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactically non-negatively charged internucleotide linkage comprises a 2' modification, such as 2' f; and (3) a 5' terminal modification selected from the group consisting of:

(a) 5' PO modifications such as, but not limited to:

(b) 5' VP modifications such as, but not limited to:

(c) 5' MeP modifications such as, but not limited to:

(d) 5'PN and 5' Trizole-P modifications, such as but not limited to:

wherein the base is selected from A, C, G, T, U, abasic, and modified nucleobases;

R ^2' is selected from H, OH, O-alkyl, F, MOE, locked Nucleic Acid (LNA) bridge, and Bridging Nucleic Acid (BNA) bridge to 4' C, such as, but not limited to:

In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide chain are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain other embodiments, the disclosure includes recognition that stereochemistry, e.g., stereochemistry at the chiral center at the 5' end nucleotide of the guide strand, can unexpectedly maintain or improve the properties of a ds oligonucleotide, wherein the guide strand of the ds oligonucleotide further comprises a phosphorothioate chiral center of Rp or Sp configuration. For example, but not limited to, the present disclosure relates in part to a ds oligonucleotide comprising a guide strand comprising: (1) a phosphorothioate chiral centre in Rp or Sp configuration; (2) Rp, sp or a stereotactically non-negatively charged internucleotide linkage, wherein the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactically non-negatively charged internucleotide linkage comprises a 2' modification, such as 2' f; and (3) a 5' terminal modification selected from the group consisting of:

(a) 5' PO nucleotides such as, but not limited to:

(b) 5' VP nucleotides, such as, but not limited to:

(c) 5' MeP nucleotides such as, but not limited to:

(d) 5'PN and 5' Trizole-P nucleotides, such as but not limited to:

(e) 5 'abasic VP and 5' abasic MeP nucleotides, such as, but not limited to:

In certain embodiments, the disclosure includes recognition of non-naturally occurring internucleotide linkages, e.g., neutral internucleotide linkages, useful in certain embodiments for ligating one or more molecules to double-stranded oligonucleotides described herein. In certain embodiments, such linked molecules may facilitate targeting and/or delivery of double-stranded oligonucleotides. Such linked molecules include, for example and without limitation, lipophilic molecules. In certain embodiments, the linked molecule is a molecule comprising one or more GalNAc moieties. In certain embodiments, the linked molecule is a receptor. In certain embodiments, the linked molecule is a receptor ligand.

In certain embodiments, the present disclosure provides techniques for incorporating a variety of additional chemical moieties into ds oligonucleotides. In certain embodiments, the present disclosure provides reagents and methods for introducing additional chemical moieties, e.g., via nucleobases (e.g., optionally introducing additional chemical moieties to sites on nucleobases via covalent bonding via linkers).

In certain embodiments, the disclosure provides techniques, e.g., ds oligonucleotide compositions and methods thereof, for achieving allele-specific suppression, wherein transcripts from one allele of a particular target gene are selectively knocked down relative to at least another allele of the same gene.

The present disclosure provides, among other things, structural elements, techniques, and/or features that can incorporate ds oligonucleotides and can confer or adjust one or more properties thereof (e.g., relative to otherwise identical ds oligonucleotides lacking the relevant techniques or features). In certain embodiments, the present disclosure demonstrates that one or more of the provided techniques and/or features can be usefully incorporated into ds oligonucleotides of various sequences.

In certain embodiments, the disclosure demonstrates that certain provided structural elements, techniques, and/or features are particularly useful for ds oligonucleotides (e.g., RNAi agents) that participate in and/or direct RNAi machinery. However, in no way is the teachings of the present disclosure limited to ds oligonucleotides that participate in or act via any particular biochemical mechanism. In certain embodiments, the disclosure relates to any ds oligonucleotide, useful for any purpose, which functions by any mechanism, and which comprises any sequence, structure, or form (or portion thereof) described herein. In certain embodiments, the present disclosure provides ds oligonucleotides that can be used for any purpose, that function by any mechanism, and that comprise any sequence, structure, or form (or portion thereof) described herein, including one or more of the following:

(3) A guide strand comprising one or more backbone phosphorothioate chiral centers at a position relative to the 3' -terminal nucleotide and penultimate

Upstream of the chiral center of the backbone phosphorothioate in the Sp configuration, i.e., in the 5' direction, between the (N-1) nucleotides and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, wherein the upstream backbone phosphorothioate chiral center is in the Rp or Sp configuration;

6) A passenger strand associated with one or more of the above-described guide strands, the passenger strand comprising a backbone phosphorothioate chiral center in the Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (i.e., 3' direction) (+2) nucleotide and between the 3' terminal nucleotide and the penultimate (N-1) nucleotide;

wherein the ds oligonucleotide further comprises one or more of the following:

(1) A guide strand, wherein one or both of the 5 'and 3' terminal dinucleotides are not joined by an inter-nucleotide linkage that is not negatively charged, i.e., the guide strand comprises one or more Rp, sp, or stereotactically non-negatively charged inter-nucleotide linkages downstream (i.e., in the 3 'direction) relative to the linkage between the 5' terminal dinucleotides and/or upstream (i.e., in the 5 'direction) relative to the linkage between the 3' terminal dinucleotides;

In certain embodiments, the provided ds oligonucleotides can participate in (e.g., direct) an RNAi machinery.

In certain embodiments, the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

(1) A guide strand, wherein one or both of the 5 'and 3' terminal dinucleotides are not joined by a non-negatively charged internucleotide linkage, i.e. the guide strand comprises one or more non-negatively charged internucleotide linkages downstream (i.e. in the 3 'direction) with respect to the linkage between the 5' terminal dinucleotides and/or upstream (i.e. in the 5 'direction) with respect to the linkage between the 3' terminal dinucleotides;

Wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' F modification, of a 3' nucleotide of a nucleotide pair linked by Rp, sp or a stereorandom non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, sp or a stereorandom non-negatively charged internucleotide linkage, wherein n is about 1 to 49,

In certain embodiments, the guide strand comprises a backbone phosphorothioate chiral center of Rp, sp, or alternating configuration between the 5' terminus (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of the following:

Wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' f modification, of a 3' nucleotide of a nucleotide pair linked by Rp, sp or a stereorandom non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, sp or stereorandom non-negatively charged internucleotide linkages, wherein n is about 1 to 49. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

In certain embodiments, the guide strand comprises one or more Rp, sp or stereotactic non-negatively charged internucleotide linkages and internucleotide linkages to the penultimate 3 '(N-1) nucleotide between the second (+2) and third (+3) nucleotides of the guide strand relative to the 5' terminal nucleotide, and one or more of the following:

Wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' f modification, of a 3' nucleotide of a nucleotide pair linked by Rp, sp or a sterically random non-negatively charged internucleotide linkage, and the passenger strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

Wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' f modification, of a 3' nucleotide of a nucleotide pair linked by Rp, sp or a stereotactic nonnegatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers of Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminus (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide and in one or both of: (a) between +3 and +4 nucleotides; and (b) (+5) and (+6) nucleotides, and one or more of:

In certain embodiments, the guide strand comprises one or more Rp, sp, or stereotactically non-negatively charged internucleotide linkages between the second (+2) nucleotide of the guide strand relative to the 5 'terminal nucleotide and any two adjacent nucleotides between the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3 'terminal nucleotide, the guide strand comprising a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp, or stereotactically non-negatively charged internucleotide linkages, and the passenger strand comprising one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide chain are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3 'terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, the guide strand comprises a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactic non-negatively charged internucleotide linkage, and the passenger strand comprises 0-N Rp, sp or a stereotactic non-negatively charged internucleotide linkage (where N is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide chain are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the guide strand comprises a backbone phosphorothioate chiral center in Rp, sp, or alternating configuration between the 5 'terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, the guide strand comprises a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp, or a stereotactic non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, sp, or a stereotactic non-negatively charged internucleotide linkage (where n is about 1 to 49) and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide chain are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3 'terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, the guide strand comprises a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactic non-negatively charged internucleotide linkage, and the passenger strand comprises 0-N Rp, sp or a stereotactic non-negatively charged internucleotide linkage (where N is about 1 to 49) and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide chain are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the guide strand comprises one or more Rp, sp or stereotactically non-negatively charged internucleotide linkages between the second (+2) nucleotide of the guide strand relative to the 5 'terminal nucleotide and any two adjacent nucleotides between the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is a 3 'terminal nucleotide, the guide strand comprises a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp or stereotactically non-negatively charged internucleotide linkages, and the passenger strand comprises 0-N Rp, sp or stereotactically non-negatively charged internucleotide linkages (wherein N is about 1 to 49) and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide chain are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the provided ds oligonucleotides may be involved in an exon skipping mechanism. In certain embodiments, the ds oligonucleotides provided may be aptamers. In certain embodiments, the provided ds oligonucleotides can bind to and inhibit the function of a protein, small molecule, nucleic acid, or cell. In certain embodiments, the provided ds oligonucleotides can participate in forming a triple helix with double stranded nucleic acids in a cell. In certain embodiments, provided ds oligonucleotides can bind genomic (e.g., chromosomal) nucleic acids. In certain embodiments, provided ds oligonucleotides can bind to genomic (e.g., chromosomal) nucleic acids, thereby preventing or reducing expression of the nucleic acids (e.g., by preventing or reducing transcription, transcription enhancement, modification, etc.). In certain embodiments, the provided ds oligonucleotides can bind DNA quadruplexes. In certain embodiments, the provided ds oligonucleotides can be immunomodulatory. In certain embodiments, the provided ds oligonucleotides can be immunostimulatory. In certain embodiments, the provided oligonucleotides may be immunostimulatory and may comprise CpG sequences. In certain embodiments, the provided ds oligonucleotides may be immunostimulatory and may contain CpG sequences and may be used as adjuvants. In certain embodiments, the provided ds oligonucleotides can be immunostimulatory and can contain CpG sequences and can be used as adjuvants for treating diseases (e.g., infectious diseases or cancers). In certain embodiments, the provided ds oligonucleotides can be therapeutic. In certain embodiments, the ds oligonucleotides provided may be non-therapeutic. In certain embodiments, the ds oligonucleotides provided may be therapeutic or non-therapeutic. In certain embodiments, the provided ds oligonucleotides can be used in therapeutic, diagnostic, research, and/or nanomaterial applications. In certain embodiments, the provided ds oligonucleotides can be used for experimental purposes. In certain embodiments, the provided ds oligonucleotides can be used for experimental purposes, e.g., as probes in microarrays, etc. In certain embodiments, the provided ds oligonucleotides can participate in more than one biological mechanism; in some such embodiments, for example, the provided ds oligonucleotides may be involved in RNAi and rnase H mechanisms.

In certain embodiments, the provided ds oligonucleotides are directed against HSD17B13 target (e.g., HSD17B13 target sequence, HSD17B13 target RNA, HSD17B13 target mRNA, HSD17B13 target pre-mRNA, HSD17B13 target gene, etc.). The HSD17B13 target gene is a gene that is intended to alter the expression and/or activity of one or more HSD17B13 gene products (e.g., HSD17B13 RNA and/or protein products) associated therewith. In certain embodiments, inhibition of the HSD17B13 target gene is intended. Thus, when a ds oligonucleotide as described herein acts on an HSD17B13 target gene, the presence and/or activity of one or more HSD17B13 gene products is altered when the ds oligonucleotide is present as compared to when the ds oligonucleotide is not present.

In certain embodiments, the HSD17B13 target is a particular HSD17B13 allele that is intended to alter the expression and/or activity of one or more products (e.g., HSD17B13 RNA and/or protein products) associated therewith. In certain embodiments, an HSD17B13 target allele is one whose presence and/or expression correlates (e.g., is associated with) the presence, incidence, and/or severity of one or more HSD17B 13-related diseases and/or disorders. Alternatively or additionally, in certain embodiments, the HSD17B13 target allele is an allele whose level and/or activity of one or more HSD17B13 gene products alters an improvement (e.g., onset delay, severity reduction, response to other therapies, etc.) in one or more aspects of the disease and/or disorder associated with HSD17B 13.

In certain embodiments, for example, when the presence and/or activity of a particular HSD17B13 allele (HSD 17B13 disease-associated allele) is correlated (e.g., associated) with the presence, incidence, and/or severity of one or more disorders, diseases, and/or conditions, different HSD17B13 alleles are present but not so correlated, or are less correlated (e.g., exhibit less significant or statistically insignificant correlation), a ds oligonucleotide and methods thereof as described herein may preferentially or specifically target the associated allele relative to the one or more less/unrelated alleles, thereby mediating allele-specific suppression.

In certain embodiments, the HSD17B13 target sequence is an HSD17B13 sequence to which the oligonucleotides described herein bind. In certain embodiments, the HSD17B13 target sequence is identical to or is a precise complement of the HSD17B13 sequence of the provided oligonucleotide or consecutive residues thereof (e.g., the provided oligonucleotide comprises a HSD17B13 target binding sequence that is identical to or is a precise complement of the HSD17B13 target sequence). In certain embodiments, the HSD17B13 target binding sequence is a precise complement of the HSD17B13 target sequence of the HSD17B13 transcript (e.g., pre-mRNA, etc.). The HSD17B13 target binding sequence/target sequence may be of various lengths to provide oligonucleotides having desired activity and/or properties. In certain embodiments, the HSD17B13 target binding sequence/target sequence comprises 5-50 (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) bases. In certain embodiments, small differences/mismatches between (relevant portions of) the oligonucleotide and its target sequence are tolerated, including but not limited to the HSD17B13 target sequence and/or the 5 'and/or 3' terminal regions of the oligonucleotide sequence. In certain embodiments, the HSD17B13 target sequence is present within the HSD17B13 target gene. In certain embodiments, the HSD17B13 target sequence is present in HSD17B13 transcripts (e.g., mRNA and/or pre-mRNA) produced from the HSD17B13 target gene.

In certain embodiments, the HSD17B13 target sequence includes one or more allelic loci (i.e., the locations within the HSD17B13 target gene where allelic variation occurs). In certain embodiments, the allele locus is a mutation. In certain embodiments, the allele locus is a SNP. In some such embodiments, the provided oligonucleotides preferentially or specifically bind to one allele relative to one or more other alleles. In certain embodiments, the provided oligonucleotides preferentially bind to disease-associated alleles. For example, in certain embodiments, the oligonucleotides (or target binding sequence portions thereof) provided herein have a sequence that is identical, or at least partially identical, or is a precise complement of a particular allelic version of the HSD17B13 target sequence.

In certain embodiments, the oligonucleotides (or target binding sequence portions thereof) provided herein have a sequence that is identical to or is exactly complementary to an HSD17B13 target sequence comprising an allele site or an allele site of a disease-associated allele. In certain embodiments, the oligonucleotides provided herein have HSD17B13 target binding sequences that are precisely complementary sequences of HSD17B13 target sequences comprising the allele site of the HSD17B13 transcript of an allele (in certain embodiments, a disease-related allele), wherein the allele site is a mutation. In certain embodiments, the oligonucleotides provided herein have HSD17B13 target binding sequences that are precisely complementary sequences of HSD17B13 target sequences comprising the allele site of an HSD17B13 transcript of an allele (in certain embodiments, a disease-related allele), wherein the allele site is a SNP. In certain embodiments, the sequence is any of the sequences disclosed herein.

Unless otherwise indicated, all sequences (including but not limited to base sequences and chemical, modification and/or stereochemical modes) are presented in 5 'to 3' order, with the 5 'terminal nucleotide identified as the "+1" position and the 3' terminal nucleotide identified by the number of nucleotides or "N" of the complete sequence, with the penultimate nucleotide identified as "N-1", and so on.

In certain embodiments, the disclosure provides compositions and methods related to oligonucleotides specific for HSD17B13 targets and having any form, structural element, or base sequence of any of the oligonucleotides disclosed herein.

In certain embodiments, the disclosure provides compositions and methods related to oligonucleotides that are specific for HSD17B13 targets and that have or comprise a base sequence of any of the oligonucleotides disclosed herein, or a region of at least 15 consecutive nucleotides of a base sequence of any of the oligonucleotides disclosed herein, wherein the first nucleotide of the base sequence or the first nucleotide of the at least 15 consecutive nucleotides can optionally be replaced with T or DNA T.

In certain embodiments, the present disclosure provides compositions and methods for RNA interference directed by RNAi agents (also referred to as RNAi oligonucleotides). In certain embodiments, the oligonucleotides of such compositions may have the form, structural elements, or base sequences of the oligonucleotides disclosed herein.

In certain embodiments, the disclosure provides compositions and methods for rnase H mediated RNA knockdown of HSD17B13 target genes guided by oligonucleotides (e.g., antisense oligonucleotides).

The provided oligonucleotides and oligonucleotide compositions may have any form, structural element, or base sequence of any of the oligonucleotides disclosed herein. In certain 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 dinucleotide, a 3' terminal cap, or any portion of any of these structures, GC content, long GC segments, and/or any modification, chemistry, stereochemistry, modification pattern, chemistry or stereochemistry, or chemical moiety (e.g., including, but not limited to, a targeting moiety, lipid moiety, galNAc moiety, carbohydrate moiety, etc.), any component, or any combination of any of the above.

In certain embodiments, the disclosure provides compositions and methods of use of oligonucleotides.

In certain embodiments, the disclosure provides compositions and methods of use of oligonucleotides that direct RNA interference and rnase H mediated knockdown of HSD17B13 target gene RNA. In certain embodiments, the oligonucleotides of such compositions may have the form, structural elements, or base sequences of the oligonucleotides disclosed herein.

In certain embodiments, an oligonucleotide that directs a particular event or activity is involved in a particular event or activity, such as a decrease in expression, level, or activity of a target gene or gene product thereof. In certain embodiments, an oligonucleotide is said to "direct" a particular event or activity when its presence in a system in which the event or activity can occur correlates with an increased detectable occurrence, frequency, intensity, and/or level of the event or activity.

In certain embodiments, provided oligonucleotides comprise any one or more structural elements of an oligonucleotide as described herein, e.g., a base sequence (or a portion thereof having at least 15 consecutive bases); an internucleotide linkage pattern (or a portion thereof having at least 5 consecutive internucleotide linkages); stereochemical mode of internucleotide linkages (or a portion thereof having at least 5 consecutive internucleotide linkages); a 5' terminal structure; a 5' terminal region; a first region; a second region; and a 3' end region (which may be a 3' terminal dinucleotide and/or a 3' end cap); and optionally an additional chemical moiety; also, in certain embodiments, at least one structural element comprises a chiral center of controlled chirality. In certain embodiments, the 3' terminal dinucleotide may comprise a total of two nucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from a targeting moiety, a carbohydrate moiety, a GalNAc moiety, a lipid moiety, and any other chemical moiety described herein or known in the art, as non-limiting examples. In certain embodiments, the moiety that binds APGR is a moiety of GalNAc, or a variant, derivative, or modified form thereof, as described herein and/or known in the art. In certain embodiments, the oligonucleotide is an RNAi agent. In some embodiments, the first region is a seed region. In some embodiments, the second region is a post-seed region.

In certain embodiments, provided oligonucleotides comprise any one or more structural elements of an RNAi agent as described herein, e.g., a 5' terminal structure; a 5' terminal region; a seed region; a post-seed region (a region between the seed region and the 3' -terminal region); and a 3' end region (which may be a 3' terminal dinucleotide and/or a 3' end cap); and optionally an additional chemical moiety; also, in certain embodiments, at least one structural element comprises a chiral center of controlled chirality. In certain embodiments, the 3' terminal dinucleotide may comprise a total of two nucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from the group consisting of a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety, as non-limiting examples. In certain embodiments, the moiety that binds APGR is any GalNAc, or variant, derivative, or modification thereof, as described herein or known in the art.

In certain embodiments, provided oligonucleotides comprise any one or more structural elements of an oligonucleotide as described herein, e.g., a 5' terminal structure, a 5' terminal region, a first region, a second region, a 3' terminal region, and optionally an additional chemical moiety, wherein at least one structural element comprises a chiral center that is chiral controlled. In certain embodiments, the oligonucleotide comprises a sequence segment (span) of at least 5 nucleotides in total without 2' -modification. In certain embodiments, the oligonucleotide further comprises an additional chemical moiety selected from the group consisting of a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety, as non-limiting examples. In certain embodiments, the provided oligonucleotides are capable of directing RNA interference. In certain embodiments, the provided oligonucleotides are capable of directing rnase H mediated knockdown. In certain embodiments, the provided oligonucleotides are capable of 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.

In certain embodiments, provided oligonucleotides comprise any one or more structural elements of an RNAi agent, e.g., 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 of controlled chirality; also, in certain embodiments, the oligonucleotides are also capable of directing rnase H mediated knockdown of target gene RNAs. In certain embodiments, the oligonucleotide comprises a sequence segment of at least 5 total 2' -deoxynucleotides. In certain embodiments, the oligonucleotide further comprises a chemical moiety selected from the group consisting of a targeting moiety, a carbohydrate moiety, a GalNAc moiety, and a lipid moiety, and any other additional chemical moiety described herein, as non-limiting examples.

In certain embodiments, the disclosure demonstrates that oligonucleotide properties can be modulated by chemical modification. In certain embodiments, the present disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, sugar, and/or base modifications. In certain embodiments, the present disclosure provides an oligonucleotide composition capable of directing RNA interference and comprising a first plurality of oligonucleotides having a common base sequence and comprising one or more internucleotide linkages, and/or one or more sugars, and/or one or more base modifications. In certain embodiments, the oligonucleotide or oligonucleotide composition is further capable of directing rnase H mediated knockdown of HSD17B13 target gene RNA. In certain embodiments, the disclosure demonstrates that oligonucleotide properties, e.g., activity, toxicity, etc., can be modulated by chemical modification of sugar, nucleobase, and/or internucleotide linkages. In certain embodiments, the present disclosure provides oligonucleotide compositions comprising a plurality of oligonucleotides having a common base sequence and comprising one or more modified internucleotide linkages (or "unnatural internucleotide linkages" found in natural DNA and RNA, which linkages may be used in place of the natural phosphate internucleotide linkages (-OP (O) (OH) O), possibly in the form of salts (-OP (O) (O ^-) O-) at the physiological pH found in natural DNA and RNA), One or more modified sugar moieties and/or one or more natural phosphate linkages. In certain embodiments, provided oligonucleotides may comprise two or more types of modified internucleotide linkages. In certain embodiments, provided oligonucleotides comprise non-negatively charged internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages are neutral internucleotide linkages. In certain embodiments, the neutral internucleotide linkage comprises a cyclic guanidine moiety. Such moieties are optionally substituted. In certain embodiments, provided oligonucleotides comprise a neutral internucleotide linkage and another internucleotide linkage that is not a neutral backbone. In certain embodiments, provided oligonucleotides comprise neutral internucleotide linkages and phosphorothioate internucleotide linkages. In certain embodiments, provided oligonucleotide compositions comprising a plurality of oligonucleotides are chirally controlled and the level of the plurality of oligonucleotides in the composition is controlled or predetermined and the plurality of oligonucleotides share a common stereochemical configuration at one or more chiral internucleotide linkages. For example, in certain embodiments, a plurality of oligonucleotides share a common stereochemical configuration at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more chiral internucleotide linkages (each independently being Rp or Sp); In certain embodiments, multiple oligonucleotides share a common stereochemical configuration at each chiral internucleotide linkage. In certain embodiments, chiral internucleotide linkages wherein a controlled level of the oligonucleotides of the composition share a common stereochemical configuration (independently Rp or Sp configuration) are referred to as chiral controlled internucleotide linkages. In certain embodiments, the modified internucleotide linkages are nonnegatively charged (neutral or cationic) internucleotide linkages, as they are predominantly (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, etc.) at pH (e.g., human physiological pH (about 7.4), the pH of the delivery site (e.g., organelle, cell, tissue, organ, organism, etc.), etc.; in certain embodiments, at least 30%; in certain embodiments, at least 40%; in certain embodiments, at least 50%; in certain embodiments, at least 60%; in certain embodiments, at least 70%; in certain embodiments, at least 80%; in certain embodiments, at least 90%; in certain embodiments, at least 99%; etc.; ) In neutral form or in cationic form (as opposed to anionic form, for example, -O-P (O) (O ^-) -O- (natural phosphate linked anionic form), -O-P (O) (S ^-) -O- (phosphorothioate linked anionic form), and the like. in certain embodiments, the modified internucleotide linkage is a neutral internucleotide linkage, since it exists predominantly in neutral form at pH. In certain embodiments, the modified internucleotide linkages are cationic internucleotide linkages, as they exist predominantly in the cationic form at pH. In certain embodiments, the pH is a physiological pH of a human (about 7.4). In certain embodiments, the modified internucleotide linkages are neutral internucleotide linkages, as at least 90% of the internucleotide linkages are present in their neutral form in aqueous solution at pH 7.4. In certain embodiments, the modified internucleotide linkages are neutral internucleotide linkages, in that at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the internucleotide linkages are present in their neutral form in aqueous solutions of oligonucleotides. In certain embodiments, the percentage is at least 90%. In certain embodiments, the percentage is at least 95%. In certain embodiments, the percentage is at least 99%. In certain embodiments, the non-negatively charged internucleotide linkages, e.g., neutral internucleotide linkages, do not have moieties having a pKa of less than 8, 9, 10, 11, 12, 13, or 14 when in its neutral form. in certain embodiments, the pKa of the internucleotide linkages in the present disclosure may be represented by the pKa of CH ₃ -internucleotide linkages-CH ₃ (i.e., replacing two nucleoside units connected by internucleotide linkages with two-CH ₃ groups). Without wishing to be bound by any particular theory, in at least some instances, neutral internucleotide linkages in an oligonucleotide may provide improved properties and/or activity, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and/or improved nuclear uptake, etc., as compared to an equivalent nucleic acid that does not comprise neutral internucleotide linkages.

In certain embodiments, the non-negatively charged internucleotide linkages have, for example, the structure ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612, etc., of the formula I-n-1, I-n-2, I-n-3, 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., as described below. In certain embodiments, the non-negatively charged internucleotide linkages comprise a cyclic guanidine moiety. In certain embodiments, the modified internucleotide linkage comprises a cyclic guanidine moiety having the structure: In certain embodiments, the neutral internucleotide linkages comprising a cyclic guanidine moiety are chirally controlled. In certain embodiments, the disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage.

In certain embodiments, the disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate internucleotide linkage is a chiral controlled internucleotide linkage of the Sp configuration.

In certain embodiments, the disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage and at least one phosphorothioate internucleotide linkage, wherein the phosphorothioate is a chirally controlled internucleotide linkage of the Rp configuration.

In certain embodiments, the disclosure relates to compositions comprising an oligonucleotide comprising at least one oligonucleotide comprising a Tmg groupNeutral internucleotide linkages of the neutral internucleotide linkages of (c) and at least one phosphorothioate.

In certain embodiments, each internucleotide linkage in an oligonucleotide is independently selected from natural phosphate linkages, phosphorothioate linkages, and nonnegatively charged internucleotide linkages (e.g., n001, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n 055). In some embodiments, each internucleotide linkage in the oligonucleotide is independently selected from a natural phosphate linkage, a phosphorothioate linkage, and a neutral internucleotide linkage (e.g., n001, n003, n004, n006, n008, n009, n013, n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n 055).

In certain embodiments, the disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage comprising a Tmg group and at least one phosphorothioate, wherein the phosphorothioate is a chiral controlled internucleotide linkage of the Sp configuration.

In certain embodiments, the disclosure relates to compositions comprising an oligonucleotide comprising at least one neutral internucleotide linkage selected from neutral internucleotide linkages comprising a Tmg group, and at least one phosphorothioate, wherein the phosphorothioate is a chirally controlled internucleotide linkage of the Rp configuration.

The various types of internucleotide linkages differ in nature. Without wishing to be bound by any theory, the present disclosure indicates that the natural phosphate linkages (phosphodiester internucleotide linkages) are anionic and may be unstable when used on their own in vivo without other chemical modifications; phosphorothioate internucleotide linkages are cationic, generally more stable in vivo than natural phosphate linkages, and generally more hydrophobic; neutral internucleotide linkages (neutral internucleotide linkages comprising a cyclic guanidine moiety as exemplified in the present disclosure) are neutral at physiological pH, can be more stable in vivo than natural phosphate linkages, and are more hydrophobic.

In certain embodiments, the chirally controlled neutral internucleotide linkages are neutral at physiological pH, chirally controlled, stable in vivo, hydrophobic, and may increase endosomal escape.

In certain embodiments, provided oligonucleotides comprise one or more regions, e.g., a segment, a wing, a core, a 5 'end, a 3' end, an intermediate, a seed, a post-seed region, and the like. In certain embodiments, the region (e.g., segment, wing, core, 5 'end, 3' end, middle region, etc.) comprises non-negatively charged internucleotide linkages ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784 and/or WO 2019/03612, e.g., of the formula I-n-1, I-n-2, I-n-3, 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., as described below. In certain embodiments, the region comprises neutral internucleotide linkages. In certain embodiments, the region comprises an internucleotide linkage comprising a cyclic guanidine. In certain embodiments, the region comprises an internucleotide linkage comprising a cyclic guanidine moiety. In some embodiments, the region comprises a structureIs a nucleotide linkage of (a). In certain embodiments, such internucleotide linkages are chirally controlled.

In certain embodiments, the nucleotide is a natural nucleotide. In certain embodiments, the nucleotide is a modified nucleotide. In certain embodiments, the nucleotide is a nucleotide analog. In certain embodiments, the base is a modified base. In certain embodiments, the base is a protected nucleobase, such as that used in oligonucleotide synthesis. In certain embodiments, the base is a base analog. In certain embodiments, the sugar is a modified sugar. In certain embodiments, the sugar is a sugar analog. In certain embodiments, the internucleotide linkage is a modified internucleotide linkage. In certain embodiments, the nucleotides comprise a base, a sugar, and an internucleotide linkage, wherein each of the base, sugar, and internucleotide linkage is independently and optionally naturally occurring or non-naturally occurring. In certain embodiments, the nucleoside comprises a base and a sugar, wherein each of the base and the sugar is independently and optionally naturally occurring or non-naturally occurring. Non-limiting examples of nucleotides include DNA (2 '-deoxy) and RNA (2' -OH) nucleotides; and those comprising one or more modifications at the base, sugar and/or internucleotide linkages. Non-limiting examples of sugars include ribose and deoxyribose; and ribose and deoxyribose having 2 '-modifications, including but not limited to 2' -F, LNA, 2'-OMe and 2' -MOE modifications. In certain embodiments, the internucleotide linkage is a moiety that does not contain phosphorus but is used to link two natural or unnatural sugars.

In certain embodiments, the composition comprises a multimer of two or more of any of the following: a first plurality of oligonucleotides and/or a second plurality of oligonucleotides, wherein the first and second plurality of oligonucleotides can independently direct knockdown of the same or different targets by RNA interference and/or rnase H mediated knockdown.

In certain embodiments, the disclosure provides an oligonucleotide composition comprising a first plurality of oligonucleotides that share:

1) A common base sequence;

2) A common backbone linkage pattern;

3) Common stereochemistry, independently 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 internucleotide linkages ("chiral controlled internucleotide linkages"); the composition is chirally controlled in that the level of the first plurality of oligonucleotides in the composition is predetermined.

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

In certain embodiments, a predetermined level of oligonucleotides of a provided composition, e.g., a first plurality of oligonucleotides of certain exemplary compositions, comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more chirally controlled internucleotide linkages.

In certain embodiments, at least 5 internucleotide linkages are chirally controlled; in certain embodiments, at least 10 internucleotide linkages are chirally controlled; in certain embodiments, at least 15 internucleotide linkages are chirally controlled; in certain embodiments, each chiral internucleotide linkage is chirally controlled.

In certain embodiments, 1% -100% of the chiral internucleotide linkages are chirally controlled. In certain 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 internucleotide linkages are chirally controlled.

1) A common base sequence;

2) A common backbone linkage pattern; and

3) The composition is a substantially pure preparation of oligonucleotides because the predetermined level of oligonucleotides in the composition have a common base sequence and length, a common backbone linkage pattern, and a common backbone chiral center pattern. In certain embodiments, the common backbone chiral center pattern comprises at least 1 internucleotide linkage comprising a chiral center of controlled chirality. In certain 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 certain 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 belonging to or comprising a common base sequence in the provided composition. In certain embodiments, all oligonucleotides belonging to or comprising a common base sequence in a 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 certain 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 belonging to or comprising a common base sequence, base modification, sugar modification, and/or modified internucleotide linkage in the provided composition. In certain embodiments, all oligonucleotides belonging to or comprising a common base sequence, base modification, sugar modification, and/or modified internucleotide linkage in a 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 certain 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 belonging to or comprising a common base sequence, base modification pattern, sugar modification pattern, and/or modified internucleotide linkage pattern in the provided composition. In certain embodiments, all oligonucleotides belonging to or comprising a common base sequence, base modification pattern, sugar modification pattern, and/or modified internucleotide linkage pattern in a 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 certain 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 linkage pattern. In certain embodiments, all oligonucleotides sharing a common base sequence, common base modification pattern, common sugar modification pattern, and/or common modified internucleotide linkage pattern in a 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 certain embodiments, the predetermined level is 1% -100%. In certain embodiments, the predetermined level is at least 1%. In certain embodiments, the predetermined level is at least 5%. In certain embodiments, the predetermined level is at least 10%. In certain embodiments, the predetermined level is at least 20%. In certain embodiments, the predetermined level is at least 30%. In certain embodiments, the predetermined level is at least 40%. In certain embodiments, the predetermined level is at least 50%. In certain embodiments, the predetermined level is at least 60%. In certain embodiments, the predetermined level is at least 10%. In certain embodiments, the predetermined level is at least 70%. In certain embodiments, the predetermined level is at least 80%. In certain embodiments, the predetermined level is at least 90%. In certain embodiments, the predetermined level is at least 5 (1/2 g), where g is the number of chiral controlled internucleotide linkages. in certain embodiments, the predetermined level is at least t0 (1/2 g), where g is the number of chirally controlled internucleotide linkages. In certain embodiments, the predetermined level is at least 100 (1/2 g), where g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.80) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.80) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.80) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.85) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.90) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0-95) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.96) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.97) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.98) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, the predetermined level is at least (0.99) g, wherein g is the number of chiral controlled internucleotide linkages. In certain embodiments, to determine the level of oligonucleotides having g chiral controlled internucleotide linkages in the composition, the product of the diastereomeric purity of each g chiral controlled internucleotide linkage: (chiral controlled internucleotide linkage 1 diastereoisomeric purity) the (chiral controlled internucleotide linkage 2 diastereoisomeric purity) of the level is used, wherein each chiral controlled internucleotide linkage of diastereoisomeric purity independently from the same internucleotide linkage and the nucleotide flanking the internucleotide linkage and in a comparable method (e.g. comparable or preferably identical oligonucleotide preparation cycle, including equivalent or preferably identical reagents and reaction conditions). In certain embodiments, the level and/or diastereoisomeric purity of the oligonucleotide may be determined by analytical methods, such as chromatography, spectroscopy, or any combination thereof. The present disclosure encompasses, among other things, the following recognition: the stereotactic oligonucleotide formulation contains a plurality of different chemical entities that differ from each other, for example, in the stereochemistry (or stereochemistry) of the individual backbone chiral centers within the oligonucleotide strand. The stereogenic random oligonucleotide formulation provides an uncontrolled composition comprising an undetermined level of oligonucleotide stereoisomers without controlling the stereochemistry of the backbone chiral center. Even though these stereoisomers may have the same base sequence and/or chemical modification, they are different chemical entities, at least due to their different backbone stereochemistry, and they may have different properties, as demonstrated herein, e.g. sensitivity to nucleases, activity, distribution, etc. In certain embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its length, its backbone linkage pattern, and its backbone chiral center pattern. In certain embodiments, the disclosure demonstrates that improvements in properties and activity achieved by controlling stereochemistry within oligonucleotides can be comparable to, or even better than, those achieved by using chemical modifications.

The present disclosure encompasses, among other things, the following recognition: the stereotactic oligonucleotide formulation contains a plurality of different chemical entities that differ from each other, for example, in the stereochemistry (or stereochemistry) of the individual backbone chiral centers within the oligonucleotide strand. The stereogenic random oligonucleotide formulation provides an uncontrolled composition comprising an undetermined level of oligonucleotide stereoisomers without controlling the stereochemistry of the backbone chiral center. Even though these stereoisomers may have the same base sequence and/or chemical modification, they are different chemical entities, at least due to their different backbone stereochemistry, and they may have different properties, as demonstrated herein, e.g. sensitivity to nucleases, activity, distribution, etc. In certain embodiments, a particular stereoisomer may be defined, for example, by its base sequence, its length, its backbone linkage pattern, and its backbone chiral center pattern. In certain embodiments, the disclosure demonstrates that improvements in properties and activity achieved by controlling stereochemistry within oligonucleotides can be comparable to, or even better than, those achieved by using chemical modifications.

In some embodiments, the ds oligonucleotide targeting HSD17B13 or the ds oligonucleotide composition targeting HSD17B13 can be used to prevent or treat a HSD17B 13-related condition, disorder or disease in a subject in need thereof. In some embodiments, the disclosure provides methods for preventing or treating a HSD17B 13-related condition, disorder or disease, the method comprising administering to a subject suffering from the condition, disorder or disease or a subject of the condition, disorder or disease a therapeutically effective amount of a provided ds oligonucleotide, or a pharmaceutical composition that may deliver or comprise a therapeutically effective amount of a provided ds oligonucleotide. In some embodiments, the disclosure provides a pharmaceutical composition comprising a provided ds oligonucleotide that targets HSD17B13 and a pharmaceutically acceptable carrier. In some embodiments, the oligonucleotides in the pharmaceutical composition are in one or more pharmaceutically acceptable salt forms, e.g., sodium salt form, ammonium salt form, and the like.

In some embodiments, the oligonucleotide or oligonucleotide composition may be used to manufacture a medicament for preventing or treating a HSD17B 13-associated condition, disorder or disease, such as NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury or hepatocyte necrosis in a subject in need thereof.

The provided techniques (e.g., oligonucleotides, compositions, methods, etc.) can be utilized to prevent and/or treat a variety of HSD17B 13-related conditions, disorders, or diseases. In some embodiments, the condition, disorder or disease is NAFLD. In some embodiments, the condition, disorder or disease is NASH. In some embodiments, the condition, disorder or disease is ASH.

Detailed Description

The techniques of the present disclosure may be understood more readily by reference to the following detailed description of certain embodiments.

Definition of the definition

As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the periodic Table of elements (Periodic Table of THE ELEMENTS), CAS version, handbook of CHEMISTRY AND PHYSICS [ Handbook of chemistry and physics ], 75 th edition. In addition, the general principles of organic chemistry are described in "Organic Chemistry [ organic chemistry ]", thomas Sorrell, university Science Books [ university science book ], soxalito (sausalato): 1999 and "March' S ADVANCED Organic Chemistry [ Ma Jigao grade organic chemistry ]", 5 th edition, editor: smith, m.b. and March, j., john Wiley & Sons [ John wili parent, new york: 2001.

As used herein in this disclosure, unless the context clearly indicates otherwise, (i) the terms "a" or "an" are to be understood to mean "at least one"; (ii) the term "or" may be understood as "and/or"; (iii) The terms "comprises," "comprising," "includes," "including," "whether used with" or not "not limited to" and "includes" are to be construed as covering the listed components or steps individually or in combination with one or more other components or steps; (iv) The term "another" may be understood to mean one or more of at least one additional/second; (v) The terms "about" and "approximately" are to be understood as allowing standard deviation, as will be appreciated by one of ordinary skill in the art; and (vi) where ranges are provided, endpoints are included.

Unless otherwise indicated, the description of the oligonucleotide and its elements (e.g., base sequence, sugar modification, internucleotide linkages, linkage phosphorus stereochemistry, modes thereof, etc.) is made from 5 'to 3'. As will be appreciated by those of skill in the art, in some embodiments, the oligonucleotides may be provided and/or used as salt forms, particularly pharmaceutically acceptable salt forms (e.g., sodium salts). Unless otherwise indicated, oligonucleotides include various forms of oligonucleotides. As will also be appreciated by those of skill in the art, in some embodiments, a single oligonucleotide in a composition may be considered to have the same composition and/or structure, even in such compositions (e.g., liquid compositions), in particular, such oligonucleotide may be in different salt form(s) at a particular time (and, e.g., when in a liquid composition, it may be dissolved and the oligonucleotide chains may be present in anionic form). For example, one of skill in the art will understand that at a given pH, a single internucleotide linkage along an oligonucleotide strand may be in the acid (H) form, or in one of a number of possible salt forms (e.g., sodium salts or salts of different cations, depending on which ions may be present in the formulation or composition), and will understand that such a single oligonucleotide may be properly considered to have the same constitution and/or structure, so long as its acid form (e.g., substitution of H ⁺ for all cations, if any) has the same constitution and/or structure.

An analog: the term "analog" includes any chemical moiety that is structurally different from a reference chemical moiety or moiety class but is capable of performing at least one function of such reference chemical moiety or moiety class. As a non-limiting example, a nucleotide analog differs in structure from a nucleotide, but is capable of performing at least one function of the nucleotide; nucleobase analogs are structurally different from nucleobases, but are capable of performing at least one function of a nucleobase; etc.

Antisense: as used herein, the term "antisense" refers to the characteristic of an oligonucleotide or other nucleic acid having a base sequence complementary or substantially complementary to a target nucleic acid to which it is capable of hybridizing. In some embodiments, the target nucleic acid is a target gene mRNA. In some embodiments, hybridization is necessary for or results in a decrease in an activity, e.g., the level, expression, or activity of a target nucleic acid or gene product thereof. As used herein, the term "antisense oligonucleotide" refers to an oligonucleotide that is complementary to a target nucleic acid. In some embodiments, the antisense oligonucleotide is capable of directing a decrease in the level, expression, or activity of a target nucleic acid or product thereof. In some embodiments, antisense oligonucleotides are capable of directing a decrease in the level, expression, or activity of a target nucleic acid or product thereof by a mechanism involving RNA interference.

Chiral control: as used herein, "chiral control" refers to controlling the stereochemical identity of chiral-bonded phosphorus in chiral internucleotide linkages within an oligonucleotide. As used herein, chiral internucleotide linkages are internucleotide linkages whose linkage phosphorus is chiral. In some embodiments, control is achieved by chiral elements not present in the sugar and base portions of the oligonucleotide, e.g., in some embodiments, by using one or more chiral auxiliary(s) during oligonucleotide preparation, as described in the present disclosure, which is typically part of the chiral phosphoramidite used during oligonucleotide preparation. In contrast to chiral control, one of ordinary skill in the art recognizes that if conventional oligonucleotide synthesis is used to form chiral internucleotide linkages, such conventional oligonucleotide synthesis without the use of chiral auxiliary agents is not capable of controlling the stereochemistry at the chiral internucleotide linkages. In some embodiments, the stereochemical identity of each chiral linkage phosphorus in each chiral internucleotide linkage within the oligonucleotide is controlled.

Chiral controlled oligonucleotide composition: as used herein, the terms "chiral controlled oligonucleotide composition," "chiral controlled nucleic acid composition," and the like refer to a composition comprising a plurality of oligonucleotides (or nucleic acids) that share: 1) a common base sequence, 2) a common backbone linkage pattern, and 3) a common backbone phosphorus modification pattern, wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry of the linkage phosphorus at one or more chiral internucleotide linkages (chiral controlled or stereodefining internucleotide linkages having chiral linkage phosphorus in the composition ("stereodefining") as Rp or Sp, rather than a random Rp and Sp mixture as with achiral controlled internucleotide linkages). the level of the plurality of oligonucleotides (or nucleic acids) in the chirally controlled oligonucleotide composition is predetermined/controlled (e.g., by chiral controlled oligonucleotide preparation to stereoselectively form one or more chiral internucleotide linkages). In some embodiments, about 1% -100% (e.g., about 5％-100％、10％-100％、20％-100％、30％-100％、40％-100％、50％-100％、60％-100％、70％-100％、80％-100％、90％-100％、95％-100％、50％-90％, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60% >, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) are the plurality of oligonucleotides. In some embodiments, about 1% -100% (e.g., about 5％-100％、10％-100％、20％-100％、30％-100％、40％-100％、50％-100％、60％-100％、70％-100％、80％-100％、90％-100％、95％-100％、50％-90％, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 5% >, all oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern in a chiral controlled oligonucleotide composition, 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 level is of all oligonucleotides in the composition; or all oligonucleotides sharing a common base sequence (e.g., base sequences of multiple oligonucleotides or one oligonucleotide type) in the composition; or all oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern in the composition; Or about 1% -100% of all oligonucleotides sharing a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and/or a common internucleotide linkage modification pattern in the composition (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%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, 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 about 1-50 chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1% -100% of the chiral internucleotide linkages. In some embodiments, the plurality of oligonucleotides (or nucleic acids) have the same composition (as understood by those of skill in the art, may exist in one or more forms, e.g., acid form, salt form, etc., in some embodiments). In some embodiments, the level of the plurality of oligonucleotides (or nucleic acids) is about 1% -100% of all oligonucleotides (or nucleic acids) in a composition sharing the same constitution as the plurality of oligonucleotides (or nucleic acids). In some embodiments, each chiral internucleotide linkage is a chiral controlled internucleotide linkage, and the composition is a completely chiral controlled oligonucleotide composition. In some embodiments, the plurality of oligonucleotides (or nucleic acids) are identical in structure. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 95%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 96%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 97%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 98%. In some embodiments, the chiral controlled internucleotide linkages have a diastereomeric purity of at least 99%. In some embodiments, the percentage (e.g., level as described herein) is or is at least (DS) ^nc, where DS is diastereoisomeric purity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) as described in the disclosure, and nc is the number of chiral controlled internucleotide linkages (e.g., 1-50、1-40、1-30、1-25、1-20、5-50、5-40、5-30、5-25、5-20、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 or more) as described in the disclosure. in some embodiments, the percentage (e.g., level as described herein) is or is at least (DS) ^nc, where DS is 95% -100%. For example, when DS is 99% and nc is 10, the percentage is or at least 90% ((99%) ¹⁰ ≡0.90=90%). In some embodiments, the level of the plurality of oligonucleotides in the composition is expressed as the product of the diastereoisomeric purity of each chiral controlled internucleotide linkage in the oligonucleotide. In some embodiments, the diastereoisomeric purity of the internucleotide linkage joining two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereoisomeric purity of the internucleotide linkage joining the same two nucleoside dimers, wherein comparable conditions are used, in some cases identical synthetic cycling conditions are used to prepare the dimers (e.g., for the linkages between Nx and Ny in an oligonucleotide..once). In some embodiments, not all chiral internucleotide linkages are chiral controlled internucleotide linkages, and the compositions are partially chiral controlled oligonucleotide compositions. In some embodiments, the achiral controlled internucleotide linkages have a diastereomeric purity of less than about 80%, 75%, 70%, 65%, 60%, 55%, or about 50%, as typically observed in stereorandom oligonucleotide compositions (e.g., from traditional oligonucleotide synthesis, such as phosphoramidite methods), as understood by those of skill in the art. In some embodiments, the plurality of oligonucleotides (or nucleic acids) are of the same type. In some embodiments, the chirally controlled oligonucleotide composition comprises a non-random level or a controlled level of individual oligonucleotide types or nucleic acid types. For example, in some embodiments, the chirally controlled oligonucleotide composition comprises one and no more than one oligonucleotide type. In some embodiments, the chirally controlled oligonucleotide composition comprises more than one oligonucleotide type. In some embodiments, the chirally controlled oligonucleotide composition comprises a plurality of oligonucleotide types. In some embodiments, the chirally controlled oligonucleotide composition is a composition of oligonucleotides of one oligonucleotide type comprising a non-random or controlled level of a plurality of oligonucleotides of that oligonucleotide type.

Internucleotide linkages: as used herein, the phrase "internucleotide linkage" generally refers to the linkage of nucleoside units that join an oligonucleotide or nucleic acid. In some embodiments, the internucleotide linkages are phosphodiester linkages, as widely found in naturally occurring DNA and RNA molecules (natural phosphate linkages (-OP (=o) (OH) O-), which may exist in salt form, as understood by those skilled in the art. In some embodiments, the internucleotide linkages are modified internucleotide linkages (not natural phosphate linkages). In some embodiments, the internucleotide linkages are "modified internucleotide linkages" in which at least one oxygen atom or-OH of the phosphodiester linkages is replaced with a different organic or inorganic moiety. In some embodiments, such organic or inorganic moiety is selected from the group consisting of =s, =se, =nr ', -SR', -SeR ', -N (R') ₂、B(R′)₃, -S-, -Se-, and-N (R ') -, wherein each R' is independently as defined and described in the disclosure. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate linkage (or a phosphorothioate diester linkage, i.e., -OP (=o) (SH) O-, which may be present in salt form as understood by those skilled in the art), or a phosphorothioate triester linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the internucleotide linkage is one of, for example, PNA (peptide nucleic acid) or PMO (phosphorodiamidate morpholino oligomer) linkages. In some embodiments, the modified internucleotide linkage is a nonnegatively charged internucleotide linkage. In some embodiments, the modified internucleotide linkage is a neutral internucleotide linkage (e.g., n001 in certain provided oligonucleotides). It is understood by one of ordinary skill in the art that internucleotide linkages may exist as anions or cations at a given pH due to the presence of acid or base moieties in the linkages. In some embodiments, the modified internucleotide linkages are those designated as s, s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 and s18, as described in WO 2017/210647.

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

In vivo: as used herein, the term "in vivo" refers to an event that occurs within an organism (e.g., an animal, plant, and/or microorganism).

Bonding phosphorus: as defined herein, the phrase "phosphorus-bonded" is used to indicate that the particular phosphorus atom referred to is a phosphorus atom present in internucleotide linkages corresponding to phosphorus atoms of phosphodiester internucleotide linkages as found in naturally occurring DNA and RNA. In some embodiments, the linking phosphorus atoms are located in modified internucleotide linkages, wherein each oxygen atom of the phosphodiester linkage is optionally and independently replaced with an organic or inorganic moiety. In some embodiments, the linking phosphorus atom is P of formula I as described herein. In some embodiments, the linking phosphorus atom is chiral. In some embodiments, the linking phosphorus atom is achiral (e.g., as in a natural phosphate linkage).

And (3) joint: the terms "linker," "linking moiety," and the like refer to any chemical moiety that connects one chemical moiety to another chemical moiety. As will be appreciated by those skilled in the art, the linker may be divalent or trivalent or higher depending on the number of chemical moieties to which the linker is attached. In some embodiments, the linker is a moiety that links one oligonucleotide to another oligonucleotide in the multimer. In some embodiments, the linker is a moiety optionally located between the terminal nucleoside and the solid support or between the terminal nucleoside and another nucleoside, nucleotide or nucleic acid. In some embodiments, in an oligonucleotide, a linker connects a chemical moiety (e.g., targeting moiety, lipid moiety, carbohydrate moiety, etc.) to the oligonucleotide strand (e.g., through its 5 'end, 3' end, nucleobase, sugar, internucleotide linkage, etc.)

Modified nucleobases: the terms "modified nucleobase", "modified base", and the like refer to a chemical moiety that is chemically different from a nucleobase but is capable of performing at least one function of the 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, e.g., forming a moiety in the polymer that can base pair with a nucleic acid comprising at least a complementary base sequence. In some embodiments, the modified nucleobase is a substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U. In some embodiments, in the case of an oligonucleotide, a modified nucleobase refers to a nucleobase that is not A, T, C, G or U.

A modified nucleoside; the term "modified nucleoside" refers to a moiety derived from or chemically similar to a natural nucleoside but comprising a chemical modification that distinguishes it from the natural nucleoside. Non-limiting examples of modified nucleosides include those comprising modifications at the base and/or sugar. Non-limiting examples of modified nucleosides include those having a 2' modification at the sugar. Non-limiting examples of modified nucleosides also include abasic nucleosides (which lack nucleobases). In some embodiments, the modified nucleoside can have at least one function of a nucleoside, e.g., forming a moiety in the polymer that is capable of base pairing with a nucleic acid comprising at least a complementary base sequence.

Modified nucleotides; the term "modified nucleotide" includes any chemical moiety that is structurally different from a natural nucleotide but is capable of performing at least one function of the natural nucleotide. In some embodiments, the modified nucleotide comprises a modification at a sugar, base, and/or internucleotide linkage. In some embodiments, the modified nucleotide comprises a modified sugar, a modified nucleobase, and/or a modified internucleotide linkage. In some embodiments, the modified nucleotide can have at least one function of a nucleotide, e.g., forming a subunit in the polymer that is capable of base pairing with a nucleic acid comprising at least a complementary base sequence.

A modified sugar; the term "modified sugar" refers to a moiety that can replace a sugar. The modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical properties of the sugar. In some embodiments, the modified sugar is a substituted ribose or deoxyribose as described in the present disclosure. In some embodiments, the modified sugar comprises a 2' -modification. Examples of useful 2' -modifications are widely used in the art and described herein. In some embodiments, the 2 '-modification is 2' or, wherein R is an optionally substituted C _1-10 aliphatic. In some embodiments, the 2 '-modification is 2' -OMe. In some embodiments, the 2 '-modification is a 2' -MOE. In some embodiments, the modified sugar is a bicyclic sugar (e.g., a sugar used in LNA, BNA, etc.). In some embodiments, in the case of oligonucleotides, the modified sugar is a sugar other than ribose or deoxyribose commonly found in natural RNA or DNA.

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

Nucleobases: the term "nucleobase" refers to a moiety in a nucleic acid that participates in hydrogen bonding that binds one nucleic acid strand to another complementary strand in a sequence-specific manner. The most common naturally occurring nucleobases are adenine (a), guanine (G), uracil (U), cytosine (C) and thymine (T). In some embodiments, the naturally occurring nucleobase is a modified adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the naturally occurring nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the nucleobase comprises a heteroaryl ring, wherein the ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to the sugar moiety. In some embodiments, the nucleobase comprises a heterocyclic ring, wherein the ring atom is nitrogen, and when in a nucleoside, the nitrogen is bonded to the sugar moiety. In some embodiments, the nucleobase is a "modified nucleobase," i.e., a nucleobase other than adenine (a), guanine (G), uracil (U), cytosine (C), and thymine (T). In some embodiments, the modified nucleobase is a substituted A, T, C, G or U. In some embodiments, the modified nucleobase is A, T, C, G or a substituted tautomer of U. In some embodiments, the modified nucleobase is a methylated adenine, guanine, uracil, cytosine, or thymine. In some embodiments, the modified nucleobases mimic the spatial arrangement, electronic properties, or some other physicochemical properties of nucleobases, and retain the properties of hydrogen bonding to bind one nucleic acid strand to another in a sequence-specific manner. In some embodiments, the modified nucleobase can pair with all five naturally occurring bases (uracil, thymine, adenine, cytosine, or guanine) without substantially affecting melting behavior, recognition by intracellular enzymes, or activity of the oligonucleotide duplex. As used herein, the term "nucleobase" also encompasses structural analogs, such as modified nucleobases and nucleobase analogs, used in place of natural nucleotides or naturally occurring nucleotides. In some embodiments, the nucleobase is an optionally substituted A, T, C, G or U, or an optionally substituted tautomer of A, T, C, G or U. In some embodiments, a "nucleobase" refers to a nucleobase unit in an oligonucleotide or nucleic acid (e.g., A, T, C, G or U in an oligonucleotide or nucleic acid).

A nucleoside: the term "nucleoside" refers to a moiety in which a nucleobase or modified nucleobase is covalently bound to a sugar or modified sugar. In some embodiments, the nucleoside is a natural nucleoside, such as adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, or deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted natural nucleoside selected from the group consisting of adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, the nucleoside is a modified nucleoside, such as a substituted tautomer of a natural nucleoside selected from the group consisting of adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, uridine, cytidine, and deoxycytidine. In some embodiments, "nucleoside" refers to a nucleoside unit in an oligonucleotide or nucleic acid.

A nucleotide; as used herein, the term "nucleotide" refers to a monomeric unit of a polynucleotide that consists of nucleobases, sugars, and one or more internucleotide linkages (e.g., phosphate linkages in natural DNA and RNA). Naturally occurring bases [ guanine (G), adenine (a), cytosine (C), thymine (T) and uracil (U) ] are derivatives of purine or pyrimidine, but are understood to also include naturally occurring and non-naturally occurring base analogs. Naturally occurring sugars are pentoses (pentoses), i.e., deoxyribose (which forms DNA) or ribose (which forms RNA), but it is understood that naturally occurring and non-naturally occurring sugar analogs are also included. Nucleotides are linked via internucleotide linkages to form nucleic acids, or polynucleotides. Many internucleotide linkages are known in the art (such as, but not limited to, phosphate esters, phosphorothioates, borane phosphate esters, and the like). Artificial nucleic acids include PNA (peptide nucleic acids), phosphotriesters, phosphorothioates, H-phosphonates, phosphoramidates, borane phosphates, methylphosphonates, phosphonoacetates (phosphonoacetate), phosphorothioates, and other variants of the phosphate backbone of natural nucleic acids, such as those described herein. In some embodiments, the natural nucleotides comprise naturally occurring bases, sugars, and internucleotide linkages. As used herein, the term "nucleotide" also encompasses structural analogs, such as modified nucleotides and nucleotide analogs, used in place of natural nucleotides or naturally occurring nucleotides. In some embodiments, "nucleotide" refers to a nucleotide unit in an oligonucleotide or nucleic acid.

An oligonucleotide: the term "oligonucleotide" refers to a polymer or oligomer of nucleotides and may comprise any combination of natural and non-natural nucleobases, sugars, and internucleotide linkages.

The oligonucleotides may be single-stranded or double-stranded. The single-stranded oligonucleotide may have a double-stranded region (formed of two parts of the single-stranded oligonucleotide), and the double-stranded oligonucleotide comprising two oligonucleotide strands may have a single-stranded region, for example a region in which the two oligonucleotide strands are not complementary to each other. Exemplary oligonucleotides include, but are not limited to, structural genes, genes comprising control and termination regions, self-replicating systems (such as viral DNA or plasmid DNA), single-and double-stranded RNAi agents and other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, micrornas, microrna mimics, supermir, aptamers, antimir, antagomir, ul adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides.

Oligonucleotides of the present disclosure may have a variety of lengths. In particular embodiments, the length of the oligonucleotide may be about 2 to about 200 nucleosides. In various related embodiments, the length of the oligonucleotide (single-stranded, double-stranded, or triplex) may range from about 4 to about 10 nucleosides, from about 10 to about 50 nucleosides, from about 20 to about 50 nucleosides, from about 15 to about 30 nucleosides, from about 20 to about 30 nucleosides. In some embodiments, the oligonucleotide is about 9 to about 39 nucleosides in length. In some embodiments, the oligonucleotide is at least 4, 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 19 nucleosides in length. In some embodiments, the oligonucleotide is at least 20 nucleosides in length. In some embodiments, the oligonucleotide is at least 25 nucleosides in length. In some embodiments, the oligonucleotide is at least 30 nucleosides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 18 nucleosides in length. In some embodiments, the oligonucleotide is a duplex of complementary strands of at least 21 nucleosides in length. In some embodiments, each nucleoside counted in the length of the oligonucleotide independently comprises A, T, C, G or U, or optionally substituted A, T, C, G or U, or A, T, C, G or an optionally substituted tautomer of U.

Oligonucleotide type; as used herein, the phrase "oligonucleotide type" is used to define an oligonucleotide having a particular base sequence, a backbone linkage pattern (i.e., a pattern of internucleotide linkage types (e.g., phosphate, phosphorothioate triester, etc.), a backbone chiral center pattern [ i.e., a linkage phosphorus stereochemical pattern (Rp/Sp) ], and a backbone phosphorus modification pattern (e.g., a pattern of "-XLR ¹" groups in formula I as described herein). In some embodiments, the common oligonucleotides of a given "type" are structurally identical to each other.

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

Optionally substituted: as described herein, a compound of the disclosure (e.g., an oligonucleotide) may contain an optionally substituted moiety and/or a substituted moiety. Generally, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents at each position may be the same or different. In some embodiments, the optionally substituted group is unsubstituted. Combinations of substituents contemplated by the present disclosure are preferably combinations that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are not substantially altered when subjected to conditions for their preparation, detection, and in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Some substituents are described below.

Suitable monovalent substituents on the substitutable atom (e.g., a suitable carbon atom) are independently halogen ;-(CH₂)_0-4R^o;-(CH₂)_0-4OR^o;-O(CH₂)_0-4R^o、-O-(CH₂)_0-4C(O)OR^o;-(CH₂)_0-4CH(OR^o)₂;-(CH₂)_0-4Ph, which may be substituted with R ^o; - (CH ₂)_0-4O(CH₂)_0-1 Ph, which may be substituted by R ^o; -ch=chph, which may be substituted by R ^o; - (CH ₂)_0-4O(CH₂)_0-1 -pyridinyl, which may be substituted by R ^o for ;-NO₂;-CN;-N₃;-(CH₂)_0-4N(R^o)₂;-(CH₂)_0-4N(R^o)C(O)R^o;-N(R^o)C(S)R^o;-(CH₂)_0-4N(R^o)C(O)NR^o ₂;-N(R^o)C(S)NR^o ₂;-(CH₂)_0-4N(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)_0-4C(O)R^o;-C(S)R^o;-(CH₂)_0-4C(O)OR^o;-(CH₂)_0-4C(O)SR^o;-(CH₂)_0-4C(O)OSiR^o ₃;-(CH₂)_0-4OC(O)R^o;-OC(O)(CH₂)_0-4SR^o、-SC(S)SR^o;-(CH₂)_0-4SC(O)R^o;-(CH₂)_0-4C(O)NR^o ₂;-C(S)NR^o ₂;-C(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)_0-4SSR^o;-(CH₂)_0-4S(O)₂R^o;-(CH₂)_0-4S(O)₂OR^o;-(CH₂)_0-4OS(O)₂R^o;-S(O)₂NR^o ₂;-(CH₂)_0-4S(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;-P(R^o)(OR^o);-OP(R^o)₂;-OP(OR^o)₂;-OP(R^o)(OR^o);-P(O)(R^o)₂;-P(O)(OR^o)₂;-OP(O)(R^o)₂;-OP(O)(OR^o)₂;-OP(O)(OR^o)(SR^o);-SP(O)(R^o)₂;-SP(O)(OR^o)₂;-N(R^o)P(O)(R^o)₂;-N(R^o)P(O)(OR^o)₂;-P(R^o)₂[B(R^o)₃];-P(OR^o)₂[B(R^o)₃];-OP(R^o)₂[B(R^o)₃];-OP(OR^o)₂[B(R^o)₃];-(C_1-4 linear or branched alkylene) O-N (R ^o)₂; Or- (C _1-4 straight or branched alkylene) C (O) O-N (R ^o)₂, wherein each R ^o may be substituted as defined herein and is independently hydrogen; A C _1-20 heteroaliphatic having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus; -CH ₂-(C_6-14 aryl); -O (CH ₂)_0-1(C_6-14 aryl); -CH ₂ - (5-14 membered heteroaryl ring); a 5-20 membered monocyclic, bicyclic, or polycyclic saturated, partially unsaturated, or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon, and phosphorus; or, regardless of the above definition, two independently occurring R ^o together with one or more intervening atoms form a 5-20 membered monocyclic, bicyclic or polycyclic saturated, partially unsaturated or aryl ring having 0 to 5 heteroatoms independently selected from nitrogen, oxygen, sulfur, silicon and phosphorus, which may be substituted as defined below.

Suitable monovalent substituents on R ^o (OR the ring formed by two independently occurring R ^o together with the atoms intervening therebetween) are independently halogen, - (CH ₂)_0-2R^·, - (halo R^·)、-(CH₂)_0-2OH、-(CH₂)_0-2OR^·、-(CH₂)_0-2CH(OR^·)₂、-O( halo R^·)、-CN、-N₃、-(CH₂)_0-2C(O)R^·、-(CH₂)_0-2C(O)OH、-(CH₂)_0-2C(O)OR^·、-(CH₂)_0-2SR^·、-(CH₂)_0-2SH、-(CH₂)_0-2NH₂、-(CH₂)_0-2NHR^·、-(CH₂)_0-2NR^· ₂、-NO₂、-SiR^· ₃、-OSiR^· ₃、-C(O)SR^·、-(C_1-4 linear OR branched alkylene) C (O) OR ^· OR-SSR ^· wherein each R ^· is unsubstituted OR substituted with only one OR more halogen if preceded by "halo" and are independently selected from C _1-4 aliphatic, -CH ₂Ph、-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.

Suitable divalent substituents on suitable carbon atoms are, for example, independently ：＝O、＝S、＝NNR^＊ ₂、＝NNHC(O)R^＊、＝NNHC(O)OR^＊、＝NNHS(O)₂R^＊、＝NR^＊、＝NOR^＊、-O(C(R^＊ ₂))_2-3O-、 or-S (C (R ^＊ ₂))_2- ₃ S-, where each independently occurring R ^＊ is selected from hydrogen, a substituted C _1-6 aliphatic group, which may be defined below, and an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur suitable divalent substituents bonded to the ortho-substitutable carbon of the "optionally substituted" group include-O (CR ^＊ ₂)_2-3 O-, where each independently occurring R ^＊ is selected from hydrogen, a C _1-6 aliphatic group, which may be substituted as defined below, and an unsubstituted 5-6 membered saturated, partially unsaturated and aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur).

Suitable substituents on the aliphatic radical of R ^＊ are independently halogen, -R ^·, - (halo R ^·)、-OH、-OR^·, -O (halo R ^·)、-CN、-C(O)OH、-C(O)OR^·、-NH₂、-NHR^·、-NR^· ₂ or-NO ₂, wherein each R ^· is unsubstituted or substituted with only one or more halogens if preceded by a "halo") and are independently C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

In some embodiments, suitable substituents on the substitutable nitrogen are independently Each of which is provided withIndependently hydrogen, may be a substituted C _1-6 aliphatic, unsubstituted-OPh or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or two independently occurring in spite of the above definitionAnd one or more intervening atoms taken together form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.

Suitable substituents on the aliphatic radical of (a) are independently halogen, -R ^·, - (halo R ^·)、-OH、-OR^·, -O (halo R ^·)、-CN、-C(O)OH、-C(O)OR^·、-NH₂、-NHR^·、-NR^· ₂ or-NO ₂), wherein each R ^· is unsubstituted or substituted with only one or more halogens with "halo" in the preceding, and are independently C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

Oral administration: the phrase "orally administered (oral administration and ADMINISTERED ORALLY)" as used herein has its art-understood meaning, referring to the administration of a compound or composition by mouth.

Parenteral: the phrase "parenteral administration (PARENTERAL ADMINISTRATION and ADMINISTERED PARENTERALLY)" as used herein has its art understood meaning, and refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

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

A pharmaceutical composition; as used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions can be specifically formulated for administration in solid or liquid form, including those suitable for use in: oral administration, e.g., infusion (drench) (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes (applied to the tongue); parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension or as a sustained release formulation; topical application, for example, as a cream, ointment, or controlled release patch or spray to the skin, lungs, or oral cavity; intravaginal or intrarectal, for example as pessaries, creams or foams; sublingual; an eye; transdermal; or nasally, pulmonary, and other mucosal surfaces.

Pharmaceutically acceptable; as used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

A pharmaceutically acceptable carrier; as used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, which involves carrying or transporting the 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 of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some 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; powdery tragacanth; malt; 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; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; a pH buffer solution; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

A pharmaceutically acceptable salt: as used herein, the term "pharmaceutically acceptable salt" refers to salts of such compounds which are suitable for use in a pharmaceutical environment, i.e., salts which are suitable for use in contact with tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al J.pharmaceutical Sciences [ journal of pharmaceutical Sciences ],66: pharmaceutically acceptable salts are described in detail in 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are salts having amino groups formed using inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids or using organic acids such as acetic, maleic, tartaric, citric, succinic or malonic acid or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulfate, borate, butyrate, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate (hemisulfate), heptanoate, caproate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate (lactobionate), lactate, laurate, Lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. In some embodiments, provided compounds (e.g., oligonucleotides) comprise one or more acidic groups, and the pharmaceutically acceptable salt is an alkali metal salt, alkaline earth metal salt, or ammonium salt (e.g., an ammonium salt of N (R) ₃, wherein each R is independently defined and described in the disclosure). Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts suitably include nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions (such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl, sulfonates, and arylsulfonates having from 1 to 6 carbon atoms). In some embodiments, provided compounds include more than one acidic group, e.g., an oligonucleotide may include two or more acidic groups (e.g., natural phosphate linkages and/or modified internucleotide linkages). In some embodiments, a pharmaceutically acceptable salt (or, in general, a salt) of such a compound comprises two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or salt in general), all of the ionizable hydrogens in the acidic groups (e.g., in aqueous solution, pKa no greater than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no greater than about 7; in some embodiments, no greater than about 6; in some embodiments, no greater than about 5; in some embodiments, no greater than about 4; in some embodiments, no greater than about 3) are replaced with cations. In some embodiments, each phosphorothioate and phosphate group is independently present in its salt form (e.g., in the case of the sodium salt, respectively, -O-P (O) (SNa) -O-and-O-P (O) (ONa) -O-). in some embodiments, each phosphorothioate and phosphate internucleotide linkage independently exists in its salt form (e.g., in the case of the sodium salt, then respectively are-O-P (O) (SNa) -O-and-O-P (O) (ONa) -O-). In some embodiments, the pharmaceutically acceptable salt is a sodium salt of the oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of the oligonucleotide, wherein each acid phosphate and modified phosphate group (e.g., phosphorothioate, phosphate, etc.), if any, is present in salt form (all as sodium salts).

Protecting group: as used herein, the term "protecting group" is well known in the art and includes those described in detail in Protecting Groups in Organic Synthesis [ protecting group in organic synthesis ] t.w.greene and p.g.m.wuts, 3 rd edition, john Wiley & Sons [ John wili parent, inc., 1999, the entire contents of which are incorporated herein by reference. Also included are those protecting groups particularly useful for nucleoside and nucleotide chemistry, which are described in Current Protocols in Nucleic ACID CHEMISTRY [ nucleic acid chemistry laboratory guidelines ] edited by Serge l. Beaucage et al, 2012, month 06, the entire contents of section 2 are incorporated herein by reference. Suitable amino protecting groups include, but are not limited to, those described herein and/or below ：WO 2018/022473、WO 2018/098264、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、 and/or WO 2019/075357, or U.S. provisional patent applications 62/825766 and 62/911339, the descriptions of each of which are individually incorporated herein by reference.

Sample: as used herein, the term "sample" generally refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or comprise a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or comprises biological tissue or body fluid. In some embodiments, the biological tissue or body fluid may be or comprise amniotic fluid, aqueous humor, ascites fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chime, prostatic fluid, endolymph, exudates, fecal matter, gastric acid, gastric fluid, lymph, mucous, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum, semen, serum, cerumen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and/or combinations or one or more components thereof. In some embodiments, the biological fluid may be or comprise an intracellular fluid, an extracellular fluid, an intravascular fluid (plasma), an interstitial fluid, a lymphatic fluid, and/or a cell-penetrating fluid. In some embodiments, the biological fluid may be or comprise plant exudates. In some embodiments, biological tissue or samples may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, washing, or lavage (e.g., bronchoalveolar, catheter, nasal, ocular, oral, uterine, vaginal, or other washing or lavage). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable method. In some embodiments, as the context clearly indicates, the term "sample" refers to a formulation 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 is performed using a semipermeable membrane. Such "treated samples'" may comprise, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the primary sample to one or more techniques such as amplification or reverse transcription of nucleic acids, isolation and/or purification of certain components, and the like.

A subject; as used herein, the term "subject" or "test subject" refers to any organism to which provided compounds (e.g., provided oligonucleotides) or compositions are administered according to the present disclosure, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; and the like) and plants. In some embodiments, the subject is a human. In some embodiments, the subject may have and/or be susceptible to a disease, disorder, and/or condition.

Basically: as used herein, the term "substantially" refers to a qualitative state that exhibits an overall or near-overall extent or degree of a feature or characteristic of interest. The base sequence substantially complementary to the second sequence is not identical to the second sequence, but is mostly identical or nearly identical to the second sequence. Furthermore, it will be appreciated by those of ordinary skill in the biological and/or chemical arts that biological and chemical phenomena (if any) rarely, if ever, reach completion and/or proceed to completion or achieve or avoid absolute results. Thus, the term "substantially" is used herein to achieve inherent completeness that is potentially lacking in many biological and/or chemical phenomena.

Sugar: the term "saccharide" 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 saccharide is a polysaccharide. Sugars include, but are not limited to, ribose, deoxyribose, pentose, and hexose moieties. As used herein, the term "sugar" also encompasses structural analogs used in place of conventional sugar molecules, such as diols, polymers forming the backbone of nucleic acid analogs, diol nucleic acids ("GNAs"), and the like. As used herein, the term "sugar" also encompasses structural analogs, such as modified sugars and nucleotide sugars, used in place of natural nucleotides or naturally occurring nucleotides. In some embodiments, the sugar is an RNA or DNA sugar (ribose or deoxyribose). In some embodiments, the sugar is a modified ribose or deoxyribose, e.g., 2 '-modified, 5' -modified, etc. As described herein, in some embodiments, the modified sugar may provide one or more desired properties, activities, etc. when used with an oligonucleotide and/or nucleic acid. In some embodiments, the sugar is optionally substituted ribose or deoxyribose. In some embodiments, "sugar" refers to a sugar unit in an oligonucleotide or nucleic acid.

Is easy to suffer from: an individual who is "susceptible to" a disease, disorder and/or condition is an individual who has a higher risk of developing the disease, disorder and/or condition than a member of the general public. In some embodiments, an individual susceptible to a disease, disorder, and/or condition is predisposed to the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not be diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

A therapeutic agent; as used herein, the term "therapeutic agent" generally refers to any agent that, when administered to a subject, causes a desired effect (e.g., a desired biological, clinical, or pharmacological effect). In some embodiments, an agent is considered a therapeutic agent if the agent exhibits a statistically significant effect throughout the appropriate population. In some embodiments, a suitable population is a population of subjects suffering from and/or susceptible to a disease, disorder, or condition. In some embodiments, the suitable population is a population of model organisms. In some embodiments, the appropriate population may be defined by one or more criteria, such as age group, gender, genetic background, pre-existing clinical conditions, previously accepted therapies. In some embodiments, a therapeutic agent, when administered to a subject in an effective amount, is an agent that reduces, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and/or reduces the incidence of: one or more symptoms or features of the disease, disorder, and/or condition of the subject. In some embodiments, a "therapeutic agent" is an agent that has been or needs to be approved by a government agency before it can be sold for administration to a human. In some embodiments, a "therapeutic agent" is an agent that requires a pharmaceutical prescription for administration to a human. In some embodiments, the therapeutic agent is a provided compound, such as a provided oligonucleotide.

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

And (3) treatment: as used herein, the term "treating (treat, treatment, or treating)" refers to any method for partially or completely alleviating, ameliorating, reducing, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. The treatment may be administered to a subject that does not exhibit signs of the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a subject that exhibits only early signs of the disease, disorder, and/or condition, e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and/or condition.

Unsaturated: as used herein, the term "unsaturated" means a moiety having one or more unsaturated units.

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

As will be appreciated by those of skill in the art, the methods and compositions described herein relating to provided compounds (e.g., ds oligonucleotides) are also generally applicable to pharmaceutically acceptable salts of such compounds.

Description of certain embodiments

Double-stranded oligonucleotides are useful tools for a variety of applications. For example, ds oligonucleotides targeted to HSD17B13 (e.g., NCBI gene ID:345275 for human HSD17B13 and related sequences from other organisms) may be used in therapeutic, diagnostic, and research applications, including the treatment of a variety of HSD17B 13-related conditions, disorders, and diseases, including but not limited to NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, due to their susceptibility to endonucleases and exonucleases. As such, various synthetic counterparts have been developed to circumvent these drawbacks and/or to further improve various properties and activities. These synthetic counterparts include synthetic oligonucleotides that contain chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., which, among other things, make these molecules less susceptible to degradation and improve other properties and/or activity of the oligonucleotide. From a structural point of view, modifications to internucleotide linkages may introduce chirality and/or change charge, and certain properties may be affected by the configuration of the linked phosphorus atoms of the oligonucleotide. For example, the chirality and/or charge of the backbone linkage atoms can affect, among other things, binding affinity, sequence-specific binding to complementary RNAs, stability to nucleases, cleavage of target nucleic acids, delivery, pharmacokinetics, and the like.

In some embodiments, the ds oligonucleotide that targets HSD17B13 comprises one or more of:

wherein the ds oligonucleotide further comprises one or more of the following:

In some embodiments, the ds oligonucleotide that targets HSD17B13 comprises: (1) a phosphorothioate chiral centre in Rp or Sp configuration; (2) Rp, sp or a stereotactically non-negatively charged internucleotide linkage, wherein the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactically non-negatively charged internucleotide linkage comprises a 2' modification, such as 2' f; and (3) a 5' terminal modification selected from the group consisting of:

(a) 5' PO modifications such as, but not limited to:

(b) 5' VP modifications such as, but not limited to:

(c) 5' MeP modifications such as, but not limited to:

(d) 5'PN and 5' Trizole-P modifications, such as but not limited to:

(a) 5' PO nucleotides such as, but not limited to:

(b) 5' VP nucleotides, such as, but not limited to:

(c) 5' MeP nucleotides such as, but not limited to:

(d) 5'PN and 5' Trizole-P nucleotides, such as but not limited to:

(e) 5 'abasic VP and 5' abasic MeP nucleotides, such as, but not limited to:

wherein the ds oligonucleotide further comprises one or more of the following:

(4) A passenger strand, wherein one or more Rp, sp or stereotactically nonnegatively charged internucleotide linkages occur upstream, i.e. 5' with respect to the central nucleotide of the passenger strand;

In some embodiments, ds oligonucleotides targeting HSD17B13 comprise non-naturally occurring internucleotide linkages, e.g., neutral internucleotide linkages, which, in certain embodiments, can be used to ligate one or more molecules to double-stranded oligonucleotides described herein. In certain embodiments, such linked molecules may facilitate targeting and/or delivery of double-stranded oligonucleotides. Such linked molecules include, for example and without limitation, lipophilic molecules. In certain embodiments, the linked molecule is a molecule comprising one or more GalNac moieties. In certain embodiments, the linked molecule is a receptor. In certain embodiments, the linked molecule is a receptor ligand.

In certain embodiments, the disclosure provides techniques (e.g., compounds, methods, etc.) for improving oligonucleotide stability while maintaining or enhancing activity, including compositions of oligonucleotides with improved stability.

In certain embodiments, the disclosure demonstrates that certain provided structural elements, techniques, and/or features are particularly useful for ds oligonucleotides (e.g., RNAi agents) that participate in and/or direct RNAi machinery. However, in no way is the teachings of the present disclosure limited to ds oligonucleotides that participate in or act via any particular biochemical mechanism.

In certain embodiments, the disclosure relates to any ds oligonucleotide, useful for any purpose, which functions by any mechanism, and which comprises any sequence, structure, or form (or portion thereof) described herein.

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a backbone phosphorothioate chiral center in Rp, sp, or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of the following:

In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 comprising one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

In some embodiments, the guide strand comprises one or more Rp, sp or stereotactic non-negatively charged internucleotide linkages and internucleotide linkages to the penultimate 3 '(N-1) nucleotide between the second (+2) and third (+3) nucleotides of the guide strand relative to the 5' terminal nucleotide, and one or more of the following:

In some embodiments, the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

In some embodiments, the guide strand comprises a backbone phosphorothioate chiral center in Rp, sp, or alternating configuration between the 5' end (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and one or more of the following:

Wherein the ds oligonucleotide further comprises a 2' modification, e.g., a 2' f modification, of a 3' nucleotide of a nucleotide pair linked by Rp, sp or a stereotactic nonnegatively charged internucleotide linkage, and the passenger strand comprises one or more backbone chiral centers of Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages. In certain embodiments disclosed herein, the backbone phosphorothioate chiral center is in the Rp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide. In certain embodiments described herein, the backbone phosphorothioate chiral center is in the Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide. In certain embodiments, the backbone phosphorothioate chiral center is in the Rp and Sp configurations between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide, and between the +2 nucleotide and the immediately downstream (+3) nucleotide, respectively. In certain embodiments described herein, the backbone phosphorothioate chiral center is in the Sp, rp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, respectively.

In some embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and one or more of the following:

In some embodiments, the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminus (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide and in one or both of: (a) between +3 and +4 nucleotides; and (b) (+5) and (+6) nucleotides, and one or more of:

In some embodiments, the guide strand comprises one or more Rp, sp, or stereotactically non-negatively charged internucleotide linkages between the second (+2) nucleotide of the guide strand relative to the 5 'terminal nucleotide and any two adjacent nucleotides between the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3 'terminal nucleotide, the guide strand comprising a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp, or stereotactically non-negatively charged internucleotide linkages, and the passenger strand comprising one or more backbone chiral centers in Rp or Sp configuration.

In certain embodiments, the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3 'terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, the guide strand comprises a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactic non-negatively charged internucleotide linkage, and the passenger strand comprises 0-N Rp, sp or a stereotactic non-negatively charged internucleotide linkage (where N is about 1 to 49) and one or more backbone chiral centers in the Rp or Sp configuration.

In certain embodiments, the guide strand comprises a backbone phosphorothioate chiral center in Rp, sp, or alternating configuration between the 5 'terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, the guide strand comprises a 2' modification, e.g., a 2'f modification, of the 3' nucleotide of the nucleotide pair linked by Rp, sp, or a stereotactic non-negatively charged internucleotide linkage, and the passenger strand comprises 0-n Rp, sp, or a stereotactic non-negatively charged internucleotide linkage (where n is about 1 to 49) and one or more backbone chiral centers in Rp or Sp configuration. In certain embodiments, one or more Rp, sp, or stereotactic non-negatively charged internucleotide linkages incorporated into the guide or passenger strand are Rp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are Sp non-negatively charged internucleotide linkages. In certain embodiments, one or more Rp, sp, or stereorandom non-negatively charged internucleotide linkages are stereorandom non-negatively charged internucleotide linkages.

In certain embodiments, the internucleotide linkages of the oligonucleotide comprise or consist of 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-40, 1-50, or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more chirally controlled internucleotide linkages. In certain embodiments, the disclosure provides dsRNAi oligonucleotide compositions, wherein the dsRNAi oligonucleotide comprises at least one chirally controlled internucleotide linkage. In certain embodiments, the disclosure provides dsRNAi oligonucleotide compositions, wherein the dsRNAi oligonucleotides are sterically random or achiral controlled. In certain embodiments, in the dsRNAi oligonucleotides, at least one internucleotide linkage is stereotactically random, and at least one internucleotide linkage is chirally controlled.

In certain embodiments, the internucleotide linkages of the oligonucleotide comprise or consist of one or more electrically neutral internucleotide linkages.

HSD17B13

In some embodiments, HSD17B13 refers to a gene from any species or gene product thereof (including but not limited to nucleic acids, including but not limited to DNA or RNA, transcripts, proteins encoded thereby; may be from any form of HSD17B13, e.g., wild-type or mutant alleles), which may be referred to as: the patent refers to the field of 'AN_SNparatus or processes for the preparation of the same'. In some embodiments, HSD17B13 refers to genes and products thereof in humans. In some embodiments, HSD17B13 refers to genes and products thereof in non-human primates. Various HSD17B13 sequences, including variants thereof, from humans, mice, rats, monkeys, etc., are readily available to those skilled in the art. In some embodiments, HSD17B13 is human or mouse HSD17B13, which is wild-type or mutant. HSD17B13 is reported to have many functions. Various techniques have also been reported, such as assays, cells, animal models, etc., and can be used to characterize and/or evaluate the provided techniques (e.g., oligonucleotides, compositions, methods, etc.) in accordance with the present disclosure.

In some embodiments, the HSD17B13 gene, transcript (e.g., mRNA before or after splicing), or protein variant or isoform comprises a mutation. In some embodiments, the HSD17B13 gene, transcript or protein is the transcriptional or translational product of an alternative splice variant or isoform.

HSD17B13 related conditions, disorders or diseases

A variety of conditions, disorders or diseases are reported to be associated with HSD17B 13. Typically, a disease, disorder, or condition is associated with HSD17B13 if the presence, level, activity, and/or form of HSD17B13 and/or its products (e.g., transcripts, encoded proteins, etc.) is associated with the incidence and/or susceptibility of the disease, disorder, or condition (e.g., in a relevant population). In some embodiments, the condition, disorder or disease associated with HSD17B13 may be treated and/or prevented by reducing expression, levels and/or activity of HSD17B13 transcripts and/or proteins.

A variety of HSD17B13 related conditions, disorders or diseases are reported. In some embodiments, the HSD17B 13-associated condition, disorder or disease is NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis. In some embodiments, the HSD17B 13-related condition, disorder or disease is NAFLD. In some embodiments, the HSD17B 13-associated condition, disorder or disease is NASH.

The provided techniques may be used to treat or prevent, among other things, conditions, disorders, or diseases associated with HSD17B13, such as NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis, among others. In some embodiments, the disclosure relates to the use of a ds oligonucleotide or composition thereof that targets HSD17B13 in the treatment of an HSD17B 13-related disorder, disease or condition, such as NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, liver steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis, and the like.

In some embodiments, treatment or prevention with the provided techniques reduces the rate of HSD17B13 production and reduces or stops or reverses the accumulation of HSD17B 13. In some embodiments, treatment or prevention with the provided techniques reduces the rate of clinical decline of a condition, disorder, or disease, or delays or prevents onset.

As will be appreciated by those of skill in the art, the mechanisms, genotypes, symptoms, biomarkers, etc., of such conditions, disorders, or diseases may be utilized in accordance with the present disclosure to characterize/evaluate the provided techniques.

Double-stranded oligonucleotides

The present disclosure provides ds oligonucleotides having a variety of designs, which may comprise a variety of nucleobases and patterns thereof, sugars and patterns thereof, internucleotide linkages and patterns thereof, and/or additional chemical moieties and patterns thereof, as described in the present disclosure, among other things. In some embodiments, provided ds oligonucleotides targeting HSD17B13 can direct reduced expression, levels, and/or activity of the HSD17B13 gene and/or one or more products thereof (e.g., transcripts, mRNA, proteins, etc.). In some embodiments, provided ds oligonucleotides targeting HSD17B13 may direct reduced expression, levels, and/or activity of the HSD17B13 gene and/or one or more products thereof in cells of a subject or patient. In some embodiments, the cell normally expresses HSD17B13 or produces HSD17B13 protein. In some embodiments, provided ds oligonucleotides targeting HSD17B13 can direct reduced expression, level, and/or activity of HSD17B13 target genes or gene products, and have a base sequence consisting of, comprising, or comprising a portion of (e.g., sequence segments of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive bases) the base sequence of a ds oligonucleotide targeting HSD17B13 disclosed herein, wherein each T can be independently substituted with U, and vice versa, and the ds oligonucleotide comprises at least one non-naturally occurring modification of a base, sugar, and/or internucleotide linkage.

In some embodiments, ds oligonucleotides targeting HSD17B13 may direct reduced expression, levels, and/or activity of a target gene, e.g., an HSD17B13 target gene or product thereof. In some embodiments, ds oligonucleotides targeting HSD17B13 may direct reduced expression, levels, and/or activity of HSD17B13 target genes or products thereof via rnase H-mediated knockdown. In some embodiments, ds oligonucleotides targeting HSD17B13 may direct reduced expression, levels, and/or activity of HSD17B13 target gene or product thereof by spatially blocking translation after binding to HSD17B13 target gene mRNA and/or by altering or interfering with mRNA splicing. However, the present disclosure is not limited to any particular mechanism in any way. In some embodiments, the disclosure provides oligonucleotides, compositions, methods, and the like, that are capable of manipulation via double stranded RNA interference.

In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating reduced expression, level and/or activity of HSD17B 13. In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating reduced levels of HSD17B13 protein. In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating reduced levels of HSD17B13 protein.

In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating reduced expression, levels, and/or activity of HSD17B13 via a mechanism involving mRNA degradation.

In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating reduced expression, levels, and/or activity of more than one HSD17B13 allele.

In some embodiments, the disclosure relates to methods of treating a disease, disorder, or condition associated with HSD17B13 (wherein HSD17B13 is expressed), comprising the step of administering a therapeutically effective amount of a ds oligonucleotide that targets HSD17B13 capable of mediating reduced expression, levels, and/or activity of HSD17B 13. In some embodiments, multiple forms (e.g., alleles) of HSD17B13 may be present, and the provided techniques may reduce expression, levels, and/or activity of two or more or all forms and products thereof.

In some embodiments, the disclosure relates to methods of treating a disease, disorder, or condition associated with HSD17B13, comprising the step of administering an effective amount of a ds oligonucleotide targeting HSD17B13 capable of mediating reduced expression, level, and/or activity of HSD17B 13.

In some embodiments, ds oligonucleotides targeting HSD17B13 are capable of mediating reduced expression, level and/or activity of HSD17B13 via mechanisms involving splice regulation (e.g., exon skipping).

In some embodiments, the ds oligonucleotide that targets HSD17B13 comprises a structural element or a portion thereof described herein, e.g., in table 1. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a base sequence (or a portion thereof) described herein (wherein each T may independently be substituted with U, and vice versa), a chemical modification or pattern of chemical modification (or a portion thereof), and/or a form described herein or a portion thereof. In some embodiments, the ds oligonucleotide that targets HSD17B13 has a base sequence comprising the base sequence (or a portion thereof) (wherein each T may be independently substituted with a U), a chemical modification pattern (or a portion thereof), and/or forms of the oligonucleotides disclosed herein (e.g., in table 1, or otherwise disclosed herein). In some embodiments, such oligonucleotides, e.g., ds oligonucleotides targeting HSD17B13, reduce expression, level and/or activity of a gene, e.g., the HSD17B13 gene or gene product thereof.

The ds oligonucleotide that targets HSD17B13 can hybridize to its target nucleic acid (e.g., pre-mRNA, mature mRNA, etc.), among other things. For example, in some embodiments, a ds oligonucleotide targeting HSD17B13 may hybridize to HSD17B13 nucleic acid derived from a DNA strand (either strand of the HSD17B13 gene). In some embodiments, ds oligonucleotides targeting HSD17B13 can hybridize to HSD17B13 transcripts. In some embodiments, ds oligonucleotides targeting HSD17B13 can hybridize to HSD17B13 nucleic acid at any stage of RNA processing, including but not limited to pre-mRNA or mature mRNA. In some embodiments, the ds oligonucleotide targeting HSD17B13 may hybridize to any element of the HSD17B13 nucleic acid or its complement, including, but not limited to: a promoter region, enhancer region, transcription termination region, translation initiation signal, translation termination signal, coding region, non-coding region, exon, intron/exon or exon/intron junction, 5'utr or 3' utr. In some embodiments, the ds oligonucleotide that targets HSD17B13 can hybridize to a target with no more than 2 mismatches thereto. In some embodiments, the ds oligonucleotide that targets HSD17B13 may hybridize to a target with no more than one mismatch thereto. In some embodiments, the ds oligonucleotide that targets HSD17B13 can hybridize to a target that does not have a mismatch with it (e.g., when all C-G and/or a-T/U base pairs).

In some embodiments, the oligonucleotide may hybridize to two or more transcript variants. In some embodiments, ds oligonucleotides targeting HSD17B13 may hybridize to two or more or all of the HSD17B13 transcript variants. In some embodiments, ds oligonucleotides targeting HSD17B13 may hybridize to two or more or all of the HSD17B13 transcript variants derived from the sense strand.

In some embodiments, the HSD17B13 target of the ds oligonucleotide that targets HSD17B13 is HSD17B13 RNA that is not mRNA.

In some embodiments, the oligonucleotide (e.g., a ds oligonucleotide targeting HSD17B 13) contains increased levels of one or more isotopes. In some embodiments, the oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) are labeled, for example, with one or more isotopes of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). In some embodiments, the oligonucleotides in the provided compositions (e.g., ds oligonucleotides targeting HSD17B 13) (e.g., oligonucleotides of various compositions) comprise base modifications, sugar modifications, and/or internucleotide linkage modifications, wherein the oligonucleotides contain enriched levels of deuterium. In some embodiments, the oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) are deuterium labeled (with- ² H instead of- ¹ H) at one or more positions. In some embodiments, one or more ¹ H of the oligonucleotide strand or any moiety conjugated to the oligonucleotide strand (e.g., targeting moiety, etc.) is substituted with ² H. Such oligonucleotides may be used in the compositions and methods described herein.

In some embodiments, the present disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides:

1) Having a common base sequence complementary to a target sequence in the transcript (e.g., HSD17B13 target sequence); and

2) Comprising one or more modified sugar moieties and/or modified internucleotide linkages.

In some embodiments, ds oligonucleotides targeting HSD17B13 that have a common base sequence may have the same pattern of nucleoside modifications (e.g., sugar modifications, base modifications, etc.). In some embodiments, the nucleoside modification pattern can be represented by a combination of position and modification. In some embodiments, the backbone linkage pattern comprises the position and type of linkage between each nucleotide (e.g., phosphate, phosphorothioate, substituted phosphorothioate, etc.).

In some embodiments, for example, the plurality of oligonucleotides in the provided compositions are the same oligonucleotide type. In some embodiments, oligonucleotides of one oligonucleotide type have a common sugar modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have a common base modification pattern. In some embodiments, oligonucleotides of one oligonucleotide type have a common nucleoside modification pattern. In some embodiments, the oligonucleotides of one oligonucleotide type have the same composition. In some embodiments, the oligonucleotides of one oligonucleotide type are identical. In some embodiments, the plurality of oligonucleotides are identical. In some embodiments, multiple oligonucleotides share the same composition.

In some embodiments, the ds oligonucleotide targeting HSD17B13 is chirally controlled, comprising one or more chirally controlled internucleotide linkages, as exemplified herein. In some embodiments, the ds oligonucleotide that targets HSD17B13 is stereochemically pure. In some embodiments, the ds oligonucleotide that targets HSD17B13 is substantially separated from the other stereoisomers.

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises one or more modified nucleobases, one or more modified sugars, and/or one or more modified internucleotide linkages.

In some embodiments, the ds oligonucleotide that targets HSD17B13 comprises one or more modified sugars. In some embodiments, an oligonucleotide of the disclosure comprises one or more modified nucleobases. According to the present disclosure, various modifications can be introduced to the sugar and/or nucleobase. For example, in some embodiments, the modification is a modification described in US 9006198. In some embodiments, the modifications are those described in US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252, the respective sugar, base and internucleotide linkage modifications of which are independently incorporated herein by reference.

As used in this disclosure, in some embodiments, "one or more" is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3,4, 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, "one or more" is one. In some embodiments, "one or more" is two. In some embodiments, "one or more" is three. In some embodiments, "one or more" is four. In some embodiments, "one or more" is five. In some embodiments, "one or more" is six. In some embodiments, "one or more" is seven. In some embodiments, "one or more" is eight. In some embodiments, "one or more" is nine. In some embodiments, "one or more" is ten. In some embodiments, "one or more" is at least one. In some embodiments, "one or more" is at least two. In some embodiments, "one or more" is at least three. In some embodiments, "one or more" is at least four. In some embodiments, "one or more" is at least five. In some embodiments, "one or more" is at least six. In some embodiments, "one or more" is at least seven. In some embodiments, "one or more" is at least eight. In some embodiments, "one or more" is at least nine. In some embodiments, "one or more" is at least ten.

As used in this disclosure, in some embodiments, "at least one" is 1-200, 1-150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, "at least one" is one. In some embodiments, "at least one" is two. In some embodiments, "at least one" is three. In some embodiments, "at least one" is four. In some embodiments, "at least one" is five. In some embodiments, "at least one" is six. In some embodiments, "at least one" is seven. In some embodiments, "at least one" is eight. In some embodiments, "at least one" is nine. In some embodiments, "at least one" is ten.

In some embodiments, the ds oligonucleotide that targets HSD17B13 is or comprises a ds oligonucleotide that targets HSD17B13 described in table 1.

As demonstrated in the present disclosure, in some embodiments, the provided oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) are characterized in that when it is contacted with a transcript in a knockdown system, knockdown of its target is achieved (e.g., HSD17B13 transcript targeting ds oligonucleotides of HSD17B 13).

In some embodiments, the ds oligonucleotide is provided in salt form. In some embodiments, ds oligonucleotides are provided in the form of salts comprising negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.) as salt forms. In some embodiments, the ds oligonucleotide is provided in the form of a pharmaceutically acceptable salt. In some embodiments, the ds oligonucleotide is provided in the form of a metal salt. In some embodiments, the oligonucleotide is provided in the form of a sodium salt. In some embodiments, the ds oligonucleotides are provided in the form of a metal salt, e.g., sodium salt, wherein each negatively charged internucleotide linkage is independently in the form of a salt (e.g., for sodium salt, for phosphorothioate internucleotide linkages, -O-P (O) (SNa) -O-, for natural phosphate linkages, -O-P (O) (ONa) -O-, etc.).

Double-stranded oligonucleotide base sequence

In some embodiments, a ds oligonucleotide targeting HSD17B13 comprises a base sequence described herein or a portion thereof having 0-5 (e.g., 0, 1, 2,3,4, or 5) mismatches (e.g., sequence segments 5-50, 5-40, 5-30, 5-20, or 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, or at least 10, at least 15 consecutive nucleobases), wherein each T may be independently substituted with U, and vice versa. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a base sequence described herein or a portion thereof, wherein a portion is a sequence segment of at least 10 consecutive nucleobases or at least 15 consecutive nucleobases with 1-5 mismatches. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a base sequence described herein or a portion thereof, wherein a portion is a sequence segment of at least 10 consecutive nucleobases or at least 10 consecutive nucleobases with 1-5 mismatches, wherein each T may be independently substituted with U, and vice versa. In some embodiments, the base sequence of the oligonucleotide comprises or consists of: 10-50 (e.g., about or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45; in some embodiments, at least 15; in some embodiments, at least 16; in some embodiments, at least 17; in some embodiments, at least 18; in some embodiments, at least 19; in some embodiments, at least 20; in some embodiments, at least 21; in some embodiments, at least 22; in some embodiments, at least 23; in some embodiments, at least 24; in some embodiments, at least 25) consecutive bases of the base sequence of the HSD17B13 gene or a transcript (e.g., mRNA) thereof (e.g., in an intron).

As will be appreciated by those of skill in the art, the base sequence of the ds oligonucleotide targeting HSD17B13 is generally of sufficient length and complementarity to its target, e.g., RNA transcript (e.g., pre-mRNA, mature mRNA, etc.), to mediate target-specific knockdown. In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 is of sufficient length and identity to the HSD17B13 transcript target to mediate target-specific knockdown. In some embodiments, the ds oligonucleotide that targets HSD17B13 is complementary to a portion of HSD17B13 transcript (HSD 17B13 transcript target sequence). In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 has 90% or greater identity to the base sequence of the oligonucleotide disclosed in table 1, wherein each T may be independently substituted with U, and vice versa. In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 has 95% or greater identity to the base sequence of the oligonucleotide disclosed in table 1, wherein each T may be independently substituted with U, and vice versa. In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 comprises a contiguous sequence segment of 15 or more bases of the oligonucleotides disclosed in table 1, wherein each T may be independently substituted with a U, and vice versa, except that one or more bases within the sequence segment are abasic (e.g., nucleobases are not present in a nucleotide). In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 comprises a contiguous stretch of 19 or more bases of the ds oligonucleotide targeting HSD17B13 disclosed herein, except that one or more bases within the stretch are abasic (e.g., nucleobases are not present in a nucleotide). In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 comprises a contiguous sequence segment of 19 or more bases of the oligonucleotides disclosed herein, wherein each T may be independently substituted with U, and vice versa, except for differences of 1 or 2 bases at the 5 'end and/or 3' end of these base sequences.

In some embodiments, the disclosure relates to oligonucleotides having a base sequence comprising a base sequence of any of the oligonucleotides disclosed herein, wherein each T can be independently replaced with U and vice versa.

In some embodiments, the disclosure relates to oligonucleotides having a base sequence of at least 15 consecutive bases comprising the base sequence of any of the oligonucleotides disclosed herein, wherein each T may be independently replaced by U and vice versa.

In some embodiments, the disclosure relates to oligonucleotides having a base sequence that is at least 90% identical to the base sequence of any of the oligonucleotides disclosed herein, wherein each T may be independently replaced by U and vice versa.

In some embodiments, the disclosure relates to oligonucleotides having a base sequence that is at least 95% identical to the base sequence of any of the oligonucleotides disclosed herein, wherein each T may be independently replaced by U and vice versa.

In some embodiments, the ds oligonucleotide that targets HSD17B13 is selected from table 1.

In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 is complementary to the base sequence of the HSD17B13 transcript or portion thereof.

In some embodiments, the base sequence of the ds oligonucleotide targeting HSD17B13 is complementary to a portion of the HSD17B13 nucleic acid sequence (e.g., HSD17B13 gene sequence, HSD17B13 transcript, HSD17B13 mRNA sequence, etc.). In some embodiments, the ds oligonucleotide targeting HSD17B13 is identical to a portion of the HSD17B13 nucleic acid sequence (e.g., HSD17B13 gene sequence, HSD17B13 transcript, HSD17B13 mRNA sequence, etc.). In some embodiments, the base sequence of such a portion is characteristic of HSD17B13, as no other genomic or transcript sequences in the system contain the same sequence as the portion. In some embodiments, no other genomic or transcript sequences in the system contain sequences that differ from such portions by no more than 1 nucleobase. In some embodiments, no other genomic or transcript sequences in the system contain sequences that differ from such portions by no more than 2 nucleobases. In some embodiments, a portion of a gene that is complementary to an oligonucleotide is referred to as a target sequence of the oligonucleotide. In some embodiments, the system is or comprises a cell, sample, tissue, organ, or species. For example, for oligonucleotides targeting human HSD17B13, the relevant species is human in many embodiments. In some embodiments, the system may be or comprise a plurality of species, for example when characterizing and/or assessing activity and/or characteristics across species. In some embodiments, such a moiety is in an exon. In some embodiments, such a moiety is in an intron. In some embodiments, such portions span introns and exons. In some embodiments, such portions span two exons. In some embodiments, such a portion is in the 5' -UTR region. In some embodiments, such a portion is in the 3' -UTR region.

In some embodiments, the ds oligonucleotide targeting HSD17B13 targets two or more or all alleles of HSD17B13 (if multiple alleles are present in the relevant system). In some embodiments, the oligonucleotide reduces expression, level and/or activity of both wild-type HSD17B13 and mutant HSD17B13 and/or transcripts and/or products thereof.

In some embodiments, the base sequence of the provided oligonucleotides is fully complementary to both human and non-human primate (NHP) HSD17B13 target sequences. In some embodiments, such sequences may be particularly useful as they can be readily evaluated in humans and non-human primates.

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a base sequence or a portion thereof (wherein each T may independently be replaced by U, and vice versa) as set forth in the table, and/or sugar, nucleobase, and/or internucleotide linkage modifications and/or patterns thereof as set forth in table 1, and/or additional chemical moieties (in addition to the oligonucleotide strand, e.g., a target moiety, a lipid moiety, a carbohydrate moiety, etc.) as set forth in table 1.

In some embodiments, the terms "complementary," "fully complementary," and "substantially complementary" may be used in terms of base matching between an oligonucleotide (e.g., a ds oligonucleotide targeting HSD17B 13) base sequence and a target sequence (e.g., an HSD17B13 target sequence), as will be appreciated by those of skill in the art from the context of use. It should be noted that substitution of U for T or vice versa does not generally alter the amount of complementarity. As used herein, an oligonucleotide that is "substantially complementary" to a target sequence is largely or mostly complementary, but not 100% complementary. In some embodiments, the substantially complementary sequence (e.g., ds oligonucleotide targeting HSD17B 13) has 1,2, 3,4, or 5 mismatches when aligned to the target sequence. In some embodiments, the ds oligonucleotide that targets HSD17B13 has a base sequence that is substantially complementary to the HSD17B13 target sequence. In some embodiments, the ds oligonucleotide that targets HSD17B13 has a base sequence that is substantially complementary to the sequence of the ds oligonucleotide that targets HSD17B13 disclosed herein. As will be appreciated by those of skill in the art, in some embodiments, the sequence of an oligonucleotide need not be 100% complementary to its target for the oligonucleotide to perform its function (e.g., knock down the target nucleic acid). Typically, when complementarity is determined, a and T (or U) are complementary nucleobases, and C and G are complementary nucleobases.

In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 comprising a sequence found in an oligonucleotide set forth in the table. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 comprising a sequence found in the oligonucleotides described in table 1, wherein one or more U is independently and optionally replaced with T, or vice versa. In some embodiments, the ds oligonucleotide targeting HSD17B13 may comprise at least one T and/or at least one U. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 comprising a sequence found in an oligonucleotide set forth in the table, wherein the sequence has greater than 50% identity to the sequence of the oligonucleotide set forth in the table. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 comprising the sequence of the oligonucleotide disclosed in table 1. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13, the base sequence of the ds oligonucleotide targeting HSD17B13 being the sequence of the oligonucleotides disclosed in table 1, wherein each T may be independently replaced with U, and vice versa. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 comprising a sequence found in the oligonucleotide in table 1, wherein the oligonucleotide has a backbone linkage pattern, a backbone chiral center pattern, and/or a backbone phosphorus modification pattern of the same oligonucleotide or another oligonucleotide in table 1.

Among other things, the disclosure presents a number of ds oligonucleotides in table 1 and elsewhere, each ds oligonucleotide having a defined base sequence. In some embodiments, the disclosure provides oligonucleotides whose base sequences are those of the oligonucleotides disclosed herein (e.g., in table 1 herein, where each T may be independently replaced with U, and vice versa), base sequences comprising the oligonucleotides disclosed herein, and base sequences comprising a portion of the base sequences of the oligonucleotides disclosed herein. In some embodiments, the disclosure provides oligonucleotides having a base sequence that is or comprises a base sequence of an oligonucleotide disclosed herein (e.g., in table 1) or comprises a portion of a base sequence of an oligonucleotide disclosed herein, wherein each T can be independently replaced with U and vice versa, wherein the oligonucleotide further comprises a chemical modification, stereochemistry, form, additional chemical moiety (e.g., targeting moiety, lipid moiety, carbohydrate moiety, etc.) and/or another structural feature described herein.

In some embodiments, a "portion" (e.g., a portion of a base sequence or modification pattern) is at least 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomer units in length (e.g., for a base sequence, at least 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases in length). In some embodiments, the "portion" of the base sequence is at least 5 bases in length. In some embodiments, the "portion" of the base sequence is at least 10 bases in length. In some embodiments, the "portion" of the base sequence is at least 15 bases in length. In some embodiments, the "portion" of the base sequence is at least 16, 17, 18, 19, or 20 bases in length. In some embodiments, the "portion" of the base sequence is at least 20 bases in length. In some embodiments, a portion of the base sequence is 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous (consecutive/consecutive) bases. In some embodiments, a portion of the base sequence is 15 or more consecutive bases. In some embodiments, a portion of the base sequence is 16, 17, 18, 19, or 20 or more consecutive bases. In some embodiments, a portion of the base sequence is 20 or more consecutive bases.

In some embodiments, the disclosure provides an oligonucleotide (e.g., a ds oligonucleotide targeting HSD17B 13) whose base sequence is the base sequence of the oligonucleotide in table 1 or a portion thereof, wherein each T may be independently replaced with U, and vice versa. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13 having the sequence of the oligonucleotide in table 1, wherein the oligonucleotide is capable of directing reduced expression, level and/or activity of the HSD17B13 gene or gene product thereof. As will be appreciated by those skilled in the art, in the base sequences provided, each U may optionally and independently be replaced by T, or vice versa, and the sequences may comprise a mixture of U and T. In some embodiments, C may optionally and independently be replaced with 5 mC.

In some embodiments, a portion is a stretch of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides. In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides with 0-3 mismatches. In some embodiments, a portion is a sequence segment of at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 total nucleotides having 0-3 mismatches, wherein the sequence segment having 0 mismatches is complementary and the sequence segment having 1 or more mismatches is a substantially complementary non-limiting example. In some embodiments, the base constitutes a characteristic portion of a nucleic acid (e.g., a gene), wherein the portion is identical or complementary to a portion of a nucleic acid or transcript thereof, and is not identical or complementary to a portion of any other nucleic acid (e.g., a gene) or transcript thereof in the same genome. In some embodiments, a portion is characteristic of human HSD17B 13.

In some embodiments, as described herein, the provided oligonucleotides, e.g., ds oligonucleotides targeting HSD17B13, are no more than about 49, 45, 40, 30, 35, 25, or 23 total nucleotides in length. In some embodiments of the sequences described herein beginning at the 5' end with U or T, U may be deleted and/or substituted with another base. In some embodiments, the oligonucleotide has a base sequence that is, or comprises, a base sequence of, or comprises a portion of a base sequence of, an oligonucleotide in the table, wherein each T may be independently replaced with U, and vice versa, having a form disclosed herein or a portion of a form disclosed herein.

In some embodiments, the oligonucleotides, e.g., ds oligonucleotides targeting HSD17B13, are stereotactic. In some embodiments, the ds oligonucleotide targeting HSD17B13 is chirally controlled. In some embodiments, the ds oligonucleotide targeting HSD17B13 is chirally pure (or "sterically pure," "stereochemically pure"), wherein the oligonucleotide exists in a single stereoisomeric form (in many cases a single diastereomeric (or "diastereomeric")) form, as multiple chiral centers may be present in the oligonucleotide, e.g., at the linkage of phosphorus, sugar carbon, etc.). As will be appreciated by those skilled in the art, a chirally pure oligonucleotide is separated from its other stereoisomeric forms (to the extent that some impurities may be present, due to chemical and biological processes, selectivity and/or purification, etc., with little if any absolute completeness being achieved). In chirally pure oligonucleotides, each chiral center is independently defined in terms of its configuration (for chirally pure oligonucleotides, each internucleotide linkage is independently defined stereoscopically or chirally controlled). In contrast to chirally controlled and chirally pure oligonucleotides comprising stereodefining, a "racemic" (or "stereostochastic", "achiral controlled") oligonucleotide comprising chiral, bonding phosphorus (e.g., from traditional phosphoramidite oligonucleotide synthesis, wherein the absence of stereochemical control during the coupling step and in combination with conventional sulfidation (formation of a sterically random phosphorothioate internucleotide linkage) refers to a random mixture of stereoisomers (typically diastereomers, because there are multiple chiral centers in the oligonucleotide; for example, from conventional oligonucleotide preparation using reagents that are free of chiral elements other than nucleosides and bridging phosphorus), for a in which a is phosphorothioate internucleotide linkage (which comprises chiral phosphorus linkages), the racemic oligonucleotide preparation comprises four diastereomers [ ² =4, each of which may exist in one of two configurations (Sp or Rp) considering two chiral phosphorus linkages ]: a S A A S A, A S A A R A, A R A A S A and A R A A R A, wherein S represents an Sp phosphorothioate internucleotide linkage and R represents an Rp phosphorothioate internucleotide linkage for chiral purity oligonucleotides, such as A S A A S A, which exist in a single stereoisomeric form, and separated from other stereoisomers (e.g., diastereomers A. Sup. S A. Sup. R A, A. Sup. R A. Sup. S A and A. Sup. R A. Sup. R A).

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more stereotactic internucleotide linkages (a mixture of Rp and Sp linkage phosphorus at internucleotide linkages, e.g., from traditional achiral controlled oligonucleotide synthesis). In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises one or more (e.g., 1-50, 1-40, 1-30, 1-25, 1-20, 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 or more) chirally controlled internucleotide linkages (Rp or Sp linkage phosphorus at the internucleotide linkages, e.g., from chirally controlled oligonucleotide synthesis). In some embodiments, the internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the internucleotide linkages are stereospecific phosphorothioate internucleotide linkages. In some embodiments, the internucleotide linkage is a chirally controlled phosphorothioate internucleotide linkage.

The present disclosure provides, among other things, techniques for preparing chiral controlled (in some embodiments, stereochemically pure) oligonucleotides. In some embodiments, the oligonucleotide is stereochemically pure. In some embodiments, an oligonucleotide of the disclosure is 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%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure. In some embodiments, the internucleotide linkages of the oligonucleotide comprise or consist of: one or more (e.g., ,1-50、1-40、1-30、1-25、1-20、5-50、5-40、5-30、5-25、5-20、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 or more) chiral internucleotide linkages, each of which independently has a diastereomeric purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. In some embodiments, an oligonucleotide of the disclosure, e.g., a DS oligonucleotide targeting HSD17B13, has a diastereoisomeric purity of (DS) ^CIL, wherein DS is diastereoisomeric purity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% or more) as described in the disclosure, and CIL is the number of chiral controlled internucleotide linkages (e.g., 1-50、1-40、1-30、1-25、1-20、5-50、5-40、5-30、5-25、5-20、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 or more). In some embodiments, DS is 95% -100%. In some embodiments, each internucleotide linkage is independently chirally controlled, and CIL is the number of chirally controlled internucleotide linkages.

By way of example, certain dsoligonucleotides targeting HSD17B13 are listed in table 1 below, and comprise certain example base sequences, nucleobase modifications and patterns, sugar modifications and patterns, internucleotide linkages and patterns, linkage phosphorus stereochemistry and patterns, linkers and/or additional chemical moieties. Oligonucleotides, such as those in table 1, may be used to target HSD17B13 transcripts, such as to reduce the level of HSD17B13 transcripts and/or products thereof, among other things.

In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise the following base sequences, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotide linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and/or additional chemical moieties: WV-42589; WV-47139; WV-47159; WV-49590; or WV-49591. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising a base sequence, nucleobase modification and pattern thereof, sugar modification and pattern thereof, internucleotide linkages and pattern thereof, linkage phosphorus stereochemistry and pattern thereof, a linker, and/or additional chemical moieties: WV-47139; WV-47159; WV-49590; or WV-49591. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a passenger strand comprising the base sequence of WV-42589, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotide linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and/or additional chemical moieties. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising a base sequence, nucleobase modification and pattern thereof, sugar modification and pattern thereof, internucleotide linkages and pattern thereof, linkage phosphorus stereochemistry and pattern thereof, a linker, and/or additional chemical moieties: WV-47139; WV-47159; WV-49590; or WV-49591, and a passenger strand comprising the base sequence of WV-42589, nucleobase modifications and patterns thereof, sugar modifications and patterns thereof, internucleotide linkages and patterns thereof, linkage phosphorus stereochemistry and patterns thereof, linkers, and/or additional chemical moieties.

In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising a base sequence of WV-47139, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety, and a passenger strand comprising a base sequence of WV-42589, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising WV-47139 and a passenger strand comprising WV-42589.

In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising a base sequence of WV-47159, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety, and a passenger strand comprising a base sequence of WV-42589, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising WV-47159 and a passenger strand comprising WV-42589.

In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising a base sequence of WV-49590, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety, and a passenger strand comprising a base sequence of WV-42589, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising WV-49590 and a passenger strand comprising WV-42589.

In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising a base sequence of WV-49591, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety, and a passenger strand comprising a base sequence of WV-42589, a nucleobase modification and pattern thereof, a sugar modification and pattern thereof, an internucleotide linkage and pattern thereof, a linkage phosphorus stereochemistry and pattern thereof, a linker, and/or an additional chemical moiety. In certain exemplary embodiments, the ds oligonucleotides of the disclosure that target HSD17B13 comprise a guide strand comprising WV-49591 and a passenger strand comprising WV-42589.

Notice that:

The descriptions, base sequences and stereochemistry/bonding in table 1 may be divided into a plurality of lines due to their length. Unless otherwise indicated, all oligonucleotides in table 1 were single stranded. As understood by those of skill in the art, unless otherwise indicated (e.g., with r, m, etc.), nucleoside units are unmodified and contain unmodified nucleobases and 2' -deoxy sugars; unless otherwise indicated, linkages are natural phosphate linkages; and the acidic/basic groups independently may be present in the form of salts. If sugar is not specified, the sugar is a natural DNA sugar; and if internucleotide linkages are not specified, the internucleotide linkages are natural phosphate linkages. Alkyl moiety and modification:

m：2′-OMe；

f：2′-F；

O, PO: phosphoric acid diesters (phosphate esters). It may be a linkage or a terminal group (or a component thereof), such as a linkage between a linker and an oligonucleotide chain, an internucleotide linkage (natural phosphate linkage), or the like. The phosphodiester is generally indicated in the stereochemistry/bonding column with "O" and is generally not labeled in the description column (if it is a terminal group, e.g., a 5' terminal group, it is indicated in the description and is generally not indicated in the stereochemistry/bonding column); if no linkage is indicated in the description column, it is typically a phosphodiester unless otherwise indicated. Note that the phosphate linkage between the linker (e.g., L001) and the oligonucleotide chain may not be labeled in the descriptive column, but may be indicated with "O" in the stereochemistry/linkage column;

P, PS: phosphorothioates. It may be a terminal group (indicated in the description column and not generally indicated in stereochemistry/bonding if it is a terminal group, e.g., a 5' terminal group), or a bond, e.g., a bond between a linker (e.g., L001) and an oligonucleotide chain, an internucleotide bond (phosphorothioate internucleotide bond), etc.;

R, rp: phosphorothioate in the Rp configuration. Note that R in the description represents a single phosphorothioate linkage in the Rp configuration;

S, sp: phosphorothioates in the Sp configuration. Note that rs in the description represents a single phosphorothioate linkage in the Sp configuration;

x: a stereorandom phosphorothioate;

n001：

nX: a stereorandom n001;

nR or n001R: n001 in Rp configuration;

nS or n001S: n001 in Sp configuration;

n009：

nX: stereotactic n009;

nR or n009R: n009 in Rp configuration;

nS or n009S: n009 in Sp configuration;

n031：

nX: a stereorandom n031;

nR or n031R: n031 in Rp configuration;

nS or n031S: n031 in Sp configuration;

n033：

nX: a stereorandom n033;

nR or n033R: n033 in Rp configuration;

nS or n033S: n033 in Sp configuration;

n037：

nX: a stereorandom n037;

nR or n037R: n037 in Rp configuration;

nS or n037S: n037 in Sp configuration:

n046：

nX: a stereorandom n046;

nR or n046R: n046 in Rp configuration;

nS or n046S: n046 in Sp configuration;

n047：

nX: a stereorandom n047;

nR or n047R: n047 in Rp configuration;

nS or n047S: n047 in Sp configuration;

n025：

nX: a stereotactic n025;

nR or n025R: n025 in Rp configuration;

nS or n025S: n025 in Sp configuration;

n054：

nX: a stereorandom n054;

nR or n054R: n054 in Rp configuration;

nS or n054S: n054 in Sp configuration;

n055：

nX: a stereorandom n055;

nR or n055R: n055 in Rp configuration;

nS or n055S: n055 in Sp configuration;

n026：

nX: a stereorandom n001;

nR or n026R: n026 in Rp configuration;

nS or n026S: n026 in Sp configuration;

n004：

nX: a stereo random n004;

nR or n004R: n004 in Rp configuration;

nS or n004S: n004 in Sp configuration;

n003：

nX: a stereo random n003;

nR or n003R: n003 in Rp configuration;

nS or n003S: n003 in Sp configuration;

n008：

nX: a stereorandom n008;

nR or n008R: n008 in Rp configuration;

nS or n008S: n008 in Sp configuration;

n029：

nX: a stereotactic n029;

nR or n029R: n029 in Rp configuration;

nS or n029S: n029 in Sp configuration;

n021：

nX: a stereorandom n021;

nR or n02IR: n021 in Rp configuration;

nS or n021S: n021 in Sp configuration;

n006：

nX: a stereotactic n006;

nR or n006R: n006 in Rp configuration;

nS or n006S: n006 in Sp configuration;

n020：

nX: a stereorandom n020;

nR or n020R: n020 in Rp configuration;

nS or n020S: n020 in Sp configuration;

x: a stereorandom phosphorothioate;

(e.g.,

(E.g.,

n013：Wherein-C (O) -is bonded to nitrogen;

sm01n013： I.e., morpholinocarbamate internucleotide linkage (sm 01n 013)

Mod015：

Mod020：

Mod029：

L001: -NH- (CH ₂)₆ -linker (C6 linker, C6 amine linker or C6 amino linker) which is linked to Mod (e.g. Mod 001) by-NH-and in the case of e.g. WV-38061 to the 5' end of the oligonucleotide chain by a phosphate linkage (O or PO).

L010：In some embodiments, when L010 is present in the middle of the oligonucleotide, it is bonded to the internucleotide linkage as an additional sugar (e.g., a DNA sugar), e.g., its 5 '-carbon is linked to another unit (e.g., 3' of the sugar), and its 3 '-carbon is independently linked to another unit (e.g., 5' -carbon of the carbon), e.g., via a linkage (e.g., phosphate linkage (O or PO) or phosphorothioate linkage (which may not be chiral controlled or chiral (Sp or Rp)));

L012: -CH ₂CH₂OCH₂CH₂OCH₂CH₂ -. When L012 is present in the middle of the oligonucleotide, each of its two ends is independently bonded to an internucleotide linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be either not chiral controlled or chiral controlled (Sp or Rp)));

L022： Wherein L022 is linked to the remainder of the molecule by a phosphate unless otherwise specified;

L023: HO- (CH ₂)₆ -where CH ₂ is linked to the remainder of the molecule by phosphoric acid unless otherwise indicated-for example, in WV-42644 (where O in OnRnRnRnRSSSSSSSSSSSSSSSSSSnRSSSSSnRSSnR represents a phosphate linkage linking L023 to the remainder of the molecule);

L025： Wherein the —ch ₂ -attachment site serves as a C5 attachment site for a sugar (e.g., a DNA sugar) and is attached to another unit (e.g., 3 'of the sugar), and the attachment site on the loop serves as a C3 attachment site and is attached to another unit (e.g., 5' -carbon of the carbon), each independently attached, for example, via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may not be chiral controlled or chiral controlled (Sp or Rp)). When L025 is at the 5' end without any modification, its-CH ₂ -attachment site is bonded to-OH. For example, L025L025L 025-of the various oligonucleotides The structure of (may exist in various salt forms) and is linked to the 5' -carbon of the oligonucleotide chain via an indicated linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be either not chiral controlled or chiral controlled (Sp or Rp)));

L016： Wherein L016 is linked to the remainder of the molecule by a phosphate unless otherwise specified; l016 and n001 are combined into L016n001, which has the structure

Double-stranded oligonucleotide Length

As will be appreciated by those of skill in the art, ds oligonucleotides targeting HSD17B13 may be of various lengths to provide desired properties and/or activity for various uses. Many techniques for assessing, selecting, and/or optimizing oligonucleotide length are available in the art and may be used in accordance with the present disclosure. As demonstrated herein, in many embodiments, the ds oligonucleotide targeting HSD17B13 is of a suitable length to hybridize to its target and reduce the level of its target and/or its encoded product. In some embodiments, the oligonucleotide is long enough to recognize a target nucleic acid (e.g., HSD17B13 mRNA). In some embodiments, the oligonucleotides are long enough to distinguish between the target nucleic acid and other nucleic acids (e.g., nucleic acids having a base sequence other than HSD17B 13) to reduce off-target effects. In some embodiments, the ds oligonucleotide targeting HSD17B13 is short enough to reduce complexity of manufacture or production and reduce product costs.

In some embodiments, the oligonucleotide has a base sequence of about 10-500 nucleobases in length. In some embodiments, the base sequence is about 10-500 nucleobases in length. In some embodiments, the base sequence is about 10-50 nucleobases in length. In some embodiments, the base sequence is about 15-50 nucleobases in length. In some embodiments, the base sequence is about 15 to about 30 nucleobases in length. In some embodiments, the base sequence is about 10 to about 25 nucleobases in length. In some embodiments, the base sequence is about 15 to about 22 nucleobases in length. In some embodiments, the base sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases in length. In some embodiments, the base sequence is about 18 nucleobases in length. In some embodiments, the base sequence is about 19 nucleobases in length. In some embodiments, the base sequence is about 20 nucleobases in length. In some embodiments, the base sequence is about 21 nucleobases in length. In some embodiments, the base sequence is about 22 nucleobases in length. In some embodiments, the base sequence is about 23 nucleobases in length. In some embodiments, the base sequence is about 24 nucleobases in length. In some embodiments, the base sequence is about 25 nucleobases in length. In some embodiments, each nucleobase is an optionally substituted A, T, C, G, U, or an optionally substituted tautomer of A, T, C, G or U.

Internucleotide linkages of double-stranded oligonucleotides

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises a base modification, a sugar modification, and/or an internucleotide linkage modification. In accordance with the present disclosure, a variety of internucleotide linkages can be utilized to link nucleobase-containing units, such as nucleosides. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises both one or more modified internucleotide linkages and one or more native phosphate ester linkages. As is well known to those skilled in the art, natural phosphoester linkages are widely found in natural DNA and RNA molecules; they have the structure of-OP (O) (OH) O-and link the sugar in the nucleosides of DNA and RNA and can be in various salt forms, for example, at physiological pH (about 7.4), the natural phosphate linkages are predominantly in the form of salts having-OP (O) (O ^-) O-anions. Modified internucleotide linkages or non-natural phosphate linkages are internucleotide linkages which are not natural phosphate linkages or salt forms thereof. Modified internucleotide linkages may also be in their salt form, depending on their structure. For example, as will be appreciated by those skilled in the art, phosphorothioate internucleotide linkages having the structure-OP (O) (SH) O-may be in the form of various salts, such as at physiological pH (about 7.4), wherein the anion is-OP (O) (S ^-) O-.

In some embodiments, the oligonucleotide comprises an internucleotide linkage that is a modified internucleotide linkage, such as phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, phosphorothioate, 3 '-phosphorothioate, or 5' -phosphorothioate.

In some embodiments, the modified internucleotide linkage is a chiral internucleotide linkage comprising a chiral linkage phosphorus. In some embodiments, the chiral internucleotide linkage is a phosphorothioate linkage. In some embodiments, the chiral internucleotide linkages are nonnegatively charged internucleotide linkages. In some embodiments, the chiral internucleotide linkage is a neutral internucleotide linkage. In some embodiments, chiral internucleotide linkages are chirally controlled with respect to their chiral phosphorus linkages. In some embodiments, the chiral internucleotide linkages are stereochemically pure with respect to their chiral linked phosphites. In some embodiments, chiral internucleotide linkages are not chirally controlled. In some embodiments, the backbone chiral center pattern comprises or consists of: the position of chiral controlled internucleotide linkages (Rp or Sp) and the position of linkage phosphorus configuration and achiral internucleotide linkages (e.g., natural phosphate linkages).

In certain embodiments, the internucleotide linkage comprises a P-modification, wherein the P-modification is a modification at the linkage phosphorus. In certain embodiments, the modified internucleotide linkages are phosphorus-free moieties useful for linking two sugars or two moieties each independently comprising a nucleobase, e.g., in Peptide Nucleic Acids (PNAs).

In certain embodiments, ds oligonucleotides comprise modified internucleotide linkages, such as those ：WO 2018/022473、WO 2018/098264、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784 and/or WO 2019/03612 having the structure of formula I, I-a, I-b, or I-c described herein and/or in the following documents, the respective internucleotide linkages (e.g., those having the formula I, I-a, I-b, I-c, etc.) are independently incorporated herein by reference. In certain embodiments, the modified internucleotide linkage is a chiral internucleotide linkage. In certain embodiments, the modified internucleotide linkage is a phosphorothioate internucleotide linkage.

In certain embodiments, the modified internucleotide linkages are nonnegatively charged internucleotide linkages. In certain embodiments, provided ds oligonucleotides comprise one or more non-negatively charged internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages are positively charged internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages are neutral internucleotide linkages. In certain embodiments, the disclosure provides ds oligonucleotides comprising one or more neutral internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages have the structure of formula I-n-1, I-n-2, I-n-3, I-n-4, 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., or salt forms thereof, as described herein and/or in the following documents ：US 9394333、US 9744183、US 9605019、US 9982257、US 20170037399、US 20180216108、US 20180216107、US 9598458、WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/022473、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/032612、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784 and/or WO 2019/03612, each of which is independently incorporated herein by reference (e.g., with those of formula I-n-1, I-n-2, I-n-3, I-n-4, 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., or suitable salt forms thereof).

In certain embodiments, non-negatively charged internucleotide linkages may improve delivery and/or activity (e.g., adenosine editing activity).

In certain embodiments, the modified internucleotide linkages (e.g., internucleotide linkages not having a negative charge) comprise an optionally substituted triazolyl group. In certain embodiments, the modified internucleotide linkages (e.g., non-negatively charged internucleotide linkages) comprise optionally substituted alkynyl groups. In certain embodiments, the modified internucleotide linkage comprises a triazole or alkyne moiety. In certain embodiments, the triazole moiety (e.g., triazolyl) is optionally substituted. In certain embodiments, the triazole moiety (e.g., triazolyl) is substituted. In certain embodiments, the triazole moiety is unsubstituted. In certain embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety. In certain embodiments, the modified internucleotide linkages haveWherein R ¹ is-L-R ', wherein L is L ^B as described herein, and R' is as described herein, and optionally chirally controlled. In certain embodiments, each R ¹ is independently R'. In certain embodiments, each R' is independently R. In certain embodiments, two R ¹ are R and taken together form a ring as described herein. In certain embodiments, two R ¹ on two different nitrogen atoms are R and taken together form a ring as described herein. In certain embodiments, R ¹ is independently optionally substituted C _1-6 aliphatic as described herein. In certain embodiments, R ¹ is methyl. In certain embodiments, two R' on the same nitrogen atom are R and taken together form a ring as described herein. In certain embodiments, the modified internucleotide linkages haveAnd optionally chirally controlled. In some embodiments of the present invention, in some embodiments,Is thatIn certain embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety, and has the structure: wherein W is O or S. In certain embodiments, W is O. In certain embodiments, W is S. In certain embodiments, the internucleotide linkages not bearing a negative charge are stereochemically controlled.

In certain embodiments, the non-negatively charged internucleotide linkage or neutral internucleotide linkage is an internucleotide linkage comprising a triazole moiety. In some embodiments, the internucleotide linkages comprising a triazole moiety (e.g., optionally substituted triazolyl) haveIs a structure of (a). In some embodiments, the internucleotide linkages comprising a triazole moiety haveIs a structure of (a). In some embodiments, the internucleotide linkages comprising a triazole moiety haveWherein W is O or S. In some embodiments, the internucleotide linkage comprising an alkyne moiety (e.g., optionally substituted alkynyl) hasWherein W is O or S. In some embodiments, the internucleotide linkages (e.g., non-negatively charged internucleotide linkages, neutral internucleotide linkages) comprise a cyclic guanidine moiety. In some embodiments, the internucleotide linkage comprising a cyclic guanidine moiety hasIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkage or neutral internucleotide linkage is or comprises a structure selected from the group consisting of: Wherein W is O or S. In certain embodiments, internucleotide linkages, e.g., nonnegatively charged internucleotide linkages, neutral internucleotide linkages, comprise cyclic guanidine moieties. In certain embodiments, the internucleotide linkages comprising a cyclic guanidine moiety have Is a structure of (a). In certain embodiments, the non-negatively charged internucleotide linkage or neutral internucleotide linkage is or comprises a structureWherein W is O or S.

In certain embodiments, the internucleotide linkages comprise a Tmg groupIn certain embodiments, the internucleotide linkages contain a Tmg group and haveThe structure of (Tmg internucleotide linkage). In certain embodiments, neutral internucleotide linkages include PNA and PMO internucleotide linkages, and Tmg internucleotide linkages.

In certain embodiments, the non-negatively charged internucleotide linkage has the structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, 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., or a salt form thereof. In certain embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, such heterocyclyl or heteroaryl groups have a 5 membered ring. In certain embodiments, such heterocyclyl or heteroaryl groups have a 6 membered ring.

In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the heteroaryl is directly bonded to the phosphorus linkage.

In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heterocyclyl having 1-10 heteroatoms. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heterocyclyl having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-6 membered heterocyclyl having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-membered heterocyclyl having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In certain embodiments, at least two heteroatoms are nitrogen. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted triazolyl group. In some embodiments, the non-negatively charged internucleotide linkages comprise an unsubstituted triazolyl group, e.g.,In some embodiments, the non-negatively charged internucleotide linkages comprise a substituted triazolyl group, e.g.,

In certain embodiments, the heterocyclyl is directly bonded to the phosphorus linkage. In certain embodiments, when the heterocyclyl is part of a guanidine moiety that is directly bonded to the phosphorus linkage via its = N-, the heterocyclyl is bonded to the phosphorus linkage via a linker (e.g., = N-). In certain embodiments, the non-negatively charged internucleotide linkages comprise optionally substitutedA group. In certain embodiments, the non-negatively charged internucleotide linkages comprise a substitutedA group. In certain embodiments, the non-negatively charged internucleotide linkages compriseA group, wherein each R ¹ is independently-L-R. In certain embodiments, each R ¹ is independently optionally substituted C _1-6 alkyl. In certain embodiments, each R ¹ is independently methyl.

In certain embodiments, the modified internucleotide linkages (e.g., non-negatively charged internucleotide linkages) comprise triazole or alkyne moieties, each of which is optionally substituted. In certain embodiments, the modified internucleotide linkage comprises a triazole moiety. In certain embodiments, the modified internucleotide linkages comprise an unsubstituted triazole moiety. In certain embodiments, the modified internucleotide linkage comprises a substituted triazole moiety. In certain embodiments, the modified internucleotide linkages comprise an alkyl moiety. In certain embodiments, the modified internucleotide linkage comprises an optionally substituted alkynyl group. In certain embodiments, the modified internucleotide linkages comprise unsubstituted alkynyl groups. In certain embodiments, the modified internucleotide linkage comprises a substituted alkynyl group. In certain embodiments, the alkynyl group is directly bonded to the phosphorus linkage.

In certain embodiments, ds oligonucleotides comprise different types of internucleotide phosphate linkages. In certain embodiments, the chirally controlled oligonucleotides comprise at least one natural phosphate linkage and at least one modified (non-natural) internucleotide linkage. In certain embodiments, the ds oligonucleotide comprises at least one natural phosphate linkage and at least one phosphorothioate. In certain embodiments, the ds oligonucleotide comprises at least one non-negatively charged internucleotide linkage. In certain embodiments, the ds oligonucleotides comprise at least one natural phosphate linkage and at least one non-negatively charged internucleotide linkage. In certain embodiments, the ds oligonucleotides comprise at least one phosphorothioate internucleotide linkage and at least one nonnegatively charged internucleotide linkage. In certain embodiments, the ds oligonucleotides comprise at least one phosphorothioate internucleotide linkage, at least one natural phosphate linkage, and at least one nonnegatively charged internucleotide linkage. In certain embodiments, the ds oligonucleotide comprises one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-15, 1-10,1, 2,3,4, 5,6, 7, 8,9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) non-negatively charged internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages are non-negatively charged, in that less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the internucleotide linkages are present as negatively charged salts at a given pH in aqueous solution. In certain embodiments, the pH is about pH 7.4. In certain embodiments, the pH is about 4-9. In certain embodiments, the percentage is less than 10%. In certain embodiments, the percentage is less than 5%. In certain embodiments, the percentage is less than 1%. In certain embodiments, the internucleotide linkages are nonnegatively charged internucleotide linkages, in that the neutral form of the internucleotide linkages does not have a pKa in water of no more than about 1,2,3,4, 5,6, or 7. In certain embodiments, none of the pKa is 7 or less. In certain embodiments, none of the pKa is 6 or less. In certain embodiments, none has a pKa of 5 or less. In certain embodiments, none of the pKa is 4 or less. In certain embodiments, none has a pKa of 3 or less. In certain embodiments, none has a pKa of 2 or less. In certain embodiments, none of the pKa is 1 or less. In certain embodiments, the pKa of the neutral form of the internucleotide linkage may be expressed as the pKa of the neutral form of the compound having the structure CH ₃ -internucleotide linkage-CH ₃. For example, the neutral form of the internucleotide linkage having the structure of formula I may be composed of a peptide having the formulaPKa representation of the neutral form of the compound of the structure of (wherein X, Y, Z are each independently-O-, -S-, -N (R ') -; L is L ^B, and R ¹ is-L-R'),The pKa of (2) may be defined byIs represented by pKa. In certain embodiments, the non-negatively charged internucleotide linkages are neutral internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages are positively charged internucleotide linkages. In certain embodiments, the non-negatively charged internucleotide linkages comprise a guanidine moiety. In certain embodiments, the non-negatively charged internucleotide linkages comprise a heteroaryl base moiety. In certain embodiments, the non-negatively charged internucleotide linkages comprise a triazole moiety. In certain embodiments, the non-negatively charged internucleotide linkage comprises an alkynyl moiety.

In certain embodiments, neutral or non-negatively charged internucleotide linkages have the structure ：US 9394333、US 9744183、US 9605019、US 9982257、US 20170037399、US 20180216108、US 20180216107、US 9598458、WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/022473、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO2019/032612、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612, 2607, WO 2019032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/or WO 2019/03612 of any of the neutral or non-negatively charged internucleotide linkages described in any of the following documents, each of which is hereby incorporated by reference.

In certain embodiments, each R' is independently an optionally substituted C _1-6 aliphatic. In certain embodiments, each R' is independently optionally substituted C _1-6 alkyl. In certain embodiments, each R' is independently-CH ₃. In certain embodiments, each R ^s is-H.

In certain embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In certain embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In certain embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkages haveIs a structure of (a). In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the neutral internucleotide linkages are nonnegatively charged internucleotide linkages as described above.

In certain embodiments, provided oligonucleotides comprise 1 or more internucleotide linkages having the formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or II-d-2, as described in ：US 9394333、US 9744183、US 9605019、US 9982257、US 20170037399、US 20180216108、US 20180216107、US 9598458、WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/022473、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO2019/032612、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612, 2607, WO 2019032612, WO 2019/055951, WO 2019/075357, WO 2019/200185, WO 2019/217784, and/or WO 2019/032512 (formulae I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II-a-1, II-a-2, II-b-1, II-b-2, or II-d-2, each of which are independently incorporated herein by reference, or a salt thereof.

In certain embodiments, the ds oligonucleotides comprise neutral internucleotide linkages and chirally controlled internucleotide linkages. In certain embodiments, the ds oligonucleotides comprise neutral internucleotide linkages and chirally controlled internucleotide linkages other than neutral internucleotide linkages. In certain embodiments, the ds oligonucleotides comprise neutral internucleotide linkages and chirally controlled phosphorothioate internucleotide linkages. In certain embodiments, the disclosure provides ds oligonucleotides comprising one or more non-negatively charged internucleotide linkages and one or more phosphorothioate internucleotide linkages, wherein each phosphorothioate internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In certain embodiments, the present disclosure provides ds oligonucleotides comprising one or more neutral internucleotide linkages and one or more phosphorothioate internucleotide linkages, wherein each phosphorothioate internucleotide linkage in the ds oligonucleotide is independently a chirally controlled internucleotide linkage. In certain embodiments, the ds oligonucleotide comprises at least 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more chirally controlled phosphorothioate internucleotide linkages. In certain embodiments, the internucleotide linkages not bearing a negative charge are chirally controlled. In certain embodiments, the internucleotide linkages not bearing a negative charge are not chirally controlled. In certain embodiments, the neutral internucleotide linkages are chirally controlled. In certain embodiments, neutral internucleotide linkages are not chirally controlled.

Without wishing to be bound by any particular theory, the present disclosure indicates that neutral internucleotide linkages may be more hydrophobic than phosphorothioate internucleotide linkages (PS), while phosphorothioate internucleotide linkages may be more hydrophobic than natural phosphate linkages (PO). Typically, unlike PS or PO, neutral internucleotide linkages have less charge. Without wishing to be bound by any particular theory, the present disclosure teaches that incorporating one or more neutral internucleotide linkages into a ds oligonucleotide can increase the ability of the ds oligonucleotide to be taken up by cells and/or to escape from endosomes. Without wishing to be bound by any particular theory, the present disclosure teaches that incorporation of one or more neutral internucleotide linkages can be used to regulate the melting temperature of the duplex formed between the ds oligonucleotide and its target nucleic acid.

Without wishing to be bound by any particular theory, the present disclosure teaches that incorporating one or more non-negatively charged internucleotide linkages (e.g., neutral internucleotide linkages) into a ds oligonucleotide may be capable of increasing the ability of the ds oligonucleotide to mediate functions such as target adenosine editing.

As will be appreciated by those of skill in the art, internucleotide linkages such as natural phosphate linkages and those having the formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or salt forms thereof, typically link two nucleosides (which may be natural or modified), as described in ：US9394333、US 9744183、US 9605019、US 9982257、US 20170037399、US 20180216108、US 20180216107、US 9598458、WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/022473、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019032612、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784 and/or WO 2019/03612 (formulae I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, I-n-4, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, or salt forms thereof, each of which are incorporated herein by reference. Typical linkages as in natural DNA and RNA are internucleotide linkages forming a bond with two sugars (which may be unmodified or modified as described herein). In many embodiments, as exemplified herein, the internucleotide linkage forms a bond through its oxygen or heteroatom (e.g., Y and Z in the formulae) with one optionally modified ribose or deoxyribose at its 5 'carbon and another optionally modified ribose or deoxyribose at its 3' carbon. In certain embodiments, each nucleoside unit linked by internucleotide linkages independently comprises a nucleobase that is independently an optionally substituted A, T, C, G or U or A, T, C, G or a substituted tautomer of U, or a nucleobase comprising an optionally substituted heterocyclyl and/or heteroaryl ring having at least one nitrogen atom.

In some embodiments, the linkage has or comprises a structure of-Y-P ^L(-X-R^L) -Z-, or a salt form thereof, wherein:

P ^L is P, P (=w), P- > B (-L ^L-R^L)₃, or P ^N;

W is O, N (-L ^L-R^L), S, or Se;

P ^N is p=n-C (-L ^L-R')(＝L^N -R') or p=n-L ^L-R^L;

l ^N is =n-L ^L1-、＝CH-L^L1 - (wherein CH is optionally substituted), or =n ⁺(R')(Q^-)-L^L1 -;

Q ^- is an anion;

each of X, Y and Z is independently -O-、-S-、-L^L-N(-L^L-R^L)-L^L-、-L^L-N＝C(-L^L-R^L)-L^L-、 or L ^L;

Each R ^L is independently -L^L-N(R')₂、-L^L-R'、-N＝C(-L^L-R')₂、-L^L-N(R')C(NR')N(R')₂、-L^L-N(R')C(O)N(R')₂、 carbohydrate, or one or more additional chemical moieties optionally linked by a linker;

L ^L1 and L ^L are each independently L;

-Cy ^IL -is-Cy-;

Each L is independently a covalent bond, or a divalent, optionally substituted, linear or branched group selected from the group consisting of a C _1-30 aliphatic group and a C ₁-₃₀ heteroaliphatic group having 1 to 10 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted group selected from the group consisting of: c _1-6 alkylene, C _1-6 alkenylene, -C≡C-, a divalent C ₁-C₆ heteroaliphatic group 、-C(R')₂-、-Cy-、-O-、-S-、-S-S-、-N(R')-、-C(O)-、-C(S)-、-C(NR')-、-C(NR')N(R')-、-N(R')C(NR')N(R')-、-C(O)N(R')-、-N(R')C(O)N(R')-、-N(R')C(O)O-、-S(O)-、-S(O)₂-、-S(O)₂N(R')-、-C(O)S-、-C(O)O-、-P(O)(OR')-、-P(O)(SR')-、-P(O)(R')-、-P(O)(NR')-、-P(S)(OR')-、-P(S)(SR')-、-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-、-OP(OR')[B(R')₃]O-、 having 1-5 heteroatoms, and- [ C (R') ₂C(R')₂ O ] n-, wherein n is 1-50, and one or more nitrogen or carbon atoms are optionally and independently replaced by Cy ^L;

each-Cy-is independently an optionally substituted divalent 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;

each Cy ^L is independently an optionally substituted trivalent or tetravalent 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;

Each R' is independently-R, -C (O) N (R) ₂, -C (O) OR, OR-S (O) ₂ R;

Each R is independently-H, or an optionally substituted group selected from: c _1-30 aliphatic, C _1-30 heteroaliphatic having 1 to 10 heteroatoms, C _6-30 aryl, C _6-30 arylaliphatic, C _6-30 arylaliphatic having 1 to 10 heteroatoms, 5 to 30 membered heteroaryl having 1 to 10 heteroatoms, and 3 to 30 membered heterocyclyl having 1 to 10 heteroatoms, or

The two R groups optionally and independently taken together form a covalent bond, or

Two or more R groups on the same atom optionally and independently form together with the atom an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atom; or alternatively

Two or more R groups on two or more atoms optionally and independently form together with the atoms interposed between them an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms interposed between them.

In some embodiments, the internucleotide linkages have the structure-O-P ^L(-X-R^L) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkages have the structure-O-P (=w) (-X-R ^L) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-O-P (=w) [ -N (-L ^L-R^L)-R^L ] -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-O-P (=w) (-NH-L ^L-R^L) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-O-P (=w) [ -N (R') ₂ ] -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkages have the structure-O-P (=w) (-NHR') -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-O-P (=w) (-NHSO ₂ R) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkages have the structure of-O-P (=w) [ -n=c (-L ^L-R')₂ ] -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-OP (=w) (-n=c (R ") ₂) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-OP (=w) (-N (R ") ₂) -O-, wherein each variable is independently as described herein. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the neutral internucleotide linkages are nonnegatively charged internucleotide linkages. In some embodiments, such internucleotide linkages are neutral internucleotide linkages.

In some embodiments, the internucleotide linkages have the structure-P ^L(-X-R^L) -Z-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkages have the structure-P ^L(-X-R^L) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=w) (-X-R ^L) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkages have the structure of-P (=W) [ -N (-L ^L-R^L)-R^L ] -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=w) (-NHR') -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=w) (-NHSO ₂ R) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=w) [ -n=c (-L ^L-R')₂ ] -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=w) [ -n=c [ N (R') ₂]₂ ] -O-, wherein each variable is independent, as described herein. In some embodiments, the internucleotide linkages have the structure-P (=w) (-n=c (R ") ₂) -O-, wherein each variable is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=w) (-N (R ") ₂) -O-, wherein each variable is independently as described herein. in some embodiments, W is O. In some embodiments, W is S. In some embodiments, the neutral internucleotide linkages are nonnegatively charged internucleotide linkages. In some embodiments, such internucleotide linkages are neutral internucleotide linkages. In some embodiments, such internucleotide-bonded P is bonded to the N of the sugar.

In some embodiments, the linkage is a phosphorylguanidine internucleotide linkage. In some embodiments, the linkage is a phosphorothioate guanidine internucleotide linkage.

In some embodiments, one or more methylene units are optionally and independently replaced with a moiety as described herein. In some embodiments, L or L ^L is or comprises-SO ₂ -. In some embodiments, L or L ^L is or comprises-SO ₂ N (R') -. in some embodiments, L or L ^L is or comprises-C (O) -. In some embodiments, L or L ^L is or comprises-C (O) O-. In some embodiments, L or L ^L is or comprises-C (O) N (R') -. in some embodiments, L or L ^L is or comprises-P (=w) (R') -. In some embodiments, L or L ^L is or comprises-P (=o) (R') -. In some embodiments, L or L ^L is or comprises-P (=s) (R') -. In some embodiments, L or L ^L is or comprises-p (R') -. In some embodiments, L OR L ^L is OR comprises-P (=w) (OR') -. In some embodiments, L OR L ^L is OR comprises-p (=o) (OR') -. In some embodiments, L OR L ^L is OR comprises-P (=s) (OR') -. In some embodiments, L OR L ^L is OR comprises-P (OR') -.

In some embodiments, -X-R ^L is-N (R') SO ₂R^L. In some embodiments, -X-R ^L is-N (R') C (O) R ^L. In some embodiments, -X-R ^L is-N (R ') P (=o) (R') R ^L.

In some embodiments, the linkage, e.g., a non-negatively charged internucleotide linkage or a neutral internucleotide linkage, has the following structure or comprises the following ：-P(＝W)(-N＝C(R")₂)-、-P(＝W)(-N(R')SO₂R")、-P(＝W)(-N(R')C(O)R")-、-P(＝W)(-N(R")₂)-、-P(＝W)(-N(R')P(O)(R")₂)-、-OP(＝W)(-N＝C(R")₂)O-、-OP(＝W)(-N(R')SO₂R")O-、-OP(＝W)(-N(R')C(O)R")O-、-OP(＝W)(-N(R")₂)O-、-OP(＝W)(-N(R')P(O)(R")₂)O-、-P(＝W)(-N＝C(R")₂)O-、-P(＝W)(-N(R')SO₂R")O-、-P(＝W)(-N(R')C(O)R")O-、-P(＝W)(-N(R")₂)O-、 or P (=w) (-N (R') P (O) (R ") ₂) O-, or salt forms thereof, wherein:

W is O or S;

Each R "is independently R ', OR', -P (=w) (R ') ₂, OR N (R') ₂;

Each R' is independently-R, -C (O) N (R) ₂, -C (O) OR, OR-S (O) ₂ R;

In some embodiments, W is O. In some embodiments, the internucleotide linkage has the structure -P(＝O)(-N＝C(R")₂)-、-P(＝O)(-N(R')SO₂R")-、-P(＝O)(-N(R')C(O)R")-、-P(＝O)(-N(R")₂)-、-P(＝O)(-N(R')P(O)(R")₂)-、-OP(＝O)(-N＝C(R")₂)O-、-OP(＝O)(-N(R')SO₂R")O-、-OP(＝O)(-N(R')C(O)R")O-、-OP(＝O)(-N(R")₂)O-、-OP(＝O)(-N(R')P(O)(R")₂)O-、-P(＝O)(-N＝C(R")₂)O-、-P(＝O)(-N(R')SO₂R")O-、-P(＝O)(-N(R')C(O)R")O-、-P(＝O)(-N(R")₂)O- or-P (=o) (-N (R') P (O) (R ") ₂) O-, or a salt form thereof. In some embodiments, the internucleotide linkage has a structure of -P(＝O)(-N＝C(R")₂)- -P(＝O)(-N(R")₂)-、-OP(＝O)(-N＝C(R")₂)-O-、-OP(＝O)(-N(R")₂)-O-、-P(＝O)(-N＝C(R")₂)-O- or-P (=o) (-N (R ") ₂) -O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-n=c (R ") ₂) -O-or-OP (=o) (-N (R") ₂) -O-or a salt form thereof. In some embodiments, the internucleotide linkage has the structure-OP (=o) (-n=c (R ") ₂) -O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure-OP (=o) (-N (R ") ₂) -O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-N (R') SO ₂ R ") O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-N (R') C (O) R ") O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-N (R') P (O) (R ") ₂) O-, or a salt form thereof. In some embodiments, the internucleotide linkage is n001.

In some embodiments, W is S. In some embodiments, the internucleotide linkage has a structure of -P(＝S)(-N＝C(R")₂)-、-P(＝S)(-N(R')SO₂R")-、-P(＝S)(-N(R')C(O)R")-、-P(＝S)(-N(R")₂)-、-P(＝S)(-N(R')P(O)(R")₂)-、-OP(＝S)(-N＝C(R")₂)O-、-OP(＝S)(-N(R')SO₂R")O-、-OP(＝S)(-N(R')C(O)R")O-、-OP(＝S)(-N(R")₂)O-、-OP(＝S)(-N(R')P(O)(R")₂)O-、-P(＝S)(-N＝C(R")₂)O-、-P(＝S)(-N(R')SO₂R")O-、-P(＝S)(-N(R')C(O)R")O-、-P(＝S)(-N(R")₂)O-、 or-P (=s) (-N (R') P (O) (R ") ₂) O-, or a salt form thereof. In some embodiments, the internucleotide linkage has a structure of -P(＝S)(-N＝C(R")₂)- -P(＝S)(-N(R")₂)-、-OP(＝S)(-N＝C(R")₂)-O-、-OP(＝S)(-N(R")₂)-O-、-P(＝S)(-N＝C(R")₂)-O- or-P (=s) (-N (R ") ₂) -O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure-OP (=s) (-n=c (R ") ₂) -O-or-OP (=s) (-N (R") ₂) -O-or a salt form thereof. In some embodiments, the internucleotide linkage has the structure-OP (=s) (-n=c (R ") ₂) -O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure-OP (=s) (-N (R ") ₂) -O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=s) (-N (R') SO ₂ R ") O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=s) (-N (R') C (O) R ") O-, or a salt form thereof. In some embodiments, the internucleotide linkage has the structure of-OP (=s) (-N (R') P (O) (R ") ₂) O-, or a salt form thereof. In some embodiments, the internucleotide linkage is n001.

In some embodiments, the internucleotide linkage has the structure-P (=o) (-N (R') SO ₂ R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure-P (=s) (-N (R') SO ₂ R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=o) (-N (R') SO ₂ R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=s) (-N (R') SO ₂ R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-N (R') SO ₂ R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-OP (=s) (-N (R') SO ₂ R ") O-, wherein R" is as described herein. In some embodiments, for example, R 'of-N (R') -is hydrogen or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is C _1-6 alkyl. In some embodiments, R' is hydrogen. In some embodiments, R "(e.g., in-SO ₂ R") is R' as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=o) (-NHSO ₂ R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=s) (-NHSO ₂ R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=o) (-NHSO ₂ R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=s) (-NHSO ₂ R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-NHSO ₂ R ") O one, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-OP (=s) (-NHSO ₂ R ") O-, wherein R" is as described herein. In some embodiments, -X-R ^L is-N (R ') SO ₂R^L, wherein each of R' and R ^L is independently as described herein. In some embodiments, R ^L is R. In some embodiments, R ^L is R'. In some embodiments, -X-R ^L is-N (R ') SO ₂ R ", wherein R' is as described herein. In some embodiments, -X-R ^L is-N (R ') SO ₂ R ', wherein R ' is as described herein. In some embodiments, -X-R ^L is-NHSO ₂ R ', where R' is as described herein. In some embodiments, R' is R as described herein. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is optionally substituted C _1-6 alkyl. In some embodiments, R' is optionally substituted phenyl. In some embodiments, R' is optionally substituted heteroaryl. In some embodiments, R "(e.g., in-SO ₂ R") is R. In some embodiments, R is an optionally substituted group selected from C _1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted C _1-6 alkenyl. In some embodiments, R is optionally substituted C _1-6 alkynyl. In some embodiments, R is optionally substituted methyl. In some embodiments, -X-R ^L is-NHSO ₂CH₃. In some embodiments, R is-CF ₃. In some embodiments, R is methyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is-CH ₂CHF₂. In some embodiments, R is-CH ₂CH₂OCH₃. In some embodiments, R is optionally substituted propyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is n-butyl. In some embodiments, R is- (CH ₂)₆NH₂). In some embodiments, R is an optionally substituted straight chain C _2-20 aliphatic. In some embodiments, R is an optionally substituted linear C _2-20 alkyl. In some embodiments, R is a linear C _2-20 alkyl group. In some embodiments, R is optionally substituted C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ aliphatic. in some embodiments, R is optionally substituted C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl. In some embodiments, R is optionally substituted straight chain C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl. In some embodiments, R is a linear C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl group. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is 4-dimethylaminophenyl. In some embodiments, R is 3-pyridinyl. In some embodiments, R isIn some embodiments, R isIn some embodiments, R is benzyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted 1, 3-diazolyl. In some embodiments, R is optionally substituted 2- (1, 3) -diazolyl. In some embodiments, R is optionally substituted 1-methyl-2- (1, 3) -diazolyl. In some embodiments, R is isopropyl. In some embodiments, R 'is-N (R') ₂. In some embodiments, R "is-N (CH ₃)₂. In some embodiments, R "(e.g., in-SO ₂ R") is-OR ', where R' is as described herein. In some embodiments, R' is R as described herein. In some embodiments, R "is-OCH ₃. In some embodiments, the linkage is-OP (=o) (-NHSO ₂ R) O-, wherein R is as described herein. In some embodiments, R is optionally substituted straight chain alkyl as described herein. In some embodiments, R is a linear alkyl group as described herein. In some embodiments, the linkage is-OP (=o) (-NHSO ₂CH₃) O-. In some embodiments, the linkage is-OP (=o) (-NHSO ₂CH₂CH₃) O-. In some embodiments, the linkage is-OP (=o) (-NHSO ₂CH₂CH₂OCH₃) O-. In some embodiments, the linkage is-OP (=o) (-NHSO ₂CH₂ Ph) O-. In some embodiments, the linkage is-OP (=o) (-NHSO ₂CH₂CHF₂) O-. In some embodiments, the linkage is-OP (=o) (-NHSO ₂ (4-methylphenyl)) O-. In some embodiments, -X-R ^L isIn some embodiments, the linkage is-OP (=o) (-X-R ^L) O-, where-X-R ^L isIn some embodiments, the linkage is-OP (=o) (-NHSO ₂CH(CH₃)₂) O-. In some embodiments, the linkage is-OP (=o) (-NHSO ₂N(CH₃)₂) O-.

In some embodiments, the internucleotide linkages have the structure-P (=o) (-N (R') C (O) R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkages have the structure-P (=s) (-N (R') C (O) R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkages have the structure-P (=o) (-N (R') C (O) R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkages have the structure-P (=s) (-N (R') C (O) R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkages have the structure-OP (=o) (-N (R') C (O) R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkages have the structure-OP (=s) (-N (R') C (O) R ") O-, wherein R" is as described herein. In some embodiments, for example, R 'of-N (R') -is hydrogen or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is C _1-6 alkyl. In some embodiments, R' is hydrogen. In some embodiments, R "(e.g., in-C (O) R") is R' as described herein. In some embodiments, the internucleotide linkage has the structure-P (=o) (-NHC (O) R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure-P (=s) (-NHC (O) R ") -wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=o) (-NHC (O) R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-P (=s) (-NHC (O) R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-OP (=o) (-NHC (O) R ") O-, wherein R" is as described herein. In some embodiments, the internucleotide linkage has the structure of-OP (=s) (-NHC (O) R ") O-, wherein R" is as described herein. In some embodiments, -X-R ^L is-N (R') COR ^L, wherein R ^L is as described herein. In some embodiments, -X-R ^L is-N (R') COR ", wherein R" is as described herein. In some embodiments, -X-R ^L is-N (R ') COR ', wherein R ' is as described herein. In some embodiments, -X-R ^L is-NHCOR ', where R' is as described herein. In some embodiments, R' is R as described herein. In some embodiments, R' is an optionally substituted C _1-6 aliphatic. In some embodiments, R' is optionally substituted C _1-6 alkyl. In some embodiments, R' is optionally substituted phenyl. In some embodiments, R' is optionally substituted heteroaryl. In some embodiments, R "(e.g., in-C (O) R") is R. In some embodiments, R is an optionally substituted group selected from C _1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted C _1-6 alkenyl. In some embodiments, R is optionally substituted C _1-6 alkynyl. In some embodiments, R is methyl. In some embodiments, -X-R ^L is-NHC (O) CH ₃. In some embodiments, R is optionally substituted methyl. In some embodiments, R is-CF ₃. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is-CH ₂CHF₂. In some embodiments, R is-CH ₂CH₂OCH₃. In some embodiments, R is an optionally substituted C _1-20 (e.g., ,C_1-6、C_2-6、C_3-6、C_1-10、C_2-10、C_3-10、C_2-20、C_3-20、C_10-20、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、 or 20, etc.) aliphatic. In some embodiments, R is optionally substituted C _1-20 (e.g., ,C_1-6、C_2-6、C_3-6、C_1-10、C_2-10、C_3-10、C_2-20、C_3-20、C_10-20、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、 or 20, etc.) alkyl. In some embodiments, R is an optionally substituted straight chain C _2-20 aliphatic. In some embodiments, R is an optionally substituted linear C _2-20 alkyl. In some embodiments, R is a linear C _2-20 alkyl group. In some embodiments, R is optionally substituted C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ aliphatic. in some embodiments, R is optionally substituted C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl. In some embodiments, R is optionally substituted straight chain C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl. In some embodiments, R is a linear C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl group. In some embodiments, R is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is benzyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted 1, 3-diazolyl. In some embodiments, R is optionally substituted 2- (1, 3) -diazolyl. In some embodiments, R is optionally substituted 1-methyl-2- (1, 3) -diazolyl. In some embodiments, R ^L is- (CH ₂)₅NH₂) in some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R 'is-N (R') ₂. In some embodiments, R 'is-N (CH ₃)₂. In some embodiments, -X-R ^L is-N (R') CON (R ^L)₂, wherein R 'and R ^L are independently as described herein, in some embodiments, -X-R ^L is-NHCON (R ^L)₂, in some embodiments, two R' or two R ^L together with the nitrogen atom to which they are attached form a ring as described herein, e.g., optionally substituted In some embodiments, R "(e.g., in-C (O) R") is-OR ', wherein R' is as described herein. In some embodiments, R' is R as described herein. In some embodiments, an optionally substituted C _1-6 aliphatic. In some embodiments, an optionally substituted C _1-6 alkyl. In some embodiments, R "is-OCH ₃. In some embodiments, -X-R ^L is-N (R ') C (O) OR ^L, wherein each of R' and R ^L is independently as described herein. In some embodiments, R isIn some embodiments, -X-R ^L is-NHC (O) OCH ₃. In some embodiments, -X-R ^L is-NHC (O) N (CH ₃)₂). In some embodiments, the linkage is-OP (O) (NHC (O) CH ₃) O-. In some embodiments, the linkage is-OP (O) (NHC (O) OCH ₃) O-. In some embodiments, the linkage is-OP (O) (NHC (O) (p-methylphenyl)) O-. In some embodiments, the linkage is-OP (O) (NHC (O) N (CH ₃)₂) O-. In some embodiments, -X-R ^L is-N (R ') R ^L, wherein each of R' and R ^L is independently as described herein. In some embodiments, -X-R ^L is-N (R ') R ^L, wherein each of R' and R ^L is independently not hydrogen. In some embodiments, -X-R ^L is-NHR ^L, wherein R ^L is as described herein. In some embodiments, R ^L is not hydrogen. In some embodiments, R ^L is optionally substituted aryl or heteroaryl. In some embodiments, R ^L is optionally substituted aryl. In some embodiments, R ^L is optionally substituted phenyl. In some embodiments, -X-R ^L is-N (R ') ₂, wherein each R' is independently as described herein. In some embodiments, -X-R ^L is-NHR ', where R' is as described herein. In some embodiments, -X-R ^L is-NHR, where R is as described herein. In some embodiments, -X-R ^L is R ^L, wherein R ^L is as described herein. In some embodiments, R ^L is-N (R ') ₂, wherein each R' is independently as described herein. In some embodiments, R ^L is-NHR ', wherein R' is as described herein. In some embodiments, R ^L is-NHR, wherein R is as described herein. In some embodiments, R ^L is-N (R ') ₂, wherein each R' is independently as described herein. In some embodiments, none of R 'in-N (R') ₂ is hydrogen. In some embodiments, R ^L is-N (R ') ₂, wherein each R' is independently C _1-6 aliphatic. In some embodiments, R ^L is-L-R ', wherein each of L and R' is independently as described herein. In some embodiments, R ^L is-L-R, wherein each of L and R is independently as described herein. In some embodiments, R ^L is-N (R ') -Cy-N (R ') -R '. In some embodiments, R ^L is-N (R ') -Cy-C (O) -R'. In some embodiments, R ^L is-N (R ') -Cy-O-R'. In some embodiments, R ^L is-N (R ') -Cy-SO ₂ -R'. In some embodiments, R ^L is-N (R') -Cy-SO ₂-N(R')₂. In some embodiments, R ^L is-N (R ') -Cy-C (O) -N (R') ₂. In some embodiments, R ^L is-N (R ') -Cy-OP (O) (R') ₂. In some embodiments, -Cy-is an optionally substituted divalent aryl group. In some embodiments, -Cy-is optionally substituted phenylene. In some embodiments, -Cy-is optionally substituted 1, 4-phenylene. In some embodiments, -Cy-is 1, 4-phenylene. In some embodiments, R ^L is-N (CH ₃)₂. In some embodiments, R ^L is-N (i-Pr) ₂. In some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L is

In some embodiments, -X-R ^L is-N (R') -C (O) -Cy-R ^L. In some embodiments, -X-R ^L is R ^L. In some embodiments, R ^L is-N (R ') -C (O) -Cy-O-R'. In some embodiments, R ^L is-N (R ') -C (O) -Cy-R'. In some embodiments, R ^L is-N (R ') -C (O) -Cy-C (O) -R'. In some embodiments, R ^L is-N (R ') -C (O) -Cy-N (R') ₂. In some embodiments, R ^L is-N (R') -C (O) -Cy-SO ₂-N(R')₂. In some embodiments, R ^L is-N (R ') -C (O) -Cy-C (O) -N (R') ₂. In some embodiments of the present invention, in some embodiments, R ^L is-N (R ') -C (O) -Cy-C (O) -N (R ') -SO ₂ -R '. In some embodiments, R' is R as described herein. In some embodiments, R ^L is

In some embodiments, one or more methylene units of L or variables comprising or being L are independently replaced by-O-, -N (R '), -C (O) -, -C (O) N (R '), -SO ₂-、-SO₂ N (R ') -, or-Cy-, as described herein. In some embodiments, the methylene units are replaced with-Cy-. In some embodiments, -Cy-is an optionally substituted divalent aryl group. In some embodiments, -Cy-is optionally substituted phenylene. In some embodiments, -Cy-is optionally substituted 1, 4-phenylene. In some embodiments, -Cy-is an optionally substituted divalent 5-20 (e.g., 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered heteroaryl group having 1-10 (e.g., 1, 2,3,4, 5,6,7,8, 9, or 10) heteroatoms. In some embodiments, -Cy-is monocyclic. In some embodiments, -Cy-is bicyclic. In some embodiments, -Cy-is polycyclic. In some embodiments, each single ring unit in-Cy-is independently 3-10 (e.g., 3,4, 5,6,7,8, 9, or 10) membered, and is independently saturated, partially saturated, or aromatic. In some embodiments, -Cy-is an optionally substituted 3-20 (e.g., 3,4, 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) membered monocyclic, bicyclic, or polycyclic aliphatic group. In some embodiments, -Cy-is an optionally substituted 3-20 (e.g., 3,4, 5,6,7,8, 9, or 10) membered monocyclic, bicyclic, or polycyclic heteroaliphatic group having 1-10 (e.g., 1, 2,3,4, 5,6,7,8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) heteroatoms.

In some embodiments, the internucleotide linkages have the structure-P (=o) (-N (R') P (O) (R ") ₂) -wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure-P (=s) (-N (R') P (O) (R ") ₂) -wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure of-P (=o) (-N (R') P (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure of-P (=s) (-N (R') P (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure-OP (=o) (-N (R') P (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure-OP (=s) (-N (R') P (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, for example, R 'of-N (R') -is hydrogen or an optionally substituted C _1-6 aliphatic. In some embodiments, R' is C _1-6 alkyl. In some embodiments, R' is hydrogen. In some embodiments, R "(e.g., in-P (O) (R") ₂) is R' as described herein. In some embodiments, the internucleotide linkage has the structure-P (=o) (-NHP (O) (R ") ₂) -wherein each R" is independently as described herein. In some embodiments, the internucleotide linkage has the structure-P (=s) (-NHP (O) (R ") ₂) -wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure of-P (=o) (-NHP (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure of-P (=s) (-NHP (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure-OP (=o) (-NHP (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the internucleotide linkages have the structure-OP (=s) (-NHP (O) (R ") ₂) O-, wherein each R" is independently as described herein. In some embodiments, the occurrence of R '(e.g., in-P (O) (R') ₂) is R. In some embodiments, R is an optionally substituted group selected from C _1-6 aliphatic, aryl, heterocyclyl, and heteroaryl. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted C _1-6 alkenyl. In some embodiments, R is optionally substituted C _1-6 alkynyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is-CF ₃. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is ethyl. In some embodiments, R is-CH ₂CHF₂. In some embodiments, R is-CH ₂CH₂OCH₃. In some embodiments, R is an optionally substituted C _1-20 (e.g., ,C_1-6、C_2-6、C_3-6、C_1-10、C_2-10、C_3-10、C_2-20、C_3-20、C_10-20、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、 or 20, etc.) aliphatic. In some embodiments, R is optionally substituted C _1-20 (e.g., ,C_1-6、C_2-6、C_3-6、C_1-10、C_2-10、C_3-10、C_2-20、C_3-20、C_10-20、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、 or 20, etc.) alkyl. In some embodiments, R is an optionally substituted straight chain C _2-20 aliphatic. In some embodiments, R is an optionally substituted linear C _2-20 alkyl. In some embodiments, R is a linear C _2-20 alkyl group. In some embodiments, R is isopropyl. in some embodiments, R is optionally substituted C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ aliphatic. In some embodiments, R is optionally substituted C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl. In some embodiments, R is optionally substituted straight chain C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl. In some embodiments, R is a linear C₁、C₂、C₃、C₄、C₅、C₆、C₇、C₈、C₉、C₁₀、C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、 or C ₂₀ alkyl group. In some embodiments, each R "is independently R as described herein, e.g., in some embodiments, each R" is methyl. In some embodiments, R "is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is p-methylphenyl. In some embodiments, R is benzyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted 1, 3-diazolyl. In some embodiments, R is optionally substituted 2- (1, 3) -diazolyl. In some embodiments, R is optionally substituted 1-methyl-2- (1, 3) -diazolyl. In some embodiments, the occurrence of R 'is-N (R') ₂. In some embodiments, R "is-N (CH ₃)₂, in some embodiments, R" (e.g., the occurrence in-P (O) (R ') ₂ is-OR ', where R ' is as described herein. In some embodiments, R' is R as described herein. In some embodiments, an optionally substituted C _1-6 aliphatic. In some embodiments, an optionally substituted C _1-6 alkyl. In some embodiments, R "is-OCH ₃. in some embodiments, each R "is-OR' as described herein. In some embodiments, each R "is-OCH ₃. In some embodiments, each R' is-OH. In some embodiments, the linkage is-OP (O) (NHP (O) (OH) ₂) O-. In some embodiments, the linkage is-OP (O) (NHP (O) (OCH ₃)₂) O-. In some embodiments, the linkage is-OP (O) (NHP (O) (CH ₃)₂) O-.

In some embodiments, -N (R ') ₂ is-N (R') ₂. In some embodiments, -N (R') ₂ is-NHR. In some embodiments, -N (R') ₂ is-NHC (O) R. In some embodiments, -N (R') ₂ is-NHC (O) OR. In some embodiments, -N (R') ₂ is-NHS (O) ₂ R.

In some embodiments, the internucleotide linkage is a phosphorylguanidine internucleotide linkage. In some embodiments, the internucleotide linkages comprise-X-R ^L as described herein. In some embodiments, -X-R ^L is-n=c (-L ^L-R^L)₂. in some embodiments, -X-R ^L is-n=c [ N (R ^L)₂]₂. In some embodiments, -X-R ^L is-n=c [ NR' R ^L]₂ ]. In some embodiments, -X-R ^L is-n=cn (R') ₂]₂. In some embodiments, -X-R ^L is-n=c [ N (R ^L)₂](CHR^L1R^L2), wherein each of R ^L1 and R ^L2 is independently as described herein. In some embodiments, -X-R ^L is-n=c (NR' R ^L)(CHR^L1R^L2), where each of R ^L1 and R ^L2 is independently as described herein. In some embodiments, -X-R ^L is-n=c (NR' R ^L)(CR'R^L1R^L2), where each of R ^L1 and R ^L2 is independently as described herein. In some embodiments, -X-R ^L is-n=c [ N (R') ₂](CHR'R^L2. In some embodiments, -X-R ^L is-n=cn (R ^L)₂](R^L). In some embodiments, -X-R ^L is-n=c (NR' R ^L)(R^L). In some embodiments, -X-R ^L is-n=c (NR 'R ^L) (R'). In some embodiments, the-X-R ^L is-N=C [ N (R ') ] ₂ (R'). In some embodiments, -X-R ^L is-n=c (NR' R ^L1)(NR'R^L2), where each R ^L1 and R ^L2 is independently R ^L, and each R' and R ^L is independently as described herein. In some embodiments, -X-R ^L is-n=c (NR' R ^L1)(NR'R^L2), where the variables are independently as described herein. In some embodiments, -X-R ^L is-n=c (NR' R ^L1)(CHR'R^L2), where the variables are independently as described herein. in some embodiments, -X-R ^L is-n=c (NR 'R ^L1) (R'), where the variables are independently as described herein. In some embodiments, each R' is independently R. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, -X-R ^L isIn some embodiments, two groups selected from R ', R ^L、R^L1、R^L2, etc. (in some embodiments, on the same atom (e.g., -N (R ') ₂, or NR ' R ^L, or-N (R ^L)², Wherein R 'and R ^L can independently be R) as described herein, etc.), or two R' on different atoms (e.g., -n=c (NR 'R ^L)(CR'R^L1R^L2) or-n=c (NR' R ^L1)(NR'R^L2); But may be two other variables that may be R, such as R ^L、R^L1、R^L2, etc.) are independent R and form together with the atoms interposed therebetween a ring as described herein. In some embodiments, two of R, R', R ^L、R^L1, or R ^L2 on the same atom, e.g., -N(R')₂、-N(R^L)₂、-NR'R^L、-NR'R^L1、-NR'R^L2、-CR'R^L1R^L2, etc., together form a ring as described herein. in some embodiments, two R ', R ^L、R^L1 or R ^L2 on two different atoms, e.g., two R ' in-n=c (NR ' R ^L)(CR'R^L1R^L2)、-N＝C(NR'R^L1)(NR'R^L2) and the like, together form a ring as described herein. In some embodiments, the ring formed is an optionally substituted 3-20 (e.g., ,3-15、3-12、3-10、3-9、3-8、3-7、3-6、4-15、4-12、4-10、4-9、4-8、4-7、4-6、5-15、5-12、5-10、5-9、5-8、5-7、5-6、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、, etc.) monocyclic, bicyclic, or tricyclic ring having 0-5 additional heteroatoms. In some embodiments, the ring formed is a single ring as described herein. In some embodiments, the ring formed is an optionally substituted 5-10 membered monocyclic ring. In some embodiments, the ring formed is bicyclic. In some embodiments, the rings formed are polycyclic. In some embodiments, two groups that are or can be R (e.g., -n=c (NR ' R ^L)(CR'R^L1R^L2) or-n=c (NR ' R ^L1)(NR'R^L2) R ', -n=c (NR ' R ^L)(CR'R^L1R^L2)、-N＝C(NR'R^L1)(NR'R^L2) R ', etc.) together form an optionally substituted divalent hydrocarbon chain, for example an optionally substituted C _1-20 aliphatic chain, optionally substituted- (CH ₂) n-, where n is 1-20 (e.g., 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is partially unsaturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, two groups that are or can be R (e.g., -n=c (NR ' R ^L)(CR'R^L1R^L2) or-n=c (NR ' R ^L1)(NR'R^L2) R ', -n=c (NR ' R ^L)(CR'R^L1R^L2)、-N＝C(NR'R^L1)(NR'R^L2) R ', etc.) together form an optionally substituted divalent heteroaliphatic chain, For example an optionally substituted C _1-20 heteroaliphatic chain having 1 to 10 heteroatoms. In some embodiments, the heteroaliphatic chain is saturated. In some embodiments, the heteroaliphatic chain is partially unsaturated. In some embodiments, the heteroaliphatic chain is unsaturated. In some embodiments, the chain is optionally substituted- (CH ₂) -. In some embodiments, the chain is optionally substituted- (CH ₂)₂ -. In some embodiments, the chain is optionally substituted- (CH ₂) -. In some embodiments, the chain is optionally substituted- (CH ₂)₂ -, in some embodiments, the chain is optionally substituted- (CH ₂)₃ -, in some embodiments, the chain is optionally substituted- (CH ₂)₄ -). In some embodiments, the chain is optionally substituted- (CH ₂)₅ -, in some embodiments, the chain is optionally substituted- (CH ₂)₆ -, in some embodiments, the chain is optionally substituted-ch=ch-, in some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, the chain is optionally substitutedIn some embodiments, two of R, R', R ^L、R^L1、R^L2, etc. on different atoms together form a ring as described herein. For example, in some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L is,-N(R')₂、-N(R)₂、-N(R^L)₂、-NR'R^L、-NR'R^L1、-NR'R^L2、-NR^L1R^L2, Etc. are loops formed in some embodiments. In some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substitutedIn some embodiments, the ring is optionally substituted

In some embodiments, R ^L1 and R ^L2 are the same. In some embodiments, R ^L1 and R ^L2 are different. In some embodiments, each of R ^L1 and R ^L2 is independently R ^L, as described herein, for example, below.

In some embodiments, R ^L is an optionally substituted C _1-30 aliphatic. In some embodiments, R ^L is optionally substituted C _1-30 alkyl. In some embodiments, R ^L is linear. In some embodiments, R ^L is optionally substituted straight chain C _1-30 alkyl. In some embodiments, R ^L is optionally substituted C _1-6 alkyl. In some embodiments, R ^L is methyl. In some embodiments, R ^L is ethyl. In some embodiments, R ^L is n-propyl. In some embodiments, R ^L is isopropyl. In some embodiments, R ^L is n-butyl. In some embodiments, R ^L is tert-butyl. In some embodiments, R ^L is (E) -CH ₂-CH＝CH-CH₂-CH₃. In some embodiments, R ^L is (Z) -CH ₂-CH＝CH-CH₂-CH₃. In some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L is CH ₃(CH₂)₂C≡CC≡C(CH₂)₃ -. In some embodiments, R ^L is CH ₃(CH₂)₅ c≡c-. In some embodiments, R ^L is optionally substituted aryl. In some embodiments, R ^L is optionally substituted phenyl. In some embodiments, R ^L is phenyl substituted with one or more halogens. In some embodiments, R ^L is phenyl optionally substituted with halogen, -N (R '), or-N (R ') C (O) R '. In some embodiments, R ^L is phenyl optionally substituted with-Cl, -Br, -F, -N (Me) ₂, or-NHCOCH ₃. In some embodiments, R ^L is-L ^L -R', where L ^L is optionally substituted C _1-20 saturated, Partially unsaturated or unsaturated hydrocarbon chains. in some embodiments, such hydrocarbon chains are straight. In some embodiments, such hydrocarbon chains are unsubstituted. In some embodiments, L ^L is (E) -CH ₂ -ch=ch-. In some embodiments, L ^L is-CH ₂-C≡C-CH₂ -. In some embodiments, L ^L is- (CH ₂)₃ -. In some embodiments, L ^L is- (CH ₂)₄ -). In some embodiments, L ^L is- (CH ₂)_n) -where n is 1-30 (e.g., 1-20, 5-30, 6-30, 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 or 30, etc.). In some embodiments, R' is optionally substituted aryl as described herein. In some embodiments, R' is optionally substituted phenyl. In some embodiments, R' is phenyl. In some embodiments, R' is optionally substituted heteroaryl as described herein. In some embodiments, R 'is 2' -pyridinyl. In some embodiments, R 'is 3' -pyridinyl. In some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L is-L ^L-N(R')₂, wherein each variable is independently as described herein. In some embodiments, each R' is independently a C _1-6 aliphatic as described herein. In some embodiments, -N (R ') ₂ is-N (CH ₃)₂. In some embodiments, -N (R') ₂ is-NH ₂. In some embodiments, R ^L is- (CH ₂)_n-N(R')₂, wherein N is 1-30 (e.g., 1-20, 5-30, 6-30, 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, etc.). In some embodiments, R ^L is- (CH ₂CH₂O)_n-CH₂CH₂-N(R')₂, wherein N is 1-30 (e.g., 1-20, 5-30, 6-30, 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, etc.) in some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L is- (CH ₂)_n-NH₂, in some embodiments, R ^L is- (CH ₂CH₂O)_n-CH₂CH₂-NH₂). In some embodiments, R ^L is- (CH ₂CH₂O)_n-CH₂CH₂ -R'), where n is 1-30 (e.g., 1-20, 5-30, 6-30, 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, 1-20, 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 or 30, etc.). In some embodiments, R ^L is- (CH ₂CH₂O)_n-CH₂CH₂CH₃) wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 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 or 30, etc.). In some embodiments, R ^L is- (CH ₂CH₂O)_n-CH₂CH₂ OH) wherein n is 1-30 (e.g., 1-20, 5-30, 6-30, 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 or 30, etc.). In some embodiments, R ^L is or comprises a carbohydrate moiety, such as GalNAc. In some embodiments, R ^L is-L ^L -GalNAc. In some embodiments, R ^L isIn some embodiments, one or more methylene units of L ^L are independently replaced with-Cy (e.g., optionally substituted 1, 4-phenylene, optionally substituted 3-to 30-membered divalent monocyclic, bicyclic, or polycyclic cycloaliphatic ring, etc.), -O-, -N (R ') - (e.g., -NH), -C (O) -, -C (O) N (R') - (e.g., -C (O) NH-), -C (NR ') - (e.g., -C (NH) -), -N (R')C (O) (N (R ') - (e.g., -NHC (O) NH-), -N (R')C (NR ') (N (R') - (e.g., -NHC (NH) NH-), - (CH ₂CH₂O)_n -, etc.), e.g., in some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isIn some embodiments, R ^L isWherein n is 0 to 20. In some embodiments, RL is or comprises one or more additional chemical moieties (e.g., carbohydrate moieties, galNAc moieties, etc.), optionally substituted and linked via a linker (the linker may be bivalent or multivalent). For example, in some embodiments, R ^L isWherein n is 0 to 20. In some embodiments, R ^L isWherein n is 0 to 20. In some embodiments, R ^L is R' as described herein. As described herein, many variables may independently be R'. In some embodiments, R' is R as described herein. As described herein, the various variables may independently be R. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted cycloaliphatic. In some embodiments, R is optionally substituted cycloalkyl. In some embodiments, R is optionally substituted aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is optionally substituted heteroaryl. In some embodiments, R is optionally substituted heterocyclyl. In some embodiments, R is an optionally substituted C _1-20 heterocyclyl having 1-5 heteroatoms, e.g., one of the heteroatoms is nitrogen. In some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substituted

In some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-RL isIn some embodiments, -X-R ^L isIn some embodiments, -X-RL isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isWherein n is 1 to 20. In some embodiments, -X-R ^L isWherein n is 1 to 20. In some embodiments, -X-R ^L is selected from:

In some embodiments, -X-R ^L is In some embodiments, -X-R ^L isIn some embodiments, -X-R ^L is

In some embodiments, R ^L is R as described herein. In some embodiments, R ^L is R as described herein.

In some embodiments, R "or R ^L is or comprises an additional chemical moiety. In some embodiments, R "or R ^L is or comprises an additional chemical moiety, wherein the additional chemical moiety is or comprises a carbohydrate moiety. In some embodiments, R "or R ^L is or comprises GalNAc. In some embodiments, R ^L or R "is replaced with or used to attach to another chemical moiety.

In some embodiments, X is-O-. In some embodiments, X is-S-. In some embodiments, X is-L ^L-N(-L^L-R^L)-L^L -. In some embodiments, X is-N (-L ^L-R^L)-L^L -. In some embodiments, X is-L ^L-N(-L^L-R^L) -. In some embodiments, X is-N (-L ^L-R^L) -. In some embodiments, X is-L ^L-N＝C(-L^L-R^L)-L^L -. In some embodiments, X is-n=c (-L ^L-R^L)-L^L -. In some embodiments, X is-L ^L-N＝C(-L^L-R^L) -. In some embodiments, X is-n=c (-L ^L-R^L) -. In some embodiments, X is L ^L. In some embodiments, X is a covalent bond.

In some embodiments, Y is a covalent bond. In some embodiments, Y is-O-. In some embodiments, Y is-N (R') -. In some embodiments, Z is a covalent bond. In some embodiments, Z is-O-. In some embodiments, Z is-N (R') -. In some embodiments, R' is R. In some embodiments, R is-H. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, R is ethyl. In some embodiments, R is propyl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is phenyl.

As described herein, the various variables in the structures in the present disclosure may be or include R. Suitable embodiments of R are broadly described in the present disclosure. As will be appreciated by those skilled in the art, the R embodiments described for a variable that may be R are also applicable to another variable that may be R. Similarly, embodiments described for a component/portion (e.g., L) of a variable are also applicable to other variables that may be or contain that component/portion.

In some embodiments, R "is R'. In some embodiments, R 'is-N (R') ₂.

In some embodiments, -X-R ^L is-SH. In some embodiments, -X-R ^L is-OH.

In some embodiments, -X-R ^L is-N (R') ₂. In some embodiments, each R' is independently an optionally substituted C _1-6 aliphatic. In some embodiments, each R' is independently methyl.

In some embodiments, the non-negatively charged internucleotide linkages have the structure-OP (=o) (-n=c ((N (R ') ₂)₂ -O) -, in some embodiments, the R ' groups of one N (R ') ₂ are R, the R ' groups of the other N (R ') ₂ are R, and the two R groups together with the atoms interposed therebetween form an optionally substituted ring, e.g., a 5-membered ring in N001.

In some embodiments, -X-R ^L is-n=c (-L ^L-R')₂. In some embodiments, -X-R ^L is-n=c (-L ^L1-L^L2-L^L3-R')₂, where each L ^L1、L^L2 and L ^L3 is independently L), wherein each L "is independently a covalent bond, or a divalent optionally substituted linear or branched radical selected from the group consisting of a C _1-10 aliphatic radical and a C _1-10 heteroaliphatic radical having 1 to 5 heteroatoms, wherein one or more methylene units are optionally and independently replaced by an optionally substituted radical selected from the group consisting of: c _1-6 alkylene, C _1-6 alkenylene, -C.ident.C-, a divalent C ₁-C₆ heteroaliphatic group 、-C(R')₂-、-Cy-、-O-、-S-、-S-S-、-N(R')-、-C(O)-、-C(S)-、-C(NR')-、-C(O)N(R')-、-N(R')C(O)N(R')-、-N(R')C(O)O-、-S(O)-、-S(O)₂-、-S(O)₂N(R')-、-C(O)S-、-C(O)O-、-P(O)(OR')-、-P(O)(SR')-、-P(O)(R')-、-P(O)(NR')-、-P(S)(OR')-、-P(S)(SR')-、-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- having 1 to 5 heteroatoms OR-OP (OR ') [ B (R') ₃ ] O-, And one or more nitrogen or carbon atoms are optionally and independently replaced by Cy ^L. In some embodiments, L ^L2 is-Cy-. In some embodiments, L ^L1 is a covalent bond. In some embodiments, L ^L3 is a covalent bond. In some embodiments, -X-R ^L is-n=c (-L ^L1-Cy-L^L3-R')₂. In some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L is

In some embodiments, L is a covalent bond, as used in the present disclosure. In some embodiments, L is a divalent optionally substituted straight or branched chain group selected from a C _1-30 aliphatic group and a C _1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one or more methylene units are optionally and independently replaced with an optionally substituted group selected from the group consisting of: c _1-6 alkylene, C _1-6 alkenylene, -C.ident.C-, a divalent C ₁-C₆ heteroaliphatic group 、-C(R')₂-、-Cy-、-O-、-S-、-S-S-、-N(R')-、-C(O)-、-C(S)-、-C(NR')-、-C(O)N(R')-、-N(R')C(O)N(R')-、-N(R')C(O)O-、-S(O)-、-S(O)₂-、-S(O)₂N(R')-、-C(O)S-、-C(O)O-、-P(O)(OR')-、-P(O)(SR')-、-P(O)(R')-、-P(O)(NR')-、-P(S)(OR')-、-P(S)(SR')-、-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- having 1 to 5 heteroatoms OR OP (OR ') [ B (R') ₃ ] O-, And one or more nitrogen or carbon atoms are optionally and independently replaced by Cy ^L. In some embodiments, L is a divalent optionally substituted straight OR branched chain group selected from the group consisting of a C _1-30 aliphatic group and a C _1-30 heteroaliphatic group having 1-10 heteroatoms, wherein one OR more methylene units are optionally and independently replaced with an optionally substituted group selected from ：-C≡C-、-C(R')₂-、-Cy-、-O-、-S-、-S-S-、-N(R')-、-C(O)-、-C(S)-、-C(NR')-、-C(O)N(R')-、-N(R')C(O)N(R')-、-N(R')C(O)O-、-S(O)-、-S(O)₂-、-S(O)₂N(R')-、-C(O)S-、-C(O)O-、-P(O)(OR')-、-P(O)(SR')-、-P(O)(R')-、-P(O)(NR')-、-P(S)(OR')-、-P(S)(SR')-、-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 nitrogen or carbon atoms are optionally and independently replaced by Cy ^L. In some embodiments, L is a divalent optionally substituted straight OR branched chain group selected from the group consisting of a C _1-10 aliphatic group and a C _1-10 heteroaliphatic group having 1-10 heteroatoms, wherein one OR more methylene units are optionally and independently replaced with an optionally substituted group selected from ：-C≡C-、-C(R')₂-、-Cy-、-O-、-S-、-S-S-、-N(R')-、-C(O)-、-C(S)-、-C(NR')-、-C(O)N(R')-、-N(R')C(O)N(R')-、-N(R')C(O)O-、-S(O)-、-S(O)₂-、-S(O)₂N(R')-、-C(O)S-、-C(O)O-、-P(O)(OR')-、-P(O)(SR')-、-P(O)(R')-、-P(O)(NR')-、-P(S)(OR')-、-P(S)(SR')-、-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 nitrogen or carbon atoms are optionally and independently replaced by Cy ^L. In some embodiments, one or more methylene units are optionally and independently replaced with an optionally substituted group selected from ：-C≡C-、-C(R')₂-、-Cy-、-O-、-S-、-S-S-、-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- or-C (O) O-.

In some embodiments, the internucleotide linkage is a phosphorylguanidine internucleotide linkage. In some embodiments, -X-R ^L is-n=cn (R') ₂]₂. In some embodiments, each R' is independently R. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is methyl. In some embodiments, -X-R ^L isIn some embodiments, one R 'on a nitrogen atom forms a ring with R' on another nitrogen atom as described herein.

In some embodiments, -X-R ^L isWherein R ¹ and R ² are independently R'. In some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, two R' on the same nitrogen together form a ring as described herein. In some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L isIn some embodiments, -X-R ^L is

In some embodiments, -X-R ^L is R as described herein. In some embodiments, R is not hydrogen. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is methyl.

In some embodiments, -X-R ^L is selected from the following table. In some embodiments, X is as described herein. In some embodiments, R ^L is as described herein. In some embodiments, the linkage has the structure of-Y-P ^L(-X-R^L) -Z-, wherein-X-R ^L is selected from the following table, and each other variable is independently as described herein. in some embodiments, the linkage has or comprises a structure of-P (O) (-X-R ^L) -wherein-X-R ^L is selected from the following table. In some embodiments, the linkage has or comprises a structure of-P (S) (-X-R ^L) -wherein-X-R ^L is selected from the following table. In some embodiments, the linkage has or comprises the structure of-P (-X-R ^L) -wherein-X-R ^L is selected from the following table. In some embodiments, the linkage has or comprises a structure of-O-P (O) (-X-R ^L) -O-, wherein-X-R ^L is selected from the following table. In some embodiments, the linkage has or comprises a structure of-O-P (S) (-X-R ^L) -O-, wherein-X-R ^L is selected from the following table. In some embodiments, the linkage has or comprises the structure of-O-P (-X-R ^L) -O-wherein-X-R ^L is selected from the following table. In some embodiments of the present invention, in some embodiments, the bond has-O-P (O) X-R ^L) -O-structure, wherein-X-R ^L is selected from the following table. In some embodiments of the present invention, in some embodiments, the bond has-O-P (S) X-R ^L) -O-structure, wherein-X-R ^L is selected from the following table. In some embodiments, the linkage has the structure of-O-P (-X-R ^L) -O-wherein-X-R ^L is selected from the following table. In some embodiments, in the following table, n is 0-20 or as described herein.

Table L-1. Certain useful moieties are bonded to the phosphorus linkage (e.g., -X-R ^L).

Wherein each R ^LS is independently R ^s. In some embodiments, each R ^LS is independently-Cl, -Br, -F, -N (Me) ₂, or-NHCOCH ₃.

Table L-2. Certain useful moieties are bonded to the phosphorus linkage (e.g., -X-R ^L).

Table L-3. Certain useful moieties are bonded to the phosphorus linkage (e.g., -X-R ^L).

Table L-4. Certain useful moieties are bonded to the phosphorus linkage (e.g., -X-RL).

Table L-5. Certain useful moieties are bonded to the phosphorus linkage (e.g., -X-RL).

Table L-6. Certain useful moieties are bonded to the phosphorus linkage (e.g., -X-RL).

In some embodiments, internucleotide linkages, such as nonnegatively charged internucleotide linkages or neutral internucleotide linkages, have the structure of-L ^L1-Cy^IL-L^L2 -. In some embodiments, L ^L1 is bonded to the 3' -carbon of the sugar. In some embodiments, L ^L2 is bonded to the 5' -carbon of the sugar. In some embodiments, L ^L1 is-O-CH ₂ -. In some embodiments, L ^L2 is a covalent bond. In some embodiments, L ^L2 is-N (R') -. In some embodiments, L ^L2 is-NH-. In some embodiments, L ^L2 is bonded to the 5 '-carbon of the sugar, which 5' -carbon is substituted with =o. In some embodiments, cy ^IL is an optionally substituted 3-10 membered saturated, partially unsaturated, or aromatic ring having 0-5 heteroatoms. In some embodiments, cy ^IL is an optionally substituted triazole ring. In some embodiments, cy ^IL isIn some embodiments, the linkage is

In some embodiments, the non-negatively charged internucleotide linkage has the structure-OP (=w) (-N (R') ₂) -O-.

In some embodiments, R' is R. In some embodiments, R' is H. In some embodiments, R' is-C (O) R. In some embodiments, R' is-C (O) OR. In some embodiments, R' is-S (O) ₂ R.

In some embodiments, R "is-NHR'. In some embodiments, -N (R ') ₂ is-NHR'.

As described herein, in some embodiments, R is H. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is methyl. In some embodiments, R is substituted methyl. In some embodiments, R is ethyl. In some embodiments, R is substituted ethyl.

In some embodiments, the non-negatively charged internucleotide linkages are neutral internucleotide linkages, as described herein.

In some embodiments, the modified internucleotide linkages (e.g., internucleotide linkages without negative charges) comprise optionally substituted triazolyl groups. In some embodiments, R' is or comprises an optionally substituted triazolyl. In some embodiments, the modified internucleotide linkages (e.g., non-negatively charged internucleotide linkages) comprise optionally substituted alkynyl groups. In some embodiments, R' is optionally substituted alkynyl. In some embodiments, R' comprises an optionally substituted triple bond. In some embodiments, the modified internucleotide linkage comprises a triazole or alkyne moiety. In some embodiments, R' is or comprises an optionally substituted triazole or alkyne moiety. In some embodiments, the triazole moiety (e.g., triazolyl) is optionally substituted. In some embodiments, the triazole moiety (e.g., triazolyl) is substituted. In some embodiments, the triazole moiety is unsubstituted. In some embodiments, the modified internucleotide linkage comprises an optionally substituted guanidine moiety. In some embodiments, the modified internucleotide linkage comprises an optionally substituted cyclic guanidine moiety. In some embodiments, R', R ^L, or-X-R ^L are or comprise an optionally substituted guanidine moiety. In some embodiments, R', R ^L, or-X-R ^L are or comprise an optionally substituted cyclic guanidine moiety. In some embodiments, R', R ^L, or-X-R ^L comprise an optionally substituted cyclic guanidine moiety and the internucleotide linkage has the structure: wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the internucleotide linkages not bearing a negative charge are stereochemically controlled.

In some embodiments, the non-negatively charged internucleotide linkage or neutral internucleotide linkage is an internucleotide linkage comprising a triazole moiety. In some embodiments, the non-negatively charged internucleotide linkages or the non-negatively charged internucleotide linkages comprise an optionally substituted triazolyl group. In some embodiments, the internucleotide linkages comprising a triazole moiety (e.g., optionally substituted triazolyl) haveIs a structure of (a). In some embodiments, the internucleotide linkages comprising a triazole moiety haveIs a structure of (a). In some embodiments, internucleotide linkages, e.g., nonnegatively charged internucleotide linkages, neutral internucleotide linkages, comprise cyclic guanidine moieties. In some embodiments, the internucleotide linkage comprising a cyclic guanidine moiety hasIs a structure of (a). In some embodiments, the non-negatively charged internucleotide linkage or neutral internucleotide linkage is or comprises a structure selected from the group consisting of: wherein W is O or S.

In some embodiments, the internucleotide linkage comprises a Tmg groupIn some embodiments, the internucleotide linkages contain a Tmg group and haveThe structure of (Tmg internucleotide linkage). In some embodiments, neutral internucleotide linkages include PNA and PMO internucleotide linkages, as well as Tmg internucleotide linkages.

In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 3-20 membered heterocyclyl or heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, such heterocyclyl or heteroaryl groups have a 5 membered ring. In some embodiments, such heterocyclyl or heteroaryl groups have a 6 membered ring.

In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heteroaryl group having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-6 membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-membered heteroaryl group having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the heteroaryl is directly bonded to the linking phosphorus. In some embodiments, the non-negatively charged internucleotide linkage comprises an optionally substituted 5-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-20 membered heterocyclyl having 1-10 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-6 membered heterocyclyl having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, the non-negatively charged internucleotide linkages comprise an optionally substituted 5-membered heterocyclyl having 1-4 heteroatoms, wherein at least one heteroatom is nitrogen. In some embodiments, at least two heteroatoms are nitrogen. In some embodiments, the heterocyclyl is directly bonded to the linking phosphorus. In some embodiments, when the heterocyclyl is part of a guanidine moiety that is directly bonded to the phosphorus linkage via its = N-, the heterocyclyl is bonded to the phosphorus linkage via a linker (e.g., = N-). In some embodiments, the non-negatively charged internucleotide linkages comprise optionally substitutedA group. In some embodiments, the non-negatively charged internucleotide linkages comprise a substitutedA group. In some embodiments, the non-negatively charged internucleotide linkages compriseA group. In some embodiments, each R ¹ is independently optionally substituted C _1-6 alkyl. In some embodiments, each R ¹ is independently methyl.

In some embodiments, the internucleotide linkages not negatively charged (e.g., neutral internucleotide linkages) are not chirally controlled. In some embodiments, the internucleotide linkages not bearing a negative charge are chirally controlled. In some embodiments, the non-negatively charged internucleotide linkage is chiral controlled and its linkage phosphorus is Rp. In some embodiments, the internucleotide linkage without negative charges is chiral and its linkage phosphorus is Sp.

In some embodiments, the internucleotide linkages do not comprise a linkage phosphorus. In some embodiments, the internucleotide linkages have the structure-C (O) - (O) -or-C (O) -N (R ') -, wherein R' is as described herein. In some embodiments, the internucleotide linkage has the structure-C (O) - (O) -. In some embodiments, the internucleotide linkages have the structure-C (O) -N (R ') -wherein R' is as described herein. In various embodiments, -C (O) -is bonded to nitrogen. In some embodiments, the internucleotide linkage is or comprises-C (O) -O-, which is part of a carbamate moiety. In some embodiments, the internucleotide linkage is or comprises-C (O) -O-, which is part of a urea moiety.

In some embodiments, the oligonucleotide comprises 1-20, 1-15, 1-10, 1-5, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or a plurality of non-negatively charged internucleotide linkages. In some embodiments, the oligonucleotide comprises 1-20, 1-15, 1-10, 1-5, or 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or a plurality of neutral internucleotide linkages. In some embodiments, the non-negatively charged internucleotide linkages and/or neutral internucleotide linkages are each optionally and independently chirally controlled. In some embodiments, each non-negatively charged internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In some embodiments, each neutral internucleotide linkage in the oligonucleotide is independently a chirally controlled internucleotide linkage. In some embodiments, at least one non-negatively charged internucleotide linkage/neutral internucleotide linkage has a formula ofIs a structure of (a). In some embodiments, the oligonucleotide comprises at least one non-negatively charged internucleotide linkage wherein the linkage phosphorus is in the Rp configuration, and at least one non-negatively charged internucleotide linkage wherein the linkage phosphorus is in the Sp configuration.

In many embodiments, as broadly demonstrated, the oligonucleotides of the disclosure comprise two or more different internucleotide linkages. In some embodiments, the oligonucleotides comprise phosphorothioate internucleotide linkages and nonnegatively charged internucleotide linkages. In some embodiments, the oligonucleotides comprise phosphorothioate internucleotide linkages, nonnegatively charged internucleotide linkages, and natural phosphate linkages. In some embodiments, the internucleotide linkage that is not negatively charged is a neutral internucleotide linkage. In some embodiments, the non-negatively charged internucleotide linkage is n001, n003, n004, n006, n008 or n009, n013, n020, n021, n025, n026, n029, n031, n037, n046, n047, n048, n054, or n 055). In some embodiments, the non-negatively charged internucleotide linkage is n001. In some embodiments, each phosphorothioate internucleotide linkage is independently chirally controlled. In some embodiments, each chiral modified internucleotide linkage is independently chirally controlled. In some embodiments, one or more non-negatively charged internucleotide linkages are not chirally controlled.

Typical linkages as in natural DNA and RNA are internucleotide linkages forming a bond with two sugars (which may be unmodified or modified as described herein). In many embodiments, the internucleotide linkage forms a bond with one optionally modified ribose or deoxyribose at its 5 'carbon and another optionally modified ribose or deoxyribose at its 3' carbon through its oxygen atom or heteroatom, as exemplified herein. In some embodiments, the internucleotide linkage is not a ribose sugar, e.g., a sugar comprising N ring atoms and an acyclic sugar as described herein.

In some embodiments, each nucleoside unit linked by internucleotide linkages independently comprises a nucleobase, which nucleobase is independently an optionally substituted A, T, C, G or U, or an optionally substituted tautomer of A, T, C, G or U.

In some embodiments, oligonucleotides comprise modified internucleotide linkages (e.g., having the structure of formula I, I-a, I-b, or I-c, I-n-1, I-n-2, I-n-3, I-n-4, 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., or salt forms )：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612 thereof, each having the negative charge of a nucleotide of formula I, I-a, I-b, or I-c, I-n-1, I-n-2, I-n-3, I-n-4, II-a-1, II-b-2, II-c-1, II-c-2, II-d-2, etc., or other than the examples of the examples provided herein include nucleotides that are independently negatively charged or are not provided by the examples of the other internucleotide linkages, the internucleotide linkages which are not negatively charged are neutral internucleotide linkages. In some embodiments, the disclosure provides oligonucleotides comprising one or more neutral internucleotide linkages. In some embodiments, non-negatively charged internucleotide linkages or neutral internucleotide linkages (e.g., having one of the formulas I-n-1, I-n-2, I-n-3, I-n-4, 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.) are as described in ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612 below. In some embodiments, the non-negatively charged internucleotide linkages or neutral internucleotide linkages have one of the formulas I-n-1, I-n-2, I-n-3, I-n-4, 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., as described in ：WO 2018/223056、WO 2019/032607、WO 2019/075357、WO 2019/032607、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612, each of which is independently incorporated herein by reference.

As described herein, the various variables may be R, such as R', R ^L, and the like. Various embodiments of R are described in this disclosure (e.g., when describing variables that may be R). Such an embodiment is generally applicable to all variables that may be R. In some embodiments, R is hydrogen. In some embodiments, R is optionally substituted C _1-30 (e.g., 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, or 30) aliphatic. In some embodiments, R is an optionally substituted C _1-20 aliphatic. In some embodiments, R is an optionally substituted C _1-10 aliphatic. In some embodiments, R is an optionally substituted C _1-6 aliphatic. In some embodiments, R is optionally substituted alkyl. In some embodiments, R is optionally substituted C _1-6 alkyl. In some embodiments, R is optionally substituted methyl. In some embodiments, R is methyl. In some embodiments, R is optionally substituted ethyl. In some embodiments, R is optionally substituted propyl. In some embodiments, R is isopropyl. In some embodiments, R is optionally substituted butyl. In some embodiments, R is optionally substituted pentyl. In some embodiments, R is optionally substituted hexyl.

In some embodiments, R is optionally substituted 3-30 membered (e.g., 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, or 30) cycloaliphatic. In some embodiments, R is optionally substituted cycloalkyl. In some embodiments, the cycloaliphatic is monocyclic, bicyclic, or polycyclic, wherein each monocyclic unit is independently saturated or partially saturated. In some embodiments, R is optionally substituted cyclopropyl. In some embodiments, R is optionally substituted cyclobutyl. In some embodiments, R is optionally substituted cyclopentyl. In some embodiments, R is optionally substituted cyclohexyl. In some embodiments, R is optionally substituted adamantyl.

In some embodiments, R is an optionally substituted C _1-30 (e.g., 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, or 30) heteroaliphatic having 1-10 heteroatoms. In some embodiments, R is an optionally substituted C _1-20 aliphatic having 1-10 heteroatoms. In some embodiments, R is an optionally substituted C _1-10 aliphatic having 1-10 heteroatoms. In some embodiments, R is an optionally substituted C _1-6 aliphatic having 1-3 heteroatoms. In some embodiments, R is optionally substituted heteroalkyl. In some embodiments, R is optionally substituted C _1-6 heteroalkyl. In some embodiments, R is an optionally substituted 3-30 membered (e.g., 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, or 30) heterocyclic aliphatic having 1-10 heteroatoms. In some embodiments, R is optionally substituted heterocycloalkyl. In some embodiments, the heterocyclic aliphatic is monocyclic, bicyclic, or polycyclic, wherein each monocyclic unit is independently saturated or partially saturated.

In some embodiments, R is optionally substituted C _6-30 aryl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is optionally substituted phenyl. In some embodiments, R is C _6-14 aryl. In some embodiments, R is optionally substituted bicyclic aryl. In some embodiments, R is an optionally substituted polycyclic aryl. In some embodiments, R is an optionally substituted C _6-30 aryl aliphatic. In some embodiments, R is a C _6-30 aryl heteroaliphatic having 1-10 heteroatoms.

In some embodiments, R is an optionally substituted 5-30 (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, or 30) membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-20 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-10 membered heteroaryl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 5-membered heteroaryl having one heteroatom. In some embodiments, R is an optionally substituted 6 membered heteroaryl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heteroaryl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heteroaryl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heteroaryl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heteroaryl having one heteroatom. In some embodiments, R is optionally substituted monocyclic heteroaryl. In some embodiments, R is optionally substituted bicyclic heteroaryl. In some embodiments, R is an optionally substituted polycyclic heteroaryl. In some embodiments, the heteroatom is nitrogen.

In some embodiments, R is optionally substituted 2-pyridinyl. In some embodiments, R is optionally substituted 3-pyridinyl. In some embodiments, R is optionally substituted 4-pyridinyl. In some embodiments, R is optionally substituted

In some embodiments, R is an optionally substituted 3-30 (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, or 30) membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 3 membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 4-membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 5-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5-10 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heterocyclyl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heterocyclyl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 5 membered heterocyclyl having one heteroatom. In some embodiments, R is an optionally substituted 6 membered heterocyclyl having 1-5 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heterocyclyl having 1-4 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heterocyclyl having 1-3 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heterocyclyl having 1-2 heteroatoms. In some embodiments, R is an optionally substituted 6 membered heterocyclyl having one heteroatom. In some embodiments, R is an optionally substituted monocyclic heterocyclyl. In some embodiments, R is an optionally substituted bicyclic heterocyclyl. In some embodiments, R is an optionally substituted polycyclic heterocyclyl. In some embodiments, R is an optionally substituted saturated heterocyclyl. In some embodiments, R is an optionally substituted partially unsaturated heterocyclyl. In some embodiments, the heteroatom is nitrogen. In some embodiments, R is optionally substitutedIn some embodiments, R is optionally substitutedIn some embodiments, R is optionally substituted

In some embodiments, two R groups are optionally and independently joined together to form a covalent bond. In some embodiments, two or more R groups on the same atom optionally and independently form together with the atom an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atom. In some embodiments, two or more R groups on two or more atoms optionally and independently form together with the atoms interposed therebetween an optionally substituted 3-30 membered monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms in addition to the atoms interposed therebetween.

Various embodiments may include an optionally substituted ring, or may form a ring with one or more atoms interposed therebetween. In some embodiments, the ring is 3-30 (e.g., 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, or 30) membered. In some embodiments, the ring is 3-20 membered. In some embodiments, the ring is 3-15 membered. In some embodiments, the ring is 3-10 membered. In some embodiments, the ring is 3-8 membered. In some embodiments, the ring is 3-7 membered. In some embodiments, the ring is 3-6 membered. In some embodiments, the ring is 4-20 membered. In some embodiments, the ring is 5-20 membered. In some embodiments, the ring is monocyclic. In some embodiments, the ring is bicyclic. In some embodiments, the rings are polycyclic. In some embodiments, each single ring or each single ring unit in a double or multiple ring is independently saturated, partially saturated, or aromatic. In some embodiments, each single ring or each single ring unit in a double or multiple ring is independently 3-10 membered and has 0-5 heteroatoms.

In some embodiments, each heteroatom is independently selected from oxygen, nitrogen, sulfur, silicon, and phosphorus. In some embodiments, each heteroatom is independently selected from oxygen, nitrogen, sulfur, and phosphorus. In some embodiments, each heteroatom is independently selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroatom is in an oxidized form.

As will be appreciated by those of skill in the art, many other types of internucleotide linkages may be utilized in accordance with the present disclosure, such as those described in the following: U.S. patent No. 3,687,808;4,469,863;4,476,301;5,177,195;5,023,243;5,034,506;5,166,315;5,185,444;5,188,897;5,214,134;5,216,141;5,235,033;5,264,423;5,264,564;5,276,019;5,278,302;5,286,717;5,321,131;5,399,676;5,405,938;5,405,939;5,434,257;5,453,496;5,455,233;5,466,677;5,466,677;5,470,967;5,476,925;5,489,677;5,519,126;5,536,821;5,541,307;5,541,316;5,550,111;5,561,225;5,563,253;5,571,799;5,587,361;5,596,086;5,602,240;5,608,046;5,610,289;5,618,704;5,623,070;5,625,050;5,633,360;5,64,562;5,663,312;5,677,437;5,677,439;6,160,109;6,239,265;6,028,188;6,124,445;6,169,170;6,172,209;6,277,603;6,326,199;6,346,614;6,444,423;6,531,590;6,534,639;6,608,035;6,683,167;6,858,715;6,867,294;6,878,805;7,015,315;7,041,816;7,273,933;7,321,029; or RE39464. In certain embodiments, the modified internucleotide linkages are modified internucleotide linkages ：US 9982257、US 20170037399、US 20180216108、WO 2017192664、WO 2017015575、WO 2017062862、WO 2018067973、WO 2017160741、WO 2017192679、WO 2017210647、WO 2018098264、PCT/US18/35687、PCT/US18/38835 or PCT/US18/51398 described in the following documents, each of which nucleobases, sugars, internucleotide linkages, chiral auxiliary/reagents, and oligonucleotide synthesis techniques (reagents, conditions, cycles, etc.) are independently incorporated herein by reference.

In certain embodiments, each internucleotide linkage in the ds oligonucleotide is independently selected from the group consisting of a natural phosphate linkage, a phosphorothioate linkage, and a non-negatively charged internucleotide linkage (e.g., n 001). In certain embodiments, each internucleotide linkage in the ds oligonucleotide is independently selected from the group consisting of a natural phosphate linkage, a phosphorothioate linkage, and a neutral internucleotide linkage (e.g., n 001).

In certain embodiments, the ds oligonucleotides comprise one or more nucleotides that independently comprise a phosphorus modification that is susceptible to "self-release" under certain conditions. That is, under certain conditions, specific phosphorus modifications are designed to self-cleave from ds oligonucleotides to provide, for example, native phosphate linkages. In certain embodiments, such phosphorus modifications have the structure of-O-L-R ¹, wherein L is L ^B as described herein, and R ¹ is R' as described herein. In certain embodiments, the phosphorus modification has the structure of-S-L-R ¹, wherein L and R ¹ are each independently as described in the disclosure. Some examples of such phosphorus modifying groups can be found in US 9982257. In certain embodiments, the self-releasing group comprises morpholino. In certain embodiments, the self-releasing group is characterized by the ability to deliver an agent to the internucleotide phosphate linker that aids in further modification of the phosphorus atom, such as desulfurization. In certain embodiments, the agent is water and the further modification is hydrolysis to form a natural phosphate linkage.

In certain embodiments, the ds oligonucleotides comprise one or more internucleotide linkages, which improve one or more pharmaceutical properties and/or activity of the oligonucleotide. It is well documented in the art that certain oligonucleotides are rapidly degraded by nucleases and exhibit poor cellular uptake by cytoplasmic cell membranes (P0 ijarvi-Virta et al, curr.Med.chem. [ contemporary pharmaceutical chemistry ] (2006), 13 (28); 3441-65; wagner et al, med.Res.Rev. [ medical research review ] (2000), 20 (6): 417-51; peyrottes et al, mini Rev.Med. Chem.) (2004), 4 (4): 395-408; gosselin et al, (1996), 43 (1): 196-208; bologna et al, (2002), antisense & Nucleic Acid Drug Development [ Antisense and nucleic acid drug development ]. 12:33-41). Vives et al (Nucleic ACIDS RESEARCH [ Nucleic acids research ] (1999), 27 (20): 4071-76) reported that under certain conditions, t-butylSATE pro-oligonucleotide (pro-oligonucleotide) exhibited significantly increased cell penetration compared to the parent oligonucleotide.

The double stranded oligonucleotide may comprise a variety of natural phosphate linkages. In certain embodiments, 5% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 10% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 15% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 20% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 25% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 30% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 35% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, 40% or more of the internucleotide linkages of the ds oligonucleotides provided are natural phosphate linkages. In certain embodiments, provided ds oligonucleotides comprise 1,2, 3,4, 5,6,7,8, 9,10, or more native phosphate linkages. In certain embodiments, provided ds oligonucleotides comprise 4, 5,6,7,8, 9,10 or more native phosphate linkages. In certain embodiments, the number of natural phosphate linkages is 2. In certain embodiments, the number of natural phosphate linkages is 3. In certain embodiments, the number of natural phosphate linkages is 4. In certain embodiments, the number of natural phosphate linkages is 5. In certain embodiments, the number of natural phosphate linkages is 6. In certain embodiments, the number of natural phosphate linkages is 7. In certain embodiments, the number of natural phosphate linkages is 8. In certain embodiments, some or all of the natural phosphate linkages are continuous.

In certain embodiments, the disclosure demonstrates that, in at least some instances, sp internucleotide linkages at the 5 'end and/or the 3' end, among other things, can improve ds oligonucleotide stability. In certain embodiments, the disclosure demonstrates, among other things, that natural phosphate linkages and/or Rp internucleotide linkages can improve ds oligonucleotide removal from the system. As will be appreciated by one of ordinary skill in the art, a variety of assays known in the art may be utilized to evaluate such characteristics in accordance with the present disclosure.

In certain embodiments, each phosphorothioate internucleotide linkage in a ds oligonucleotide or portion thereof (e.g., domain, subdomain, etc.) is independently chirally controlled. In certain embodiments, each is independently Sp or Rp. In certain embodiments, the high level is Sp as described herein. In certain embodiments, each phosphorothioate internucleotide linkage in the ds oligonucleotide or portion thereof is chirally controlled and is Sp. In certain embodiments, one or more, for example about 1-5 (e.g., about 1, 2, 3,4, or 5) are Rp.

In certain embodiments, as shown in certain examples, the ds oligonucleotides or portions thereof comprise one or more non-negatively charged internucleotide linkages, each of which is optionally and independently chirally controlled. In certain embodiments, each non-negatively charged internucleotide linkage is independently n001. In certain embodiments, the chiral non-negatively charged internucleotide linkages are not chiral controlled. In certain embodiments, each chiral non-negatively charged internucleotide linkage is not chirally controlled. In certain embodiments, chiral non-negatively charged internucleotide linkages are chiral controlled. In certain embodiments, the chiral non-negatively charged internucleotide linkage is chiral controlled and Rp. in certain embodiments, the chiral non-negatively charged internucleotide linkage is chiral controlled and Sp. In certain embodiments, each chiral non-negatively charged internucleotide linkage is chirally controlled. In certain embodiments, the number of non-negatively charged internucleotide linkages in the ds oligonucleotide or portion thereof is about 1-10, or about 1, 2, 3,4, 5,6, 7, 8, 9, or 10. In certain embodiments, it is about 1. In certain embodiments, it is about 2. In certain embodiments, it is about 3. In certain embodiments, it is about 4. In certain embodiments, it is about 5. In certain embodiments, it is about 6. In certain embodiments, it is about 7. In certain embodiments, it is about 8. In certain embodiments, it is about 9. In certain embodiments, it is about 10. In certain embodiments, two or more non-negatively charged internucleotide linkages are contiguous. In certain embodiments, no two non-negatively charged internucleotide linkages are contiguous. In certain embodiments, all non-negatively charged internucleotide linkages in the ds oligonucleotide or portion thereof are contiguous (e.g., 3 contiguous non-negatively charged internucleotide linkages). In certain embodiments, a non-negatively charged internucleotide linkage, or two or more (e.g., about 2, about 3, about 4, etc.) consecutive non-negatively charged internucleotide linkages, are at the 3' terminus of the ds oligonucleotide or portion thereof. In certain embodiments, the last two or three or four internucleotide linkages of the ds oligonucleotide or portion thereof comprise at least one internucleotide linkage other than a non-negatively charged internucleotide linkage. In certain embodiments, the last two or three or four internucleotide linkages of the ds oligonucleotide or portion thereof comprise at least one internucleotide linkage other than n 001. In certain embodiments, the internucleotide linkage joining the first two nucleosides of the ds oligonucleotide or portion thereof is a non-negatively charged internucleotide linkage. In certain embodiments, the internucleotide linkage joining the last two nucleosides of the ds oligonucleotide or portion thereof is a non-negatively charged internucleotide linkage. in certain embodiments, the internucleotide linkage joining the first two nucleosides of the ds oligonucleotide or portion thereof is a phosphorothioate internucleotide linkage. In certain embodiments, it is Sp. In certain embodiments, the internucleotide linkage joining the last two nucleosides of the ds oligonucleotide or portion thereof is a phosphorothioate internucleotide linkage. In certain embodiments, it is Sp.

In certain embodiments, one or more chiral internucleotide linkages are chirally controlled, and one or more chiral internucleotide linkages are not chirally controlled. In certain embodiments, each phosphorothioate internucleotide linkage is independently chirally controlled, and one or more non-negatively charged internucleotide linkages are not chirally controlled. In certain embodiments, each phosphorothioate internucleotide linkage is independently chirally controlled, and each non-negatively charged internucleotide linkage is not chirally controlled. In certain embodiments, the internucleotide linkage between the first two nucleosides of the ds oligonucleotide is a non-negatively charged internucleotide linkage. In certain embodiments, the internucleotide linkages between the last two nucleosides are each independently non-negatively charged internucleotide linkages. In certain embodiments, both are independently non-negatively charged internucleotide linkages. In certain embodiments, each non-negatively charged internucleotide linkage is independently a neutral internucleotide linkage. In certain embodiments, each non-negatively charged internucleotide linkage is independently n001.

In certain embodiments, a controlled level of ds oligonucleotide in the composition is a desired ds oligonucleotide. In certain embodiments, the level of desired ds oligonucleotides (which may be present in multiple forms (e.g., salt forms) and which generally differ only at achiral controlled internucleotide linkages (for which the various forms of the same stereoisomer may be considered identical)) in all ds oligonucleotides in a composition that share a common base sequence (e.g., a desired sequence for a certain purpose), or in all ds oligonucleotides in a composition, is 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％,%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. In certain embodiments, the level is at least about 50%. In certain embodiments, the level is at least about 60%. In certain embodiments, the level is at least about 70%. In certain embodiments, the level is at least about 75%. In certain embodiments, the level is at least about 80%. In certain embodiments, the level is at least about 85%. In certain embodiments, the level is at least about 90%. In certain embodiments, the level is or at least is (DS) ^nc, where DS is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, and nc is the number of chiral controlled internucleotide linkages (e.g., ,1-50、1-40、1-30、1-25、1-20、5-50、5-40、5-30、5-25、5-20,1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25 or more) as described in the present disclosure. In certain embodiments, the level is or at least (DS) ^nc, where DS is 95% -100%.

Various types of internucleotide linkages can be used in combination with other structural elements, such as sugars, to achieve desired ds oligonucleotide characteristics and/or activity. For example, the present disclosure generally utilizes modified internucleotide linkages and modified sugars, optionally with natural phosphate linkages and natural sugars, in designing ds oligonucleotides. In certain embodiments, the disclosure provides ds oligonucleotides comprising one or more modified sugars. In certain embodiments, the disclosure provides ds oligonucleotides comprising one or more modified sugars and one or more modified internucleotide linkages, wherein one or more are natural phosphate linkages.

Double-stranded oligonucleotide compositions

The present disclosure provides, among other things, a variety of ds oligonucleotide compositions. In some embodiments, the disclosure provides ds oligonucleotide compositions of the ds oligonucleotides described herein. In some embodiments, the ds oligonucleotide composition (e.g., a ds oligonucleotide composition that targets HSD17B 13) comprises a plurality of oligonucleotides described in the present disclosure. In some embodiments, the ds oligonucleotide composition (e.g., a ds oligonucleotide composition that targets HSD17B 13) is chirally controlled. In some embodiments, the ds oligonucleotide composition (e.g., the ds oligonucleotide composition that targets HSD17B 13) is not chirally controlled (is stereorandom).

The natural phosphate-bonded phosphorus linkages are achiral. Many modified internucleotide linkages, such as phosphorothioate internucleotide-linked phosphorus, are chiral. In some embodiments, during preparation of the oligonucleotide composition (e.g., in traditional phosphoramidite oligonucleotide synthesis), the configuration of the chiral linkage is not purposefully designed or controlled, resulting in an achiral controlled (stereorandom) oligonucleotide composition (essentially racemic formulation), which is a complex random mixture of various stereoisomers (diastereomers) -typically 2 ⁿ stereoisomers (e.g., 2 ¹⁰ = 1,032 when n is 10; 2 ²⁰ =1, 048,576 when n is 20) for oligonucleotides having n chiral internucleotide linkages (linkage phosphorus is chiral). These stereoisomers have the same composition, but differ in stereochemical pattern of their bonded phosphorus.

In some embodiments, the stereorandom oligonucleotide composition has properties and/or activity sufficient for certain purposes and/or applications. In some embodiments, the stereorandom oligonucleotide composition may be less expensive, easier and/or simpler to produce than the chirally controlled oligonucleotide composition. However, stereoisomers in the stereogenic compositions may have different properties, activity and/or toxicity, resulting in inconsistent therapeutic effects and/or unexpected side effects, particularly compared to chiral controlled oligonucleotide compositions of certain oligonucleotides of the same constitution.

Chirally controlled double stranded oligonucleotide compositions

In some embodiments, the disclosure encompasses techniques for designing and preparing chirally controlled ds oligonucleotide compositions. In some embodiments, the chirally controlled ds oligonucleotide composition comprises a controlled/predetermined (not random as in a stereorandom composition) level of multiple ds oligonucleotides, wherein the ds oligonucleotides share the same linkage phosphorus stereochemistry at one or more chiral internucleotide linkages (chirally controlled internucleotide linkages). In some embodiments, multiple ds oligonucleotides share the same backbone chiral center pattern (stereochemistry of the linkage phosphate). In some embodiments, the backbone chiral center pattern is as described in the present disclosure. In some embodiments, multiple ds oligonucleotides share a common composition. In some embodiments, the ds oligonucleotides are structurally identical.

For example, in some embodiments, the disclosure provides a ds oligonucleotide composition comprising a plurality of ds oligonucleotides, wherein the plurality of oligonucleotides share:

1) A common base sequence, and

2) Independently the same stereochemistry of the bonded phosphorus at one or more (e.g., about 1-50、1-40、1-30、1-25、1-20、1-15、1-10、5-50、5-40、5-30、5-25、5-20、5-15、5-10,1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24 or 25 or more) chiral internucleotide linkages ("chirally controlled internucleotide linkages");

wherein the levels of the plurality of ds oligonucleotides in the composition are non-random (e.g., controlled/predetermined as described herein).

In some embodiments, the disclosure provides a ds oligonucleotide composition comprising a plurality of oligonucleotides, wherein the plurality of oligonucleotides share:

1) A common base sequence, and

wherein the composition enriches the plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides sharing the common base sequence.

In some embodiments, the disclosure provides a ds oligonucleotide composition comprising a plurality of ds oligonucleotides, wherein the plurality of ds oligonucleotides share:

1) A common base sequence, and

Wherein 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%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all ds oligonucleotides sharing a common base sequence in the composition is the plurality of oligonucleotides.

In some embodiments, the percentage/level of the plurality of DS oligonucleotides is or is at least (DS) ^nc, wherein DS is 90% -100%, and nc is the number of chirally controlled internucleotide linkages. In some embodiments, nc is 5, 6, 7, 8, 9, 10, or greater. In some embodiments, the percentage/level is at least 10%. In some embodiments, the percentage/level is at least 20%. In some embodiments, the percentage/level is at least 30%. In some embodiments, the percentage/level is at least 40%. In some embodiments, the percentage/level is at least 50%. In some embodiments, the percentage/level is at least 60%. In some embodiments, the percentage/level is at least 65%. In some embodiments, the percentage/level is at least 70%. In some embodiments, the percentage/level is at least 75%. In some embodiments, the percentage/level is at least 80%. In some embodiments, the percentage/level is at least 85%. In some embodiments, the percentage/level is at least 90%. In some embodiments, the percentage/level is at least 95%.

In some embodiments, multiple ds oligonucleotides share a common backbone linkage pattern. In some embodiments, each ds oligonucleotide of the plurality of ds oligonucleotides independently has an internucleotide linkage of a particular composition (e.g., -O-P (O) (SH) -O-) or a salt form thereof (e.g., -O-P (O) (SNa) -O-) at each internucleotide linkage site. In some embodiments, the internucleotide linkages at each internucleotide linkage site are of the same form. In some embodiments, the internucleotide linkages at each internucleotide linkage site have a different form.

In some embodiments, multiple ds oligonucleotides share a common composition. In some embodiments, the plurality of ds oligonucleotides have the same form of common composition. In some embodiments, the plurality of ds oligonucleotides have two or more forms of common constitution. In some embodiments, the plurality of ds oligonucleotides each independently has a specific ds oligonucleotide or a pharmaceutically acceptable salt thereof, or independently has a ds oligonucleotide of the same composition as a specific ds oligonucleotide or a pharmaceutically acceptable salt thereof. In some embodiments, about 1% -100% (e.g., ,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%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all ds oligonucleotides sharing a common composition in a composition is the plurality of oligonucleotides. In some embodiments, the percentage of levels is or at least is (DS) ^nc, where DS is 90% -100%, and nc is the number of chirally controlled internucleotide linkages. In some embodiments, nc is 5, 6, 7, 8, 9,10, or greater. In some embodiments, the level is at least 10%. In some embodiments, the level is at least 20%. In some embodiments, the level is at least 30%. In some embodiments, the level is at least 40%. In some embodiments, the level is at least 50%. In some embodiments, the level is at least 60%. In some embodiments, the level is at least 65%. In some embodiments, the level is at least 70%. In some embodiments, the level is at least 75%. In some embodiments, the level is at least 80%. In some embodiments, the level is at least 85%. In some embodiments, the level is at least 90%. In some embodiments, the level is at least 95%.

In some embodiments, each phosphorothioate internucleotide linkage is independently a chirally controlled internucleotide linkage.

In some embodiments, the present disclosure provides a chirally controlled ds oligonucleotide composition comprising a plurality of oligonucleotides of a particular oligonucleotide type characterized by:

a) A common base sequence;

b) A common backbone linkage pattern;

c) A common backbone chiral center pattern;

Wherein the composition enriches ds oligonucleotides of the particular ds oligonucleotide type relative to a substantially racemic preparation of ds oligonucleotides having the same common base sequence.

In some embodiments, the present disclosure provides a chirally controlled ds oligonucleotide composition comprising a plurality of ds oligonucleotides of a particular ds oligonucleotide type characterized by:

a) A common base sequence;

b) A common backbone linkage pattern;

c) A common backbone chiral center pattern;

wherein the plurality of ds oligonucleotides comprises at least one internucleotide linkage comprising a common linkage phosphorus in the Sp configuration;

Wherein the composition enriches ds oligonucleotides of the particular ds oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same common base sequence.

As understood by those skilled in the art, a common backbone chiral center pattern includes at least one Rp or at least one Sp. Some backbone chiral center patterns are shown, for example, in table 1.

In some embodiments, the chirally controlled ds oligonucleotide composition is enriched for oligonucleotides of a particular ds oligonucleotide type relative to a substantially racemic preparation of ds oligonucleotides sharing the same common base sequence and common backbone linkage pattern.

In some embodiments, a plurality of ds oligonucleotides (e.g., a particular ds oligonucleotide type) have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, the plurality of ds oligonucleotides have a common sugar modification pattern. In some embodiments, multiple ds oligonucleotides have a common base modification pattern. In some embodiments, a plurality of ds oligonucleotides have a common nucleoside modification pattern. In some embodiments, the plurality of ds oligonucleotides have the same composition. In many embodiments, the plurality of ds oligonucleotides are identical. In some embodiments, multiple ds oligonucleotides have the same oligonucleotide (as will be appreciated by those skilled in the art, such ds oligonucleotides may each independently exist in one of a plurality of forms of ds oligonucleotides, and may be the same or different forms of oligonucleotides). In some embodiments, the plurality of ds oligonucleotides each independently have the same ds oligonucleotide or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure provides chirally controlled ds oligonucleotide compositions, e.g., the chirally controlled ds oligonucleotide compositions of table 1 containing a plurality of ds oligonucleotides of S and/or R in their "stereochemistry/linkage". In some embodiments, the plurality of ds oligonucleotides are each independently a particular oligonucleotide (which "stereochemistry/linkage" contains S and/or R) in table 1, optionally in various forms. In some embodiments, each of the plurality of oligonucleotides is independently a particular oligonucleotide (whose "stereochemistry/linkage" contains S and/or R) in table 1 or a pharmaceutically acceptable salt thereof.

In some embodiments, the level of a plurality of ds oligonucleotides in a composition can be determined as the product of the diastereoisomeric purity of each chiral controlled internucleotide linkage in the oligonucleotide. In some embodiments, the diastereoisomeric purity of the internucleotide linkage linking two nucleosides in an oligonucleotide (or nucleic acid) is represented by the diastereoisomeric purity of the internucleotide linkage linking the same two nucleoside dimer, wherein comparable conditions (in some cases, the same synthetic cycling conditions) are used to prepare the dimer.

In some embodiments, all chiral internucleotide linkages are independently chirally controlled, and the composition is a completely chirally controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chiral controlled internucleotide linkages, and the compositions are partially chiral controlled oligonucleotide compositions.

Oligonucleotides may comprise or consist of a plurality of modes of backbone chiral centers (stereochemical modes of chiral linkage phosphorus). Certain useful modes of backbone chiral centers are described in this disclosure. In some embodiments, multiple oligonucleotides share a common backbone chiral center pattern that is or comprises the patterns described in the disclosure (e.g., backbone chiral center patterns of chirally controlled oligonucleotides in table 1, as described in "stereochemistry and backbone chiral center pattern", etc.).

In some embodiments, the chirally controlled oligonucleotide composition is a chirally pure (or stereopure, stereochemically pure) oligonucleotide composition, wherein the oligonucleotide composition comprises a plurality of oligonucleotides, wherein the oligonucleotides independently have the same stereoisomer [ including each chiral element of the oligonucleotide, including each chiral linkage phosphorus, are independently defined (stereodefining) ]. The chirally pure (or stereopure, stereochemically pure) oligonucleotide composition of oligonucleotide stereoisomers does not comprise other stereoisomers (as understood by those skilled in the art, one or more undesired stereoisomers may be present as impurities from preparation, storage, etc.).

Chiral controlled oligonucleotide compositions may exhibit a number of advantages over stereorandom oligonucleotide compositions. Among other things, the chirally controlled oligonucleotide composition is more homogeneous with respect to the oligonucleotide structure than the corresponding stereotactic oligonucleotide composition. By controlling stereochemistry, compositions of individual stereoisomers can be prepared and evaluated, such that chiral controlled oligonucleotide compositions of stereoisomers having desired properties and/or activity can be developed. In some embodiments, the chiral controlled oligonucleotide composition provides better delivery, stability, clearance, activity, selectivity, and/or toxicity characteristics than, for example, a corresponding stereorandom oligonucleotide composition. In some embodiments, the chirally controlled oligonucleotide composition provides better efficacy, fewer side effects, and/or a more convenient and effective dosage regimen. Backbone chiral center patterns as described herein can be used to provide, among other things, controlled cleavage of an oligonucleotide target (e.g., a transcript, such as a pre-mRNA, mature mRNA, etc., including control of cleavage sites, cleavage rate and/or extent at cleavage sites, and/or total cleavage rate and extent, etc.).

In some embodiments, the oligonucleotides in the provided compositions, e.g., chiral controlled oligonucleotide compositions, are ds oligonucleotides targeting HSD17B13 as described herein.

In some embodiments, the disclosure provides a stereotactic oligonucleotide composition, e.g., a stereotactic ds oligonucleotide composition targeting HSD17B 13. In some embodiments, the disclosure provides a stereoscopic, random HSD17B 13-targeting ds oligonucleotide composition capable of reducing the level, activity, or expression of the HSD17B13 gene or gene product thereof. In some embodiments, the disclosure provides a stereospecific ds oligonucleotide composition targeting HSD17B13 capable of reducing the level, activity or expression of the HSD17B13 gene or gene product thereof, and wherein the base sequence of the ds oligonucleotide targeting HSD17B13 is a base sequence disclosed herein (e.g., a base sequence in table 1, wherein each T can be independently replaced with a U, and vice versa), comprising a base sequence disclosed herein or a sequence segment (e.g., at least 10 or 15 consecutive bases) comprising a base sequence disclosed herein.

In some embodiments, the oligonucleotide composition comprises one or more stereocontrolled (chiral controlled; in some embodiments, stereopure) internucleotide linkages and one or more stereorandom internucleotide linkages. In some embodiments, the ds oligonucleotide composition that targets HSD17B13 comprises one or more stereotactically controlled (chirally controlled; in some embodiments, stereotactically pure) internucleotide linkages and one or more stereotactically random internucleotide linkages.

In some embodiments, the oligonucleotide composition comprises one or more stereotactic (e.g., chirally controlled or stereotactic pure) internucleotide linkages and one or more stereotactic random internucleotide linkages. Such oligonucleotides may target various targets and may have various base sequences, and may be capable of manipulation by one or more of a variety of means (e.g., rnase H mechanism, steric hindrance, double or single stranded RNA interference, exon skipping regulation, CRISPR, aptamers, etc.).

In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B 13. In some embodiments, provided chiral controlled oligonucleotide compositions comprise a plurality of identically configured oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) and have one or more internucleotide linkages. In some embodiments, for example, the plurality of oligonucleotides in the chiral controlled oligonucleotide composition is a plurality of oligonucleotides selected from table 1, wherein the oligonucleotides comprise at least one Rp or Sp linkage phosphorus in a chiral controlled internucleotide linkage. In some embodiments, for example, the plurality of oligonucleotides in the chirally controlled oligonucleotide composition is a plurality of oligonucleotides selected from table 1, wherein each phosphorothioate internucleotide linkage in the oligonucleotide is independently chirally controlled (each phosphorothioate internucleotide linkage is independently Rp or Sp). In some embodiments, an oligonucleotide composition, e.g., a ds oligonucleotide composition that targets HSD17B13, is a substantially pure preparation of a single oligonucleotide, because in some cases, after certain purification procedures, oligonucleotides in the composition that are not the single oligonucleotide are impurities from the preparation of the single oligonucleotide. In some embodiments, a single oligonucleotide is an oligonucleotide of table 1, wherein each chiral internucleotide linkage of the oligonucleotide is chirally controlled (e.g., denoted S or R, but not X in "stereochemistry/linkage").

In some embodiments, the chirally controlled oligonucleotide compositions may have increased activity and/or stability, increased delivery, and/or reduced ability to cause adverse effects such as complement, TLR9 activation, and the like, relative to corresponding stereotactic random oligonucleotide compositions. In some embodiments, a stereotactic (achiral controlled) oligonucleotide composition differs from a chiral controlled oligonucleotide composition in that its corresponding plurality of oligonucleotides does not contain any chiral controlled internucleotide linkages, but the stereotactic oligonucleotide composition is otherwise identical to the chiral controlled oligonucleotide composition.

In some embodiments, the disclosure relates to chirally controlled ds oligonucleotide compositions targeting HSD17B13 capable of reducing the level, activity, or expression of the HSD17B13 gene or gene product thereof.

In some embodiments, the disclosure provides chirally controlled ds oligonucleotide compositions targeting HSD17B13 capable of reducing the level, activity, or expression of the HSD17B13 gene or gene product thereof, and comprising a plurality of oligonucleotides sharing a common base sequence that is or comprises a base sequence disclosed herein (e.g., in table 1, wherein each T may be independently replaced by a U, and vice versa).

In some embodiments, the provided chirally controlled oligonucleotide composition is a chirally controlled ds oligonucleotide composition targeting HSD17B13 comprising a plurality of ds oligonucleotides targeting HSD17B 13. In some embodiments, the chirally controlled oligonucleotide composition is a chirally pure (or "stereochemically pure") oligonucleotide composition. In some embodiments, the disclosure provides chiral pure oligonucleotide compositions of the oligonucleotides in table 1, wherein each chiral internucleotide linkage of the oligonucleotide is independently chirally controlled (Rp or Sp, e.g., R or S but not X in "stereochemistry/linkage"). As will be appreciated by one of ordinary skill in the art, little, if any, chemoselectivity achieves completeness (absolute 100%). In some embodiments, the chirally pure oligonucleotide composition comprises a plurality of oligonucleotides, wherein the plurality of oligonucleotides are structurally identical and all have the same structure (identical stereoisomeric forms; in the case of oligonucleotides, diastereoisomeric forms that are generally identical to the multichiral centers that are typically present in oligonucleotides), and the chirally pure oligonucleotide composition does not contain any other stereoisomers (in the case of oligonucleotides, diastereoisomers of multichiral centers that are typically present as in oligonucleotides; the extent thereof can be achieved, for example, by stereoselective preparation). As understood by those of skill in the art, a stereorandom (or "racemic," "achiral controlled") oligonucleotide composition is a random mixture of many stereoisomers (e.g., 2 ⁿ diastereomers, where n is the number of chiral-linked phosphites of the oligonucleotide, where the other chiral centers (e.g., carbon chiral centers in the sugar) are chirally controlled, each independently exist in one configuration, and only the chiral-linked phosphites centers are not chirally controlled.

Some data are shown, for example, in the examples section herein, which data show the properties and/or activity of a chirally controlled oligonucleotide composition, e.g., a chirally controlled ds oligonucleotide composition targeting HSD17B13, in reducing the level, activity, and/or expression of the HSD17B13 target gene or gene product thereof.

In some embodiments, the disclosure provides oligonucleotide compositions comprising oligonucleotides comprising at least one chiral linkage phosphorus. In some embodiments, the disclosure provides a ds oligonucleotide composition that targets HSD17B13 comprising a ds oligonucleotide that targets HSD17B13, the ds oligonucleotide that targets HSD17B13 comprising at least one chiral linkage phosphorus. In some embodiments, the disclosure provides ds oligonucleotide compositions that target HSD17B13, wherein the ds oligonucleotide that targets HSD17B13 comprises a chirally controlled phosphorothioate internucleotide linkage, wherein the linkage phosphorus has the Rp configuration. In some embodiments, the disclosure provides ds oligonucleotide compositions that target HSD17B13, wherein the ds oligonucleotide that targets HSD17B13 comprises a chirally controlled phosphorothioate internucleotide linkage, wherein the linkage phosphorus has the Sp configuration.

In some embodiments, the provided chiral controlled oligonucleotide compositions (e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B 13) are unexpectedly effective compared to reference oligonucleotide compositions. In some embodiments, the desired biological effect (e.g., as measured by targeted reduced levels of mRNA, protein, etc.) can be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100-fold (e.g., measured by residual levels of mRNA, protein, etc.). In some embodiments, the change is measured by a decrease in undesired mRNA levels compared to a reference condition. In some embodiments, the change is measured by an increase in the desired mRNA level compared to a reference condition. In some embodiments, the change is measured by a decrease in undesired mRNA levels compared to a reference condition. In some embodiments, the reference condition is not treated, e.g., by a chirally controlled oligonucleotide composition. In some embodiments, the reference condition is a corresponding oligonucleotide stereorandom composition having the same composition.

In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein at least one chiral controlled internucleotide-bonded linking phosphorus is Sp. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein the majority of the linkage phosphorus of the chiral controlled internucleotide linkages is Sp. In some embodiments, about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all chiral controlled phosphorothioate internucleotide linkages are Sp. In some embodiments, about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all phosphorothioate internucleotide linkages are chirally controlled and are Sp. In some embodiments, about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all chiral internucleotide linkages (or all chiral internucleotide linkages or all internucleotide linkages) are Sp. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein a majority of chiral internucleotide linkages are chiral controlled and Sp at their linkage phosphorus. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein each chiral internucleotide linkage is chiral controlled and each chiral linked phosphorus is Sp. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein at least one chiral controlled internucleotide linkage has Rp linkage with rplinkage phosphorus. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein at least one chiral controlled internucleotide linkage comprises Rp linkage phosphorus and at least one chiral controlled internucleotide linkage comprises Sp linkage phosphorus. In some embodiments, at least one phosphorothioate internucleotide linkage is chirally controlled and Rp. In some embodiments, about 1-5, e.g., about 1,2,3, 4, or 5 phosphorothioate internucleotide linkages are chirally controlled and are Rp. In some embodiments, about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all chiral controlled non-negatively charged internucleotide linkages (e.g., n 001) are Rp. in some embodiments, each chirally controlled n001 is Rp.

Stereochemistry and pattern of backbone chiral centers

In contrast to natural phosphate linkages, the linkage phosphorus of a chiral modified internucleotide linkage (e.g., phosphorothioate internucleotide linkage) is chiral. Among other things, the present disclosure provides techniques (e.g., oligonucleotides, compositions, methods, etc.) that include controlling the stereochemistry of chiral linkage phosphates in chiral internucleotide linkages. In some embodiments, as demonstrated herein, control of stereochemistry may provide improved properties and/or activity, including desired stability, reduced toxicity, improved target nucleic acid reduction, and the like. In some embodiments, the present disclosure provides a backbone chiral center pattern useful for oligonucleotides and/or regions thereof that is a combination of stereochemistry for each chiral phosphorus (Rp or Sp) of chiral phosphorus linkages, each achiral phosphorus linkage (Op, if present), etc., indicated from 5 'to 3'. In some embodiments, the backbone chiral center pattern can control the cleavage pattern of a target nucleic acid when contacted with a provided oligonucleotide or a composition thereof in a cleavage system (e.g., in vitro assay, cell, tissue, organ, organism, subject, etc.). In some embodiments, the backbone chiral center pattern improves cleavage efficiency and/or selectivity of a target nucleic acid when contacted with a provided oligonucleotide or a composition thereof in a cleavage system.

In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is any (Np) n (Op) m, wherein Np is Rp or Sp, op represents that the linkage phosphorus is achiral (e.g., for a natural phosphate linkage phosphorus), and n and m are each independently as defined and described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Sp) n (Op) m, wherein each variable is independently as defined and described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Rp) n (Op) m, wherein each variable is independently as defined and described in the disclosure. In some embodiments, n is 1. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Sp) (Op) m, wherein m is 1,2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Rp) (Op) m, wherein m is 1,2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the backbone chiral center mode of the 5' -wing is or comprises (Np) n (Op) m. In some embodiments, the backbone chiral center mode of the 5' -wing is or comprises (Sp) n (Op) m. In some embodiments, the backbone chiral center mode of the 5' -wing is or comprises (Rp) n (Op) m. In some embodiments, the backbone chiral center mode of the 5' -wing is or comprises (Sp) (Op) m. In some embodiments, the backbone chiral center mode of the 5' -wing is or comprises (Rp) (Op) m. In some embodiments, the backbone chiral center mode of the 5' -wing is (Sp) (Op) m. In some embodiments, the backbone chiral center mode of the 5' -wing is (Rp) (Op) m. In some embodiments, the backbone chiral center pattern of the 5 '-wing is (Sp) (Op) m, where Sp is the bonding phosphorus configuration from the first internucleotide linkage of the 5' -terminal oligonucleotide. In some embodiments, the backbone chiral center pattern of the 5 '-wing is (Rp) (Op) m, where Rp is the bonding phosphorus configuration of the first internucleotide linkage from the 5' -terminal oligonucleotide. In some embodiments, as described in the present disclosure, m is 2; in some embodiments, m is 3; in some embodiments, m is 4; in some embodiments, m is 5; in some embodiments, m is 6.

In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Op) m (Np) n, wherein Np is Rp or Sp, op represents that the linkage phosphorus is achiral (e.g., linkage phosphorus for a natural phosphate linkage), and n and m are each independently as defined and described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Op) m (Sp) n, wherein each variable is independently as defined and described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Op) m (Rp) n, wherein each variable is independently as defined and described in the disclosure. In some embodiments, n is 1. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Op) m (Sp), wherein m is 1,2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof comprises or is (Op) m (Rp), where m is 1,2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the backbone chiral center mode of the 3' -wing is or comprises (Op) m (Np) n. In some embodiments, the backbone chiral center mode of the 3' -wing is or comprises (Op) m (Sp) n. In some embodiments, the backbone chiral center mode of the 3' -wing is or comprises (Op) m (Rp) n. In some embodiments, the backbone chiral center mode of the 3' -wing is or comprises (Op) m (Sp). In some embodiments, the backbone chiral center mode of the 3' -wing is or comprises (Op) m (Rp). In some embodiments, the backbone chiral center mode of the 3' -wing is (Op) m (Sp). In some embodiments, the backbone chiral center mode of the 3' -wing is (Op) m (Rp). In some embodiments, the backbone chiral center mode of the 3 '-wing is (Op) m (Sp), where Sp is the bonding phosphorus configuration of the last internucleotide linkage of the oligonucleotide from the 5' terminus. In some embodiments, the backbone chiral center pattern of the 3 '-wing is (Op) m (Rp), where Rp is the last internucleotide-bonded phosphorus configuration of the oligonucleotide from the 5' -terminus. In some embodiments, as described in the present disclosure, m is 2; in some embodiments, m is 3; in some embodiments, m is 4; in some embodiments, m is 5; in some embodiments, m is 6.

In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises either (Sp) m (Rp/Op) n or (Rp/Op) n (Sp) m, wherein each variable is independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises either (Sp) m (Rp) n or (Rp) n (Sp) m, wherein each variable is independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises either (Sp) m (Op) n or (Op) n (Sp) m, wherein each variable is independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises either (Np) t [ (Rp/Op) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y (Np) t, wherein y is 1-50, and the other variables are each independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises either (Np) t [ (Rp) n (Sp) m ] y or [ (Rp) n (Sp) m ] y (Np) t, wherein each variable is independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises or is [ (Rp/Op) n (Sp) m ] y (Rp) k, [ (Rp/Op) n (Sp) m ] y, (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k, where k is 1-50, and the other variables are each independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises or is [ (Op) n (Sp) m ] y (Rp) k, [ (Op) n (Sp) m ] y, (Sp) t [ (Op) n (Sp) m ] y (Rp) k, wherein each variable is independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of an oligonucleotide or region thereof (e.g., core) comprises or is [ (Rp) n (Sp) m ] y (Rp) k, [ (Rp) n (Sp) m ] y, (Sp) t [ (Rp) n (Sp) m ] y (Rp) k, wherein each variable is independently as described in the disclosure. In some embodiments, the oligonucleotide comprises a core region. In some embodiments, the oligonucleotide comprises a core region, wherein each sugar in the core region does not contain 2' -OR ¹, wherein R ¹ is as described in the disclosure. In some embodiments, the oligonucleotide comprises a core region, wherein each sugar in the core region is independently a natural DNA sugar. In some embodiments, the backbone chiral center mode of the core comprises or is (Rp) (Sp) m. In some embodiments, the backbone chiral center mode of the core comprises or is (Op) (Sp) m. In some embodiments, the backbone chiral center pattern of the core comprises either (Np) t [ (Rp/Op) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y (Np) t. In some embodiments, the backbone chiral center pattern of the core comprises either (Np) t [ (Rp/Op) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y (Np) t. In some embodiments, the backbone chiral center pattern of the core comprises either (Np) t [ (Rp) n (Sp) m ] y or [ (Rp) n (Sp) m ] y (Np) t. In some embodiments, the backbone chiral center pattern of the core comprises or is [ (Rp/Op) n (Sp) m ] y (Rp) k, [ (Rp/Op) n (Sp) m ] y, (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern of the core comprises either [ (Op) n (Sp) m ] y (Rp) k, [ (Op) n (Sp) m ] y, (Sp) t [ (Op) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern of the core comprises either [ (Rp) n (Sp) m ] y (Rp) k, [ (Rp) n (Sp) m ] y, (Sp) t [ (Rp) n (Sp) m ] y, or (Sp) t [ (Rp) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern of the core comprises [ (Rp) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern of the core comprises [ (Rp) n (Sp) m ] y (Rp). In some embodiments, the backbone chiral center pattern of the core comprises [ (Rp) n (Sp) m ] y. In some embodiments, the backbone chiral center pattern of the core comprises (Sp) t [ (Rp) n (Sp) m ] y. In some embodiments, the backbone chiral center pattern of the core comprises (Sp) t [ (Rp) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern of the core comprises (Sp) t [ (Rp) n (Sp) m ] y (Rp). In some embodiments, the backbone chiral center mode of the core is [ (Rp) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center mode of the core is [ (Rp) n (Sp) m ] y (Rp). In some embodiments, the backbone chiral center mode of the core is [ (Rp) n (Sp) m ] y. In some embodiments, the backbone chiral center mode of the core is (Sp) t [ (Rp) n (Sp) m ] y. In some embodiments, the backbone chiral center mode of the core is (Sp) t [ (Rp) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center mode of the core is (Sp) t [ (Rp) n (Sp) m ] y (Rp). In some embodiments, each n is 1. In some embodiments, each t is 1. In some embodiments, t is 2, 3, 4, 5, 6, 7, 8, 9, or 10. in some embodiments, t and n are each 1. In some embodiments, each m is 2 or greater. In some embodiments, k is 1. In some embodiments, k is 2-10.

In some embodiments, the backbone chiral center pattern comprises either (Sp)m(Rp)n、(Rp)n(Sp)m、(Np)t(Rp)n(Sp)m、(Sp)t(Rp)n(Sp)m、(Np)t[(Rp)n(Sp)m]2、(Sp)t[(Rp)n(Sp)m]2、(Np)t(Op)n(Sp)m、(Sp)t(Op)n(Sp)m、(Np)t[(Op)n(Sp)m]2、 or (Sp) t [ (Op) n (Sp) m ]2. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) 1-5 (Op/Rp) n (Sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) 2-5 (Op/Rp) n (Sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) 2 (Op/Rp) n (Sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) 3 (Op/Rp) n (Sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) 4 (Op/Rp) n (Sp) m. In some embodiments, the pattern is (Np) t (Op/Rp) n (Sp) 5 (Op/Rp) n (Sp) m.

In some embodiments, np is Sp. In some embodiments, (Op/Rp) is Op. In some embodiments, (Op/Rp) is Rp. In some embodiments, np is Sp and (Op/Rp) is Rp. In some embodiments, np is Sp and (Op/Rp) is Op. In some embodiments, np is Sp and at least one (Op/Rp) is Rp and at least one (Op/Rp) is Op. In some embodiments, the backbone chiral center pattern comprises either (Rp) n (Sp) m, (Np) t (Rp) n (Sp) m, or (Sp) t (Rp) n (Sp) m, where m >2. In some embodiments, the backbone chiral center pattern comprises either (Rp) n (Sp) m, (Np) t (Rp) n (Sp) m, or (Sp) t (Rp) n (Sp) m, where n is 1, at least one t >1, and at least one m >2.

In some embodiments, oligonucleotides comprising a core region (whose backbone chiral center pattern starts with Rp) may provide high activity and/or improved properties. In some embodiments, oligonucleotides comprising a core region (whose backbone chiral center pattern ends with Rp) may provide high activity and/or improved properties. In some embodiments, an oligonucleotide comprising a core region (whose backbone chiral center pattern begins with Rp) provides high activity (e.g., target cleavage) without significantly affecting its properties (e.g., stability). In some embodiments, an oligonucleotide comprising a core region (whose backbone chiral center pattern ends with Rp) provides high activity (e.g., target cleavage) without significantly affecting its properties (e.g., stability). In some embodiments, the backbone chiral center mode starts with Rp and ends with Sp. In some embodiments, the backbone chiral center mode starts with Rp and ends with Rp. In some embodiments, the backbone chiral center mode starts with Sp and ends with Rp. Typically, for backbone chiral center modes, internucleotide linkages linking the core nucleoside and the winged nucleoside are included in the core region mode. In many embodiments as described in the present disclosure (e.g., the various oligonucleotides in table 1), the winged saccharides that are linked by such linked internucleotide linkages have a different structure than the core saccharides that are linked by the same linked internucleotide linkages (e.g., in some embodiments, the winged saccharides comprise 2 '-modifications, while the core saccharides do not comprise the same 2' -modification or have two-H's at the 2' position). In some embodiments, the winged sugar comprises a sugar modification not comprised by the core sugar. In some embodiments, the winged sugar is a modified sugar and the core sugar is a natural DNA sugar. In some embodiments, the winged sugar comprises a sugar modification at the 2 'position (less than two-H's at the 2 'position) and the core sugar has no modification at the 2' position (two-H's at the 2' position).

In some embodiments, as demonstrated herein, additional Rp internucleotide linkages connect a sugar that does not contain a 2' -substituent (e.g., a core sugar) and a sugar that comprises a 2' -modification (e.g., 2' -OR ', where R ' is an optionally substituted C _1-6 aliphatic (e.g., 2' -OMe, 2' -MOE, etc.), which may be a winged sugar). In some embodiments, the internucleotide linkage connecting a sugar containing no 2 '-substituent to the 5' -terminus (e.g., to the 3 '-carbon of the sugar) and connecting a sugar containing a 2' -modification to the 3 '-terminus (e.g., to the 5' -carbon of the sugar) is Rp internucleotide linkage. In some embodiments, the internucleotide linkage connecting a sugar containing no 2 '-substituent to the 3' -terminus (e.g., to the 5 '-carbon of the sugar) and connecting a sugar containing a 2' -modification to the 5 '-terminus (e.g., to the 3' -carbon of the sugar) is Rp internucleotide linkage. In some embodiments, each internucleotide linkage connecting a sugar containing no 2 '-substituents and a sugar containing a 2' -modification is independently an Rp internucleotide linkage. In some embodiments, the Rp internucleotide linkage is an Rp phosphorothioate internucleotide linkage.

In some embodiments, the backbone chiral center pattern of the ds oligonucleotide or region thereof (e.g., core) that targets HSD17B13 comprises or is (Op) [ (Rp/Op) n (Sp) m ] y (Rp) k (Op), (Op) [ (Rp/Op) n (Sp) m ] y (Op), (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Op), or (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op), where k is 1-50, and the other variables are each independently as described in the disclosure. In some embodiments, the backbone chiral center pattern of the ds oligonucleotide targeting HSD17B13 comprises either (Op) [ (Rp/Op) n (Sp) m ] y (Rp) k (Op), (Op) [ (Rp/Op) n (Sp) m ] y (Op), (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Op), or (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op), wherein f, g, h, and j are each independently 1-50, and the other variables are each independently as described in the disclosure, and the oligonucleotide comprises a core region, The backbone chiral center pattern of the core region comprises either [ (Rp/Op) n (Sp) m ] y (Rp) k, [ (Rp/Op) n (Sp) m ] y, (Sp) t [ (Rp/Op) n (Sp) m ] y, or (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k as described in the present disclosure. In some embodiments, the backbone chiral center pattern is or comprises (Op) [ (Rp/Op) n (Sp) m ] y (Rp) k (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) [ (Rp/Op) n (Sp) m ] y (Rp) (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) [ (Rp/Op) n (Sp) m ] y (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) [ (Rp) n (Sp) m ] y (Rp) k (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) [ (Rp) n (Sp) m ] y (Rp) (Op). In some embodiments, the backbone chiral center mode is or comprises (Op) [ (Rp) n (Sp) m ] y (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) (Sp) t [ (Rp) n (Sp) m ] y (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) (Sp) t [ (Rp) n (Sp) m ] y (Rp) k (Op). In some embodiments, the backbone chiral center pattern is or comprises (Op) (Sp) t [ (Rp) n (Sp) m ] y (Rp) (Op). In some embodiments, each n is 1. In some embodiments, k is 1. In some embodiments, k is 2-10.

In some embodiments, the backbone chiral center pattern of the ds oligonucleotide or region thereof (e.g., core) that targets HSD17B13 comprises either (Np)f(Op)g[(Rp/Op)n(Sp)m]y(Rp)k(Op)h(Np)j、(Np)f(Op)g[(Rp/Op)n(Sp)m]y(Op)h(Np)j、(Np)f(Op)g(Sp)t[(Rp/Op)n(Sp)m]y(Op)h(Np)j、 or (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j, wherein f, g, h, and j are each independently 1-50, and the other variables are each independently as described in the disclosure. in some embodiments, the backbone chiral center pattern of the ds oligonucleotide targeting HSD17B13 comprises either (Np)f(Op)g[(Rp/Op)n(Sp)m]y(Rp)k(Op)h(Np)j、(Np)f(Op)g[(Rp/Op)n(Sp)m]y(Op)h(Np)j、(Np)f(Op)g(Sp)t[(Rp/Op)n(Sp)m]y(Op)h(Np)j、 or (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j, and the oligonucleotide comprises a core region comprising either [ (Rp/Op) n (Sp) m ] y (Rp) k, [ (Rp/Op) n (Sp) m ] y, (Sp) t [ (Rp/Op) n (Sp) m ] y, or (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern of the ds oligonucleotide targeting HSD17B13 is (Np)f(Op)g[(Rp/Op)n(Sp)m]y(Rp)k(Op)h(Np)j、(Np)f(Op)g[(Rp/Op)n(Sp)m]y(Op)h(Np)j、(Np)f(Op)g(Sp)t[(Rp/Op)n(Sp)m]y(Op)h(Np)j、 or (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j, and the oligonucleotide comprises a core region comprising or as described in the disclosure [ (Rp/Op) n (Sp) m ] y (Rp) k, [ (Rp/Op) n (Sp) m ] y, (Sp) t [ (Rp/Op) n (Sp) m ] y, or (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g [ (Rp/Op) n (Sp) m ] y (Rp) (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g [ (Rp/Op) n (Sp) m ] y (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Op) h (Np) j. in some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g [ (Rp) n (Sp) m ] y (Rp) k (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g [ (Rp) n (Sp) m ] y (Rp) (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g [ (Rp) n (Sp) m ] y (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Rp) k (Op) h (Np) j. In some embodiments, the backbone chiral center pattern is or comprises (Np) f (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Rp) (Op) h (Np) j. In some embodiments, at least one Np is Sp. In some embodiments, at least one Np is Rp. In some embodiments, the 5' maximum Np is Sp. In some embodiments, the 3' maximum Np is Sp. In some embodiments, each Np is Sp. In some embodiments, (Np) f (Op) g [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j is (Sp) (Op) g [ (Rp) n (Sp) m ] y (Rp) k (Op) h (Sp). In some embodiments, (Np) f (Op) g [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j is (Sp) (Op) g [ (Rp) n (Sp) m ] y (Rp) (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises (Sp) (Op) g [ (Rp) n (Sp) m ] y (Rp) (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is (Sp) (Op) g [ (Rp) n (Sp) m ] y (Rp) (Op) h (Sp). In some embodiments, (Np) f (Op) g [ (Rp/Op) n (Sp) m ] y (Op) h (Np) j is (Sp) (Op) g [ (Rp) n (Sp) m ] y (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises (Sp) (Op) g [ (Rp) n (Sp) m ] y (Op) h (Sp). in some embodiments, the backbone chiral center pattern of the oligonucleotide is (Sp) (Op) g [ (Rp) n (Sp) m ] y (Op) h (Sp). In some embodiments, (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Op) h (Np) j is (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Op) h (Sp). In some embodiments, (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j is (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Rp) k (Op) h (Sp). In some embodiments, (Np) f (Op) g (Sp) t [ (Rp/Op) n (Sp) m ] y (Rp) k (Op) h (Np) j is (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Rp) (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Rp) (Op) h (Sp). In some embodiments, the backbone chiral center pattern of the oligonucleotide is (Sp) (Op) g (Sp) t [ (Rp) n (Sp) m ] y (Rp) (Op) h (Sp). In some embodiments, each n is 1. In some embodiments, f is 1. In some embodiments, g is 1. In some embodiments, g is greater than 1. In some embodiments, g is 2. In some embodiments, g is 3. In some embodiments, g is 4. In some embodiments, g is 5. In some embodiments, g is 6. In some embodiments, g is 7. In some embodiments, g is 8. In some embodiments, g is 9. In some embodiments, g is 10. In some embodiments, h is 1. In some embodiments, h is greater than 1. In some embodiments, h is 2. In some embodiments, h is 3. In some embodiments, h is 4. In some embodiments, h is 5. In some embodiments, h is 6. In some embodiments, h is 7. In some embodiments, h is 8. In some embodiments, h is 9. in some embodiments, h is 10. In some embodiments, j is 1. In some embodiments, k is 1. In some embodiments, k is 2-10.

In some embodiments, the backbone chiral center pattern of the ds oligonucleotide or region (e.g., core) thereof that targets HSD17B13 comprises or [(Rp/Op)n(Sp)m]y、(Sp)t[(Rp/Op)n(Sp)m]y、(Sp)t[(Rp/Op)n(Sp)m]yRp、[(Rp/Op)n(Sp)m]y(Rp)k、(Sp)t[(Rp/Op)n(Sp)m]y(Rp)k、(Sp)t[(Rp/Op)n(Sp)m]y(Rp)k(Op)h、(Sp)t[(Rp/Op)n(Sp)m]y(Rp)k(Op)h(Np)j, wherein each variable is independently as described in the disclosure.

In some embodiments, in the provided backbone chiral center mode, at least one (Rp/Op) is Rp. In some embodiments, at least one (Rp/Op) is Op. In some embodiments, each (Rp/Op) is Rp. In some embodiments, each (Rp/Op) is Op. In some embodiments, at least one of the [ (Rp) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y of the pattern is RpSp. In some embodiments, at least one of the [ (Rp) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y of the pattern is or comprises RpSpSp. In some embodiments, at least one of [ (Rp) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y in the mode is RpSp and at least one of [ (Rp) n (Sp) m ] y or [ (Rp/Op) n (Sp) m ] y in the mode is or comprises RpSpSp. For example, in some embodiments [ (Rp) n (Sp) m ] y is (RpSp) [ (Rp) n (Sp) m ] _(y-1) in the pattern; in some embodiments, [ (Rp) n (Sp) m ] y in the pattern is (RpSp) [ Rp spsp (Sp) _(m-2)][(Rp)n(Sp)m]_(y-2). In some embodiments, (Sp) t [ (Rp) n (Sp) m ] y (Rp) is (Sp) t (Rp Sp) [ (Rp) n (Sp) m ] _(y-1) (Rp). In some embodiments, (Sp) t [ (Rp) n (Sp) m ] y (Rp) is (Sp) t (RpSp) [ rpspp (Sp) _(m-2)][(Rp)n(Sp)m]_(y-2) (Rp). In some embodiments, each [ (Rp/Op) n (Sp) m ] is independently [ Rp (Sp) m ]. In some embodiments, the first Sp of (Sp) t represents the first internucleotide-bonded phosphorus stereochemistry of the 5 'to 3' oligonucleotide. In some embodiments, the first Sp of (Sp) t represents the first internucleotide-bonded phosphorus stereochemistry of the 5 'to 3' region (e.g., core). In some embodiments, the last Np of (Np) j represents the last internucleotide-bonded phosphorus stereochemistry of the 5 'to 3' oligonucleotide. In some embodiments, the last Np is Sp.

In some embodiments, the backbone chiral center pattern (e.g., of the 5' -wing) of the oligonucleotide or region is or comprises Sp (Op) ₃. In some embodiments, the backbone chiral center pattern (e.g., of the 5' -wing) of the oligonucleotide or region is or comprises Rp (Op) ₃. In some embodiments, the backbone chiral center pattern (e.g., of the 3' -wing) of the oligonucleotide or region is or comprises (Op) ₃ Sp. In some embodiments, the backbone chiral center pattern (e.g., of the 3' -wing) of the oligonucleotide or region is or comprises (Op) ₃ Rp. In some embodiments, the backbone chiral center pattern (e.g., of the core) of the oligonucleotide or region is or comprises Rp (Sp) ₄Rp(Sp)₄ Rp. In some embodiments, the backbone chiral center pattern (e.g., of the core) of the oligonucleotide or region is or comprises (Sp) ₅Rp(Sp)₄ Rp. In some embodiments, the backbone chiral center pattern (e.g., of the core) of the oligonucleotide or region is or comprises (Sp) ₅Rp(Sp)₅. In some embodiments, the backbone chiral center pattern (e.g., of the core) of the oligonucleotide or region is or comprises Rp (Sp) ₄Rp(Sp)₅. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Np (Op) ₃Rp(Sp)₄Rp(Sp)₄Rp(Op)₃ Np. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Np (Op) ₃(Sp)₅Rp(Sp)₄Rp(Op)₃ Np. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Np (Op) ₃(Sp)₅Rp(Sp)₅(Op)₃ Np. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Np (Op) ₃Rp(Sp)₄Rp(Sp)₅(Op)₃ Np. In some embodiments, the backbone chiral center mode of the oligonucleotide is or comprises Sp (Op) ₃Rp(Sp)₄Rp(Sp)₄Rp(Op)₃ Sp. In some embodiments, the backbone chiral center mode of the oligonucleotide is or comprises Sp (Op) ₃(Sp)₅Rp(Sp)₄Rp(Op)₃ Sp. In some embodiments, the backbone chiral center mode of the oligonucleotide is or comprises Sp (Op) ₃(Sp)₅Rp(Sp)₅(Op)₃ Sp. In some embodiments, the backbone chiral center mode of the oligonucleotide is or comprises Sp (Op) ₃Rp(Sp)₄Rp(Sp)₅(Op)₃ Sp. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Rp (Op) ₃Rp(Sp)₄Rp(Sp)₄Rp(Op)₃ Rp. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Rp (Op) ₃(Sp)₅Rp(Sp)₄Rp(Op)₃ Rp. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Rp (Op) ₃(Sp)₅Rp(Sp)₅(Op)₃ Rp. In some embodiments, the backbone chiral center pattern of the oligonucleotide is or comprises Rp (Op) ₃Rp(Sp)xRp(Sp)₅(Op)₃ Rp.

In some embodiments m, y, t, n, k, f, g, h, and j are each independently 1-25.

In some embodiments, m is 1-25. In some embodiments, m is 1-20. In some embodiments, m is 1-15. In some embodiments, m is 1-10. In some embodiments, m is 1-5. In some embodiments, m is 2-20. In some embodiments, m is 2-15. In some embodiments, m is 2-10. In some embodiments, m is 2-5. In some embodiments, m is 1, 2,3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, in the backbone chiral center mode, each m is independently 2 or greater. In some embodiments, each m is independently 2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each m is independently 2-3, 2-5, 2-6, or 2-10. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, where there are two or more m, they may be the same or different, and each independently as described in the present disclosure.

In some embodiments, y is 1-25. In some embodiments, y is 1-20. In some embodiments, y is 1-15. In some embodiments, y is 1-10. In some embodiments, y is 1-5. In some embodiments, y is 2-20. In some embodiments, y is 2-15. In some embodiments, y is 2-10. In some embodiments, y is 2-5. In some embodiments, y is 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, y is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10.

In some embodiments, t is 1-25. In some embodiments, t is 1-20. In some embodiments, t is 1-15. In some embodiments, t is 1-10. In some embodiments, t is 1-5. In some embodiments, t is 2-20. In some embodiments, t is 2-15. In some embodiments, t is 2-10. In some embodiments, t is 2-5. In some embodiments, t is 1,2,3,4, 5,6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, each t is independently 1,2,3,4, 5,6, 7, 8, 9, or 10. In some embodiments, t is 2 or greater. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, t is 4. In some embodiments, t is 5. In some embodiments, t is 6. In some embodiments, t is 7. In some embodiments, t is 8. In some embodiments, t is 9. In some embodiments, t is 10. In some embodiments, where there are two or more t, they may be the same or different, and each independently as described in the present disclosure.

In some embodiments, n is 1-25. In some embodiments, n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. In some embodiments, where there are two or more n, they may be the same or different, and each is independently as described in the present disclosure. In many embodiments, at least one occurrence of n is 1 in the backbone chiral center mode; in some cases, each n is 1.

In some embodiments, k is 1-25. In some embodiments, k is 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, k is 1. In some embodiments, k is 2. In some embodiments, k is 3. In some embodiments, k is 4. In some embodiments, k is 5. In some embodiments, k is 6. In some embodiments, k is 7. In some embodiments, k is 8. In some embodiments, k is 9. In some embodiments, k is 10.

In some embodiments, f is 1-25. In some embodiments, f is 1-20. In some embodiments, f is 1-10. In some embodiments, f is 1-5. In some embodiments, f is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, f is 1. In some embodiments, f is 2. In some embodiments, f is 3. In some embodiments, f is 4. In some embodiments, f is 5. In some embodiments, f is 6. In some embodiments, f is 7. In some embodiments, f is 8. In some embodiments, f is 9. In some embodiments, f is 10.

In some embodiments, g is 1-25. In some embodiments, g is 1-20. In some embodiments, g is 1-10. In some embodiments, g is 1-5. In some embodiments, g is 2-10. In some embodiments, g is 2-5. In some embodiments, g is 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, g is 1. In some embodiments, g is 2. In some embodiments, g is 3. In some embodiments, g is 4. In some embodiments, g is 5. In some embodiments, g is 6. In some embodiments, g is 7. In some embodiments, g is 8. In some embodiments, g is 9. In some embodiments, g is 10.

In some embodiments, h is 1-25. In some embodiments, h is 1-10. In some embodiments, h is 1-5. In some embodiments, h is 2-10. In some embodiments, h is 2-5. In some embodiments, h is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, h is 1. In some embodiments, h is 2. In some embodiments, h is 3. In some embodiments, h is 4. In some embodiments, h is 5. In some embodiments, h is 6. In some embodiments, h is 7. In some embodiments, h is 8. In some embodiments, h is 9. In some embodiments, h is 10.

In some embodiments, j is 1-25. In some embodiments, j is 1-10. In some embodiments, j is 1-5. In some embodiments, j is 1,2,3, 4,5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, j is 1. In some embodiments, j is 2. In some embodiments, j is 3. In some embodiments, j is 4. In some embodiments, j is 5. In some embodiments, j is 6. In some embodiments, j is 7. In some embodiments, j is 8. In some embodiments, j is 9. In some embodiments, j is 10.

In some embodiments, at least one n is 1 and at least one m is not less than 2. In some embodiments, at least one n is 1, at least one t is not less than 2, and at least one m is not less than 3. In some embodiments, each n is 1. In some embodiments, t is 1. In some embodiments, at least one t >1. In some embodiments, at least one t >2. In some embodiments, at least one t >3. In some embodiments, at least one t >4. In some embodiments, at least one m >1. In some embodiments, at least one m >2. In some embodiments, at least one m >3. In some embodiments, at least one m >4. In some embodiments, the backbone chiral center pattern comprises one or more achiral natural phosphate linkages. In some embodiments, the sum of m, t, and n (or the sum of m and n without t in one mode) is not less than 5,6,7,8, 9,10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the sum is 5. In some embodiments, the sum is 6. In some embodiments, the sum is 7. In some embodiments, the sum is 8. In some embodiments, the sum is 9. In some embodiments, the sum is 10. In some embodiments, the sum is 11. In some embodiments, the sum is 12. In some embodiments, the sum is 13. In some embodiments, the sum is 14. In some embodiments, the sum is 15.

In some embodiments, the plurality of linkage phosphites in the chiral controlled internucleotide linkage is Sp. In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the chirally controlled internucleotide linkages have an Sp linkage phosphorus. In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all chiral internucleotide linkages are chiral controlled internucleotide linkages with Sp-linked phosphorus. In some embodiments, at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of all internucleotide linkages are chirally controlled internucleotide linkages with Sp-linked phosphorus. In some embodiments, the percentage is at least 20%. In some embodiments, the percentage is at least 30%. In some embodiments, the percentage is at least 40%. In some embodiments, the percentage is at least 50%. In some embodiments, the percentage is at least 60%. In some embodiments, the percentage is at least 65%. In some embodiments, the percentage is at least 70%. In some embodiments, the percentage is at least 75%. In some embodiments, the percentage is at least 80%. In some embodiments, the percentage is at least 90%. In some embodiments, the percentage is at least 95%. In some embodiments, at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 internucleotide linkages are chirally controlled internucleotide linkages having an Sp linkage phosphorus. In some embodiments, at least 5 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 6 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 7 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 8 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 9 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 10 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 11 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 12 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 13 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 14 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 15 internucleotide linkages are chirally controlled internucleotide linkages with Sp linkage phosphorus. In some embodiments, at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 internucleotide linkages are chirally controlled internucleotide linkages having an Rp linkage phosphorus. In some embodiments, no more than 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 internucleotide linkages are chirally controlled internucleotide linkages having an Rp linkage phosphorus. In some embodiments, one and no more than one internucleotide linkage in the oligonucleotide is a chirally controlled internucleotide linkage with Rp linkage phosphorus. In some embodiments, 2 and no more than 2 internucleotide linkages in the oligonucleotide are chirally controlled internucleotide linkages having Rp linkage phosphorus. In some embodiments, 3 and no more than 3 internucleotide linkages in the oligonucleotide are chirally controlled internucleotide linkages having Rp linkage phosphorus. In some embodiments, 4 and no more than 4 internucleotide linkages in the oligonucleotide are chirally controlled internucleotide linkages having Rp linkage phosphorus. In some embodiments, 5 and no more than 5 internucleotide linkages in the oligonucleotide are chirally controlled internucleotide linkages having Rp linkage phosphorus.

In some embodiments, all, substantially all, or a majority of the internucleotide linkages in an oligonucleotide are in the Sp configuration (e.g., about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all chiral internucleotide linkages, or all chiral internucleotide linkages in an oligonucleotide, or about 35%, or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all chiral internucleotide linkages in an oligonucleotide), except for one or a few internucleotide linkages (e.g., 1,2, 3,4, or 5, and/or less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of all chiral internucleotide linkages in an oligonucleotide) are in the Rp configuration. In some embodiments, all, substantially all, or a majority of the internucleotide linkages in the core are in the Sp configuration (e.g., about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more of all chiral internucleotide linkages, or all chiral internucleotide linkages in the core, and/or less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of all chiral internucleotide linkages), except for one or a few internucleotide linkages (e.g., 1,2, 3,4, or 5 of all chiral internucleotide linkages, or all chiral internucleotide linkages in the core) in the Rp configuration. In some embodiments, all, substantially all, or a majority of the internucleotide linkages in the core are phosphorothioates in the Sp configuration (e.g., about 50％-100％、55％-100％、60％-100％、65％-100％、70％-100％、75％-100％、80％-100％、85％-100％、90％-100％、55％-95％、60％-95％、65％-95％、 or about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of all chiral internucleotide linkages, or all chiral internucleotide linkages in the core, or all chiral internucleotide linkages, or 1,2, 3,4, or 5, and/or less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the internucleotide linkages) are phosphorothioates in the Rp configuration. In some embodiments, each internucleotide linkage in the core is a phosphorothioate in the Sp configuration, except for one in the Rp configuration. In some embodiments, each internucleotide linkage in the core is a phosphorothioate in the Sp configuration, except for one in the Rp configuration.

In some embodiments, the oligonucleotide comprises one or more Rp internucleotide linkages. In some embodiments, the oligonucleotide comprises one and no more than one Rp internucleotide linkage. In some embodiments, the oligonucleotide comprises two or more Rp internucleotide linkages. In some embodiments, the oligonucleotide comprises three or more Rp internucleotide linkages. In some embodiments, the oligonucleotide comprises four or more Rp internucleotide linkages. In some embodiments, the oligonucleotide comprises five or more Rp internucleotide linkages. In some embodiments, about 5% -50% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp. In some embodiments, about 5% -40% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp. In some embodiments, about 10% -40% of all chiral controlled internucleotide linkages in an oligonucleotide are Rp. In some embodiments, about 15% -40% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp. In some embodiments, about 20% -40% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp. In some embodiments, about 25% -40% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp. In some embodiments, about 30% -40% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp. In some embodiments, about 35% -40% of all chiral controlled internucleotide linkages in the oligonucleotide are Rp.

In some embodiments, instead of Rp internucleotide linkages, natural phosphate linkages may be similarly utilized, optionally with modifications such as sugar modifications (e.g., 5' -modifications such as R ^5s described herein). In some embodiments, the modification improves the stability of the natural phosphate linkage.

In some embodiments, the disclosure provides oligonucleotides having backbone chiral center patterns as described herein. In some embodiments, the oligonucleotides in the chirally controlled oligonucleotide composition share a common backbone chiral center pattern as described herein.

In some embodiments, at least about 25% of the internucleotide linkages of the ds oligonucleotide targeting HSD17B13 are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 30% of the internucleotide linkages of the oligonucleotide are chiral controlled and have Sp-linked phosphorus. In some embodiments, at least about 40% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 50% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 60% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 65% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 70% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 75% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 80% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 85% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 90% of the internucleotide linkages of the provided oligonucleotides are chirally controlled and have Sp-linked phosphorus. In some embodiments, at least about 95% of the internucleotide linkages of the provided oligonucleotides are chiral controlled and have an Sp

And (3) bonding phosphorus.

In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions, e.g., chiral controlled ds oligonucleotide compositions targeting HSD17B13, wherein the composition comprises a non-random or controlled level of a plurality of oligonucleotides, wherein the plurality of oligonucleotides share a common base sequence and independently share the same configuration of a linkage phosphorus at 1-50、1-40、1-30、1-25、1-20、1-15、1-10、5-50、5-40、5-30、5-25、5-20、5-15、5-10、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、 or 25 or more chiral internucleotide linkages.

In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises 2-30 chirally controlled internucleotide linkages. In some embodiments, oligonucleotide compositions are provided comprising 5-30 chirally controlled internucleotide linkages. In some embodiments, oligonucleotide compositions are provided that comprise 10-30 chirally controlled internucleotide linkages.

In some embodiments, the percentage is about 5% -100%. In some embodiments, the percentage is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or 99%. In some embodiments, the percentage is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or 99%.

In some embodiments, the backbone chiral center pattern in the ds oligonucleotide targeting HSD17B13 comprises pattern i^o-i^s-i^°-i^s-i^o、i^o-i^s-i^s-i^s-i^o、i^o-i^s-i^s-i^s-i^o-i^s、i^s-i^°-i^s-i^o、i^s-i^o-i^s-i^o、i^s-i^o-i^s-i^o-i^s、i^s-i^o-i^s-i^o-i^s-i^o、i^s-i^o-i^s-i^o-i^s-i^o-i^s-i^o、i^s-i^o-i^s-i^s-i^s-i^o、i^s-i^s-i^o-i^s-i^s-i^s-i^o-i^s-i^s、i^s-i^s-i^s-i^°-i^s-i^o-i^s-i^s-i^s、i^s-i^s-i^s-i^s-i^o-i^s-i^o-i^s-i^s-i^s-i^s、i^s-i^s-i^s-i^s-i^s、i^s-i^s-i^s-i^s-i^s-i^s、i^s-i^s-i^s-i^s-i^s-i^s-i^s、i^s-i^s-i^s-i^s-i^s-i^s-i^s-i^s、i^s-i^s-i^s-i^s-i^s-i^s-i^s-i^s-is or i ^r-i^r-i^r, wherein i ^s represents an internucleotide linkage in the Sp configuration; i ^o represents achiral internucleotide linkages; and i ^r represents an internucleotide linkage in the Rp configuration.

In some embodiments, the internucleotide linkage in the Sp configuration (with Sp linkage phosphorus) is a phosphorothioate internucleotide linkage. In some embodiments, the achiral internucleotide linkages are natural phosphate linkages. In some embodiments, the internucleotide linkage in the Rp configuration (with Rp linkage phosphorus) is a phosphorothioate internucleotide linkage. In some embodiments, each internucleotide linkage in the Sp configuration is a phosphorothioate internucleotide linkage. In some embodiments, each achiral internucleotide linkage is a natural phosphate linkage. In some embodiments, each internucleotide linkage in the Rp configuration is a phosphorothioate internucleotide linkage. In some embodiments, each internucleotide linkage in the Sp configuration is a phosphorothioate internucleotide linkage, each achiral internucleotide linkage is a natural phosphate linkage, and each internucleotide linkage in the Rp configuration is a phosphorothioate internucleotide linkage.

In some embodiments, the backbone chiral center pattern (e.g., in the core or in the wings or in both wings of an oligonucleotide (e.g., a ds oligonucleotide targeting HSD17B 13) or an oligonucleotide (e.g., a ds oligonucleotide targeting HSD17B 13)) comprises pattern OpSpOpSpOp、OpSpSpSpOp、OpSpSpSpOpSp、SpOpSpOp、SpOpSpOp、SpOpSpOpSp、SpOpSpOpSpOp、SpOpSpOpSpOpSpOp、SpOpSpSpSpOp、SpSpOpSpSpSpOpSpSp、SpSpSpOpSpOpSpSpSp、SpSpSpSpOpSpOpSpSpSpSp、SpSpSpSpSp、SpSpSpSpSpSp、SpSpSpSpSpSpSp、SpSpSpSpSpSpSpSp、SpSpSpSpSpSpSpSpSp or RpRpRp, wherein Rp and Sp are each independently a chiral-controlled internucleotide-bonded phosphorus configuration (in some embodiments, rp and Sp are each independently a chiral-controlled phosphorothioate internucleotide-bonded phosphorus configuration), and each Op independently represents a non-chiral phosphorus linkage in a natural phosphate linkage.

In some embodiments, the backbone chiral center pattern (e.g., of an oligonucleotide (e.g., a ds oligonucleotide or a portion thereof that targets HSD17B 13)) is or comprises the backbone chiral center pattern described in table 1.

In some embodiments, for example, when describing the type, modification, number, and/or pattern of core internucleotide linkages, the internucleotide linkages bonded to the winged nucleoside and the core nucleoside are considered to be one of the core internucleotide linkages. In some embodiments, for example, when describing the type, modification, number, and/or pattern of core internucleotide linkages, each internucleotide linkage bonded to a winged nucleoside and a core nucleoside is considered one of the core internucleotide linkages. In some embodiments, the core internucleotide linkage is bonded to two core nucleosides. In some embodiments, the core internucleotide linkages are bonded to core nucleosides and wing nucleosides. In some embodiments, each core internucleotide linkage is independently bonded to two core nucleosides, or a core nucleoside and a winged nucleoside. In some embodiments, each winged internucleotide linkage is independently bonded to two winged nucleosides.

In some embodiments, the ds oligonucleotides targeting HSD17B13 in the chiral controlled oligonucleotide composition each comprise a different type of internucleotide linkage. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least one modified internucleotide linkage. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least two modified internucleotide linkages. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least three modified internucleotide linkages. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least four modified internucleotide linkages. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least five modified internucleotide linkages. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, each modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate triester internucleotide linkage. In some embodiments, each modified internucleotide linkage is a phosphorothioate triester internucleotide linkage. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive modified internucleotide linkages. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive phosphorothioate internucleotide linkages. In some embodiments, the ds oligonucleotide targeting HSD17B13 comprises at least one natural phosphate linkage and at least 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive phosphorothioate triester internucleotide linkages.

In some embodiments, the oligonucleotides in the chiral controlled oligonucleotide composition each comprise at least two different stereochemistry and/or different P-modified internucleotide linkages relative to each other. In some embodiments, at least two internucleotide linkages have different stereochemistry relative to each other, and the oligonucleotides each comprise a backbone chiral center pattern comprising alternating linkage phosphorus stereochemistry.

In some embodiments, the linkage comprises a chiral auxiliary, for example, for controlling the stereoselectivity of the reaction (e.g., a coupling reaction in an oligonucleotide synthesis cycle). In some embodiments, the phosphorothioate triester linkages are free of chiral auxiliary. In some embodiments, phosphorothioate triester linkages are intentionally maintained until administration of the oligonucleotide composition to a subject, and/or phosphorothioate triester linkages are intentionally maintained during administration of the oligonucleotide composition to a subject.

In some embodiments, the purity, particularly the stereochemical purity, and particularly the diastereoisomeric purity of a number of oligonucleotides and their compositions, wherein all other chiral centers in the oligonucleotide, except for the chiral phosphorus center, have been stereodefining (e.g., carbon chiral centers in a sugar, which are defined in phosphoramidites for oligonucleotide synthesis, for example), can be controlled by the stereoselectivity at the chiral phosphorus bond when forming chiral internucleotide linkages in a coupling step (diastereoselectivity in many cases of oligonucleotide synthesis, wherein the oligonucleotide comprises more than one chiral center, as understood by those skilled in the art). In some embodiments, the coupling step has a 60% stereoselectivity at the phosphorus linkage (diastereoselectivity when other chiral centers are present). After such a coupling step, the new internucleotide linkages formed can be considered to have a stereochemical purity of 60% (for oligonucleotides, in view of the presence of other chiral centers, usually diastereoisomeric purity). In some embodiments, each coupling step independently has a stereoselectivity of at least 60%. In some embodiments, each coupling step independently has a stereoselectivity of at least 70%. In some embodiments, each coupling step independently has a stereoselectivity of at least 80%. In some embodiments, each coupling step independently has a stereoselectivity of at least 85%. In some embodiments, each coupling step independently has a stereoselectivity of at least 90%. In some embodiments, each coupling step independently has a stereoselectivity of at least 91%. In some embodiments, each coupling step independently has a stereoselectivity of at least 92%. In some embodiments, each coupling step independently has a stereoselectivity of at least 93%. In some embodiments, each coupling step independently has a stereoselectivity of at least 94%. In some embodiments, each coupling step independently has a stereoselectivity of at least 95%. In some embodiments, each coupling step independently has a stereoselectivity of at least 96%. In some embodiments, each coupling step independently has a stereoselectivity of at least 97%. In some embodiments, each coupling step independently has a stereoselectivity of at least 98%. In some embodiments, each coupling step independently has a stereoselectivity of at least 99%. In some embodiments, each coupling step independently has a stereoselectivity of at least 99.5%. In some embodiments, each coupling step independently has a stereoselectivity of nearly 100%. In some embodiments, the coupling step has a stereoselectivity of nearly 100% because each detectable product from the coupling step has the desired stereoselectivity as analyzed according to the analytical method (e.g., NMR, HPLC, etc.). In some embodiments, chiral controlled internucleotide linkages are typically formed with a stereoselectivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5%, or nearly 100% (in some embodiments, at least 90%, in some embodiments, at least 95%, in some embodiments, at least 96%, in some embodiments, at least 97%, in some embodiments, at least 98%, in some embodiments, at least 99%). In some embodiments, the chirally controlled internucleotide linkages have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5% or almost 100% (in some embodiments, at least 90%) at their chiral linkage phosphorus; In some embodiments, at least 95%; in some embodiments, at least 96%; in some embodiments, at least 97%; in some embodiments, at least 98%; in some embodiments, at least 99%) of stereochemical purity (typically diastereoisomeric purity for oligonucleotides having multiple chiral centers). In some embodiments, each chiral controlled internucleotide linkage independently has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5% or almost 100% (in some embodiments, at least 90%) at its chiral linkage phosphorus; in some embodiments, at least 95%; in some embodiments, at least 96%; in some embodiments, at least 97%; in some embodiments, at least 98%; in some embodiments, at least 99%) of stereochemical purity (typically diastereoisomeric purity for oligonucleotides having multiple chiral centers). In some embodiments, the achiral controlled internucleotide linkages are typically at less than 60%, 70%, 80%, 85%, or 90% (in some embodiments, less than 60%, in some embodiments, less than 70%, in some embodiments, less than 80%, in some embodiments, less than 85%; In some embodiments, less than 90%) of stereoselective formation. In some embodiments, each achiral controlled internucleotide linkage independently forms with a stereoselectivity of less than 60%, 70%, 80%, 85%, or 90% (in some embodiments, less than 60%, in some embodiments, less than 70%, in some embodiments, less than 80%, in some embodiments, less than 85%, in some embodiments, less than 90%). In some embodiments, the achiral controlled internucleotide linkages have less than 60%, 70%, 80%, 85%, or 90% (in some embodiments, less than 60%) at their chiral linkage phosphorus; In some embodiments, less than 70%; in some embodiments, less than 80%; in some embodiments, less than 85%; in some embodiments, less than 90%) of stereochemical purity (typically diastereoisomeric purity for oligonucleotides having multiple chiral centers). In some embodiments, each achiral controlled internucleotide linkage independently has less than 60%, 70%, 80%, 85%, or 90% (in some embodiments, less than 60%, in some embodiments, less than 70%, in some embodiments, less than 80%, in some embodiments, less than 85%) at its chiral linkage phosphorus; In some embodiments, less than 90%) of stereochemical purity (typically diastereoisomeric purity for oligonucleotides having multiple chiral centers).

In some embodiments, at least 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 couplings of a monomer (in many embodiments, phosphoramidite for oligonucleotide synthesis) independently have a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90% (for oligonucleotide synthesis, typically diastereoselectivity in terms of one or more formed chiral centers of linked phosphorus). In some embodiments, at least one coupling has a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, at least two couplings independently have a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, at least three couplings independently have a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, at least four couplings independently have a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, at least five couplings independently have a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, each coupling independently has a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, each achiral controlled internucleotide linkage independently forms with a stereoselectivity of less than about 60%, 70%, 80%, 85%, or 90%. In some embodiments, the stereoselectivity is less than about 60%. In some embodiments, the stereoselectivity is less than about 70%. In some embodiments, the stereoselectivity is less than about 80%. In some embodiments, the stereoselectivity is less than about 90%. In some embodiments, at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 couplings independently have a stereoselectivity of less than about 90%. In some embodiments, at least one coupling has a stereoselectivity of less than about 90%. In some embodiments, at least two couplings have a stereoselectivity of less than about 90%. In some embodiments, at least three couplings have a stereoselectivity of less than about 90%. In some embodiments, at least four couplings have a stereoselectivity of less than about 90%. In some embodiments, at least five couplings have a stereoselectivity of less than about 90%. In some embodiments, each coupling independently has a stereoselectivity of less than about 90%. In some embodiments, at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 couplings independently have a stereoselectivity of less than about 85%. In some embodiments, each coupling independently has a stereoselectivity of less than about 85%. In some embodiments, at least 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 couplings independently have a stereoselectivity of less than about 80%. In some embodiments, each coupling independently has a stereoselectivity of less than about 80%. In some embodiments, at least 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 couplings independently have a stereoselectivity of less than about 70%. In some embodiments, each coupling independently has a stereoselectivity of less than about 70%.

In some embodiments, the oligonucleotides and compositions of the disclosure have high purity. In some embodiments, the oligonucleotides and compositions of the disclosure have high stereochemical purity. In some embodiments, the stereochemical purity, e.g., diastereomeric purity, is about 60% -100%. In some embodiments, the diastereomeric purity is about 60% -100%. In some embodiments, the percentage is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage is at least 80%, 85%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the percentage is at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric purity is at least 60%. In some embodiments, the diastereomeric purity is at least 70%. In some embodiments, the diastereomeric purity is at least 80%. In some embodiments, the diastereomeric purity is at least 85%. In some embodiments, the diastereomeric purity is at least 90%. In some embodiments, the diastereomeric purity is at least 91%. In some embodiments, the diastereomeric purity is at least 92%. In some embodiments, the diastereomeric purity is at least 93%. In some embodiments, the diastereomeric purity is at least 94%. In some embodiments, the diastereomeric purity is at least 95%. In some embodiments, the diastereomeric purity is at least 96%. In some embodiments, the diastereomeric purity is at least 97%. In some embodiments, the diastereomeric purity is at least 98%. In some embodiments, the diastereomeric purity is at least 99%. In some embodiments, the diastereomeric purity is at least 99.5%.

In some embodiments, compounds of the present disclosure (e.g., oligonucleotides, chiral auxiliary agents, etc.) comprise a plurality of chiral elements (e.g., a plurality of carbon and/or phosphorus (e.g., chiral internucleotide-bonded, linked phosphorus) chiral centers. In some embodiments, at least 1,2, 3,4, 5,6, 7, 8, 9, or more chiral elements of a provided compound (e.g., oligonucleotide) each independently have a diastereomeric purity as described herein. In some embodiments, at least 1,2, 3,4, 5,6, 7, 8, 9, or more chiral carbon centers of a provided compound each independently have a diastereomeric purity as described herein. In some embodiments, at least 1,2, 3,4, 5,6, 7, 8, 9, or more chiral phosphorus centers of a provided compound each independently have a diastereomeric purity as described herein. In some embodiments, each chiral element independently has a diastereomeric purity as described herein. In some embodiments, each chiral center independently has a diastereomeric purity as described herein. In some embodiments, each chiral carbon center independently has a diastereomeric purity as described herein. In some embodiments, each chiral phosphorus center independently has a diastereomeric purity as described herein. In some embodiments, each chiral phosphorus center independently has a diastereomeric purity of at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% or more.

As will be appreciated by one of ordinary skill in the art, in some embodiments, the diastereoselectivity of the coupled diastereoselectivity or diastereoisomeric purity of the chiral-bonded phosphorus center can be assessed by the diastereoselectivity of dimer formation and diastereoselectivity of the produced dimer under the same or comparable conditions, wherein the dimer has the same 5 '-and 3' -nucleosides and internucleotide linkages.

Various techniques can be used to identify or confirm stereochemistry (e.g., configuration of chiral phosphorus linkages) and/or backbone chiral center patterns of chiral elements, and/or to evaluate stereoselectivity (e.g., diastereoselectivity of coupling steps in oligonucleotide synthesis) and/or stereochemical purity (e.g., diastereoisomeric purity of internucleotide linkages, compounds (e.g., oligonucleotides), etc.). Exemplary techniques include NMR [ e.g., 1D (one-dimensional) and/or 2D (two-dimensional) ¹H-³¹ P HETCOR (heteronuclear correlation Spectrum) ], HPLC, RP-HPLC, mass spectrometry, LC-MS, cleavage of internucleotide linkages by stereospecific nucleases, and the like, which may be used alone or in combination. Exemplary useful nucleases include benzoate, micrococcus nuclease, and svPDE (snake venom phosphodiesterase), which are specific for certain internucleotide linkages having Rp linkage phosphorus (e.g., rp phosphorothioate linkages); and nucleases P1, mung bean nuclease, and nuclease S1, which are specific for internucleotide linkages having an Sp linkage phosphorus (e.g., an Sp phosphorothioate linkage). Without wishing to be bound by any particular theory, the present disclosure indicates that, in at least some instances, cleavage of an oligonucleotide by a particular nuclease may be affected by a structural element such as a chemical modification (e.g., a 2' modification of a sugar), a base sequence, or a stereochemical environment. For example, it was observed that in some cases, the separated Rp phosphorothioate internucleotide linkages flanking the Sp phosphorothioate internucleotide linkages were not cleaved by the benzoate and micrococcus nucleases specific for internucleotide linkages having Rp phosphorothioate.

In some embodiments, oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern share a common backbone phosphorus modification pattern and a common base modification pattern. In some embodiments, oligonucleotide compositions sharing a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern share a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides sharing a common base sequence, a common backbone linkage pattern, and a common backbone chiral center pattern have the same structure.

In some embodiments, the disclosure provides an oligonucleotide composition comprising a plurality of oligonucleotides capable of directing HSD17B13 knockdown, wherein the plurality of oligonucleotides are of a particular oligonucleotide type, the composition being chirally controlled in that the composition is enriched for oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides of the same base sequence.

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

In some embodiments, the disclosure provides ds oligonucleotide compositions comprising a plurality of oligonucleotides that target HSD17B 13. In some embodiments, the disclosure provides chiral controlled oligonucleotide compositions of ds oligonucleotides targeting HSD17B 13. In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13, the base sequence of the ds oligonucleotide targeting HSD17B13 being or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13, the base sequence of the ds oligonucleotide targeting HSD17B13 comprising a base sequence that is or is complementary to: the HSD17B13 sequences disclosed herein, or portions thereof (e.g., the various base sequences in table 1). In some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13, the base sequence of the ds oligonucleotide targeting HSD17B13 comprising 15 consecutive bases of the base sequence that are or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). in some embodiments, the disclosure provides a ds oligonucleotide targeting HSD17B13, the base sequence of the ds oligonucleotide targeting HSD17B13 comprising 15 consecutive bases with 0-3 mismatches as or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides ds oligonucleotide compositions targeting HSD17B13, wherein the ds oligonucleotides targeting HSD17B13 comprise at least one achiral controlled chiral internucleotide linkage. In some embodiments, the disclosure provides a ds oligonucleotide comprising an achiral controlled chiral internucleotide linkage that targets HSD17B13, wherein the base sequence of the ds oligonucleotide that targets HSD17B13 comprises a base sequence that is or is complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide composition comprising an achiral controlled chiral internucleotide linkage targeting HSD17B13, wherein the base sequence of the ds oligonucleotide targeting HSD17B13 is as or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide comprising an achiral controlled chiral internucleotide linkage that targets HSD17B13, wherein the base sequence of the ds oligonucleotide that targets HSD17B13 comprises 15 consecutive bases that are the following or complementary base sequences: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide comprising an achiral controlled chiral internucleotide linkage targeting HSD17B13, wherein the base sequence of the ds oligonucleotide targeting HSD17B13 comprises 15 consecutive bases with 0-3 mismatches as or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide comprising a chirally controlled chiral internucleotide linkage targeting HSD17B13, wherein the base sequence of the ds oligonucleotide targeting HSD17B13 comprises a base sequence that is or is complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides ds oligonucleotide compositions comprising chiral controlled chiral internucleotide linkages targeting HSD17B13, wherein the base sequence of the ds oligonucleotide targeting HSD17B13 is as or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide comprising a chirally controlled chiral internucleotide linkage targeting HSD17B13, wherein the base sequence of the ds oligonucleotide targeting HSD17B13 comprises 15 consecutive bases that are the following or complementary base sequences: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa). In some embodiments, the disclosure provides a ds oligonucleotide comprising a chirally controlled chiral internucleotide linkage targeting HSD17B13, wherein the base sequence of the ds oligonucleotide targeting HSD17B13 comprises 15 consecutive bases with 0-3 mismatches as or complementary to: the HSD17B13 sequences disclosed herein, or a portion thereof (e.g., the various base sequences in table 1, wherein each T may be independently replaced by U, and vice versa).

In some embodiments, oligonucleotides of the same oligonucleotide type have a common backbone phosphorus modification pattern and a common nucleoside modification pattern. In some embodiments, oligonucleotides of the same oligonucleotide type have a common sugar modification pattern. In some embodiments, oligonucleotides of the same oligonucleotide type have a common base modification pattern. In some embodiments, oligonucleotides of the same oligonucleotide type have a common nucleoside modification pattern. In some embodiments, oligonucleotides of the same oligonucleotide type have the same composition. In many embodiments, the oligonucleotides of the same oligonucleotide type are identical. In some embodiments, oligonucleotides of the same oligonucleotide type have the same oligonucleotide (as will be appreciated by those skilled in the art, such oligonucleotides may each independently exist in one of a plurality of forms of the oligonucleotide, and may be the same or different forms of the oligonucleotide). In some embodiments, the oligonucleotides of the same oligonucleotide type each independently have the same oligonucleotide or a pharmaceutically acceptable salt thereof.

In some embodiments, the plurality of oligonucleotides or oligonucleotides of a particular oligonucleotide type in the provided oligonucleotide composition are ds oligonucleotides targeting HSD17B 13. In some embodiments, the disclosure provides chirally controlled ds oligonucleotide compositions targeting HSD17B13 comprising a plurality of ds oligonucleotides targeting HSD17B13, wherein the oligonucleotides share:

1) A common base sequence;

2) A common backbone linkage pattern; and

3) The same linkage phosphorus stereochemistry at one or more chiral internucleotide linkages (chirally controlled internucleotide linkages),

Wherein the composition enriches the plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides sharing a common base sequence and backbone linkage pattern.

In some embodiments, as used herein, "one or more" or "at least one" is 1-50、1-40、1-30、1-25、1-20、1-15、1-10、5-50、5-40、5-30、5-25、5-20、5-15、5-10、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、 or more.

In some embodiments, the oligonucleotide type is further defined by: 4) Additional chemical moieties, if any.

In some embodiments, the percentage is at least about 10%. In some embodiments, the percentage is at least about 20%. In some embodiments, the percentage is at least about 30%. In some embodiments, the percentage is at least about 40%. In some embodiments, the percentage is at least about 50%. In some embodiments, the percentage is at least about 60%. In some embodiments, the percentage is at least about 70%. In some embodiments, the percentage is at least about 75%. In some embodiments, the percentage is at least about 80%. In some embodiments, the percentage is at least about 85%. In some embodiments, the percentage is at least about 90%. In some embodiments, the percentage is at least about 91%. In some embodiments, the percentage is at least about 92%. In some embodiments, the percentage is at least about 93%. In some embodiments, the percentage is at least about 94%. In some embodiments, the percentage is at least about 95%. In some embodiments, the percentage is at least about 96%. In some embodiments, the percentage is at least about 97%. In some embodiments, the percentage is at least about 98%. In some embodiments, the percentage is at least about 99%. In some embodiments, the percentage is (DS) ^nc or greater, where DS and nc are each independently as described in the present disclosure.

In some embodiments, multiple oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) share the same composition. In some embodiments, the plurality of oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) are identical (same stereoisomers). In some embodiments, the chirally controlled oligonucleotide composition, e.g., the chirally controlled ds oligonucleotide composition targeting HSD17B13, is a stereomerically pure oligonucleotide composition, wherein the plurality of oligonucleotides are identical (same stereoisomers) and the composition does not contain any other stereoisomers. One skilled in the art will appreciate that one or more other stereoisomers may be present as impurities because the process, selectivity, purification, etc. may not achieve completeness.

In some embodiments, the provided compositions are characterized in that when it is contacted with a target nucleic acid (e.g., HSD17B13 transcript (e.g., pre-mRNA, mature mRNA, other types of RNAs that hybridize to oligonucleotides of the composition, etc.)), the level of the target nucleic acid and/or product encoded thereby is reduced compared to that observed under reference conditions. In some embodiments, the reference condition is selected from the group consisting of: the absence of a composition, the presence of a reference composition, and combinations thereof. In some embodiments, the reference condition is the absence of a composition. In some embodiments, the reference condition is the presence of a reference composition. In some embodiments, the reference composition is a composition whose oligonucleotides do not hybridize to the target nucleic acid. In some embodiments, the reference composition is a composition whose oligonucleotides do not contain sequences sufficiently complementary to the target nucleic acid. In some embodiments, the provided compositions are chirally controlled oligonucleotide compositions, while the reference composition is an achiral controlled oligonucleotide composition that is otherwise identical but not chirally controlled (e.g., a racemic preparation of oligonucleotides having the same constitution as the plurality of oligonucleotides in the chirally controlled oligonucleotide composition).

In some embodiments, the disclosure provides chirally controlled ds oligonucleotide compositions targeting HSD17B13 comprising a plurality of ds oligonucleotides targeting HSD17B13 capable of directing the knockdown of HSD17B13, wherein the oligonucleotides share:

1) A common base sequence;

2) A common backbone linkage pattern; and

3) The same stereochemistry of the phosphorus linkages at one or more (e.g., ,1-50、1-40、1-30、1-25、1-20、1-15、1-10、5-50、5-40、5-30、5-25、5-20、5-15、5-10、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20 or more) chiral internucleotide linkages (chirally controlled internucleotide linkages),

Wherein the composition enriches the plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides sharing a common base sequence and backbone linkage pattern,

The oligonucleotide composition is characterized in that: when contacted with HSD17B13 transcript in an HSD17B13 knockdown system, the knockdown of the transcript is improved relative to that observed under reference conditions selected from the group consisting of: the absence of the composition, the presence of a reference composition, and combinations thereof.

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

As demonstrated herein, oligonucleotide structural elements (e.g., sugar modification pattern, backbone linkage pattern, backbone chiral center pattern, backbone phosphorus modification pattern, etc.), and combinations thereof, can provide unexpectedly improved properties and/or biological activity.

In some embodiments, the oligonucleotide composition is capable of reducing expression, level and/or activity of a target gene or gene product thereof. In some embodiments, the oligonucleotide composition is capable of reducing expression, level and/or activity of a target gene or gene product thereof by sterically blocking translation after annealing to the target gene mRNA, by cleaving the mRNA (pre-mRNA or mature mRNA) and/or by altering or interfering with mRNA splicing. In some embodiments, provided ds oligonucleotide compositions targeting HSD17B13 are capable of reducing expression, level and/or activity of HSD17B13 target gene or gene product thereof. In some embodiments, the provided ds oligonucleotide compositions targeting HSD17B13 are capable of reducing expression, level and/or activity of HSD17B13 target gene or gene product thereof by sterically blocking translation after annealing to HSD17B13 target gene mRNA, by cleaving HSD17B13 mRNA (pre-mRNA or mature mRNA) and/or by altering or interfering with mRNA splicing.

In some embodiments, the oligonucleotide composition (e.g., the ds oligonucleotide composition that targets HSD17B 13) is a substantially pure preparation of a single oligonucleotide stereoisomer (e.g., the ds oligonucleotide stereoisomer that targets HSD17B 13), because in some cases, after certain purification procedures, the oligonucleotides in the composition that do not belong to the oligonucleotide stereoisomer are impurities from the preparation of the oligonucleotide stereoisomer.

In some embodiments, the disclosure provides chirally controlled oligonucleotides and oligonucleotide compositions, and in some embodiments, stereopure oligonucleotides and oligonucleotide compositions. For example, in some embodiments, provided compositions contain a non-random level or a controlled level of one or more individual oligonucleotide types as described herein. In some embodiments, the oligonucleotides of the same oligonucleotide type are identical.

Sugar

According to the present disclosure, a variety of sugars may be used, including modified sugars. In some embodiments, the disclosure provides sugar modifications and patterns thereof, optionally in combination with other structural elements (e.g., internucleotide linkage modifications and patterns thereof, backbone chiral center patterns thereof, etc.), which may provide improved properties and/or activity when incorporated into oligonucleotides.

The most common naturally occurring nucleosides include ribose (e.g., in RNA) or deoxyribose (e.g., in DNA) linked to the nucleobases adenosine (a), cytosine (C), guanine (G), thymine (T), or uracil (U). In some embodiments, the sugar, e.g., each of the many oligonucleotides in Table 1 (unless otherwise specified), is a natural DNA sugar (in a DNA nucleic acid or oligonucleotide, having a structureWherein the nucleobases are attached to the 1' position and the 3' and 5' positions are attached to internucleotide linkages (as understood by those skilled in the art, the 5' position may be attached to a 5' end group (e.g., -OH) if at the 5' end of the oligonucleotide, and the 3' position may be attached to a 3' end group (e.g., -OH) if at the 3' end of the oligonucleotide). In some embodiments, the sugar is a natural RNA sugar in an RNA nucleic acid or oligonucleotide havingWherein nucleobases are attached to the 1' position and 3' and 5' positions are attached to internucleotide linkages (as understood by those skilled in the art, if at the 5' end of an oligonucleotide, the 5' position may be attached to a 5' end group (e.g., -OH), and if at the 3' end of an oligonucleotide, the 3' position may be attached to a 3' end group (e.g., -OH)). In some embodiments, the sugar is a modified sugar because it is not a natural DNA sugar or a natural RNA sugar. Among other things, the modified sugar may provide improved stability. In some embodiments, the modified sugar can be used to alter and/or optimize one or more hybridization characteristics. In some embodiments, modified sugars can be used to alter and/or optimize target recognition. In some embodiments, modified sugars can be used to optimize Tm. In some embodiments, modified sugars can be used to improve oligonucleotide activity.

The sugar may be bound to the internucleotide linkages at various positions. As non-limiting examples, the internucleotide linkages may be bonded to the 2', 3', 4', or 5' positions of the sugar. In some embodiments, as is most common in natural nucleic acids, unless otherwise indicated, internucleotide linkages are attached at the 5 'position to one sugar and at the 3' position to another sugar.

In some embodiments, the sugar is an optionally substituted natural DNA or RNA sugar. In some embodiments, the sugar is optionally substitutedIn some embodiments, the 2' position is optionally substituted. In some embodiments, the sugar isIn some embodiments, the sugar hasWherein R ^1s、R^2s、R^3s、R^4s, and R ^5s are each independently-H, a suitable substituent or a suitable sugar modification (e.g., those described in ,US 9394333、US 9744183、US 9605019、US 9982257、US 20170037399、US 20180216108、US 20180216107、US 9598458、WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/022473、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/032612 and/or WO 2020/191252, the respective substituents, sugar modifications, descriptions of R ^1s、R^2s、R^3s、R^4s, and R ^5s, and modified sugars of which are independently incorporated herein by reference). In some embodiments, the sugar hasIs a structure of (a). In some embodiments, R ^4s is —h. In some embodiments, the sugar hasWherein R ^2s is-H, halogen OR-OR, wherein R is optionally substituted C _1-6 aliphatic. In some embodiments, R ^2s is —h. In some embodiments, R ^2s is-F. In some embodiments, R ^2s is —ome. In some embodiments, R ^2s is-OCH ₂CH₂ OMe.

In some embodiments, the sugar hasWherein R ^2s and R ^4s together form-L ^s, wherein L ^s is a covalent bond or an optionally substituted divalent C _1-6 aliphatic or heteroaliphatic having 1-4 heteroatoms. In some embodiments, each heteroatom is independently selected from nitrogen, oxygen, or sulfur). In some embodiments, L ^s is optionally substituted C2-O-CH ₂ -C4. In some embodiments, L ^s is C2-O-CH ₂ -C4. In some embodiments, L ^s is C2-O- (R) -CH (CH ₂CH₃) -C4. In some embodiments, L ^s is C2-O- (S) -CH (CH ₂CH₃) -C4.

In some embodiments, the sugar is a bicyclic sugar, e.g., as described in the disclosure wherein R ^2s and R ^4s together form a linked sugar. In some embodiments, the sugar is selected from LNA sugar, BNA sugar, cEt sugar, and the like. In some embodiments, the bridge is between the 2 '-carbon atom and the 4' -carbon atom (corresponding to R ^2s and R ^4s together with the atoms interposed therebetween form an optionally substituted ring as described herein). Examples of bicyclic sugars include α -L-methyleneoxy (4 '-CH ₂ -O-2') LNA, β -D-methyleneoxy (4 '-CH ₂ -O-2') LNA, ethyleneoxy (4 '- (CH ₂)₂ -O-2') LNA, aminooxy (4 '-CH ₂ -O-N (R) -2') LNA, and oxyamino (4 '-CH ₂ -N (R) -O-2') LNA in some embodiments, a bicyclic sugar such as LNA or BNA sugar, is a sugar having at least one bridge between the two sugar carbons.

In some embodiments, the bicyclic sugar is an alpha-L-methyleneoxy (4 '-CH ₂ -O-2') BNA, beta-D-methyleneoxy (4 '-CH ₂ -O-2') BNA, ethyleneoxy (4 '- (CH ₂)₂ -O-2') BNA, aminooxy (4 '-CH ₂ -O-N (R) -2') BNA, oxyamino (4 '-CH ₂ -N (R) -O-2') BNA, methyl (methyleneoxy) (4 '-CH (CH ₃) -O-2') BNA (also known as restricted ethyl or cEt), methylene-thio (4 '-CH ₂ -S-2') BNA, methylene-amino (4 '-CH ₂ -N (R) -2') BNA, methyl carbocycle (4 '-CH ₂-CH(CH₃) -2') BNA, propylene carbocycle (4 '- (CH ₂)₃ -2') BNA, or vinyl BNA sugar.

In some embodiments, the sugar modification is 2' -OMe, 2' -MOE, 2' -LNA, 2' -F, 5' -vinyl or S-cEt. In some embodiments, the modified sugar is FRNA sugar, FANA sugar, or morpholino sugar. In some embodiments, the oligonucleotide comprises a nucleic acid analog, such as GNA, LNA, PNA, TNA, F-HNA (F-THP or 3' -fluorotetrahydropyran), MNA (mannitol nucleic acid, such as Leumann 2002Bioorg. Med. Chem. [ J. Bioorganic chemistry and medical chemistry ] 10:841-854), ANA (aniytol (anitol) nucleic acid), or morpholino, or a portion thereof. In some embodiments, the sugar modification replaces the natural sugar with another cyclic or acyclic moiety. Examples of such moieties are well known in the art, such as those used in morpholino, diol nucleic acids, and the like, and may be used in accordance with the present disclosure. As will be appreciated by those skilled in the art, when used with modified sugars, in some embodiments, the internucleotide linkages may be modified, e.g., as in morpholino, PNA, etc.

In some embodiments, the sugar is a 6' -modified bicyclic sugar having (R) or (S) chirality at the 6-position, such as those described in US 7399845. In some embodiments, the sugar is a 5' -modified bicyclic sugar having (R) or (S) chirality at the 5-position, such as those described in US 20070287831.

In some embodiments, the modified sugar contains one or more substituents (typically one substituent, and typically at an axial position) at the 2' position, which are independently selected from-F; -CF ₃、-CN、-N₃、-NO、-NO₂, -OR ', -SR', OR-N (R ') ₂, wherein each R' is independently an optionally substituted C _1-10 aliphatic; -O- (C ₁-C₁₀ alkyl), -S- (C ₁-C₁₀ alkyl), -NH- (C ₁-C₁₀ alkyl), or-N (C ₁-C₁₀ alkyl) ₂;-O-(C₂-C₁₀ alkenyl), -S- (C ₂-C₁₀ alkenyl), -NH- (C ₂-C₁₀ alkenyl), or-N (C ₂-C₁₀ alkenyl) ₂;-O-(C₂-C₁₀ alkynyl), -S- (C ₂-C₁₀ alkynyl), -NH- (C ₂-C₁₀ alkynyl), or-N (C ₂-C₁₀ alkynyl) ₂; or-O- (C ₁-C₁₀ alkylene) -O- (C ₁-C₁₀ alkyl), -O- (C ₁-C₁₀ alkylene) -NH- (C ₁-C₁₀ alkyl) or-O- (C ₁-C₁₀ alkylene) -NH (C ₁-C₁₀ alkyl) ₂、-NH-(C₁-C₁₀ alkylene) -O- (C ₁-C₁₀ alkyl), or-N (C ₁-C₁₀ alkyl) - (C ₁-C₁₀ alkylene) -O- (C ₁-C₁₀ alkyl), wherein alkyl, alkylene, alkenyl and alkynyl are each independently and optionally substituted. In some embodiments, the substituent is-O (CH ₂)_nOCH₃、-O(CH₂)_nNH₂, MOE, DMAOE, or DMAEOE, where n is 1 to about 10. In some embodiments, the modified sugar is a sugar described in WO 2001/088198; and Martin et al, helv.Chim. Acta, proc. Swiss chemistry, 1995, 78, 486-504. In some embodiments, the modified sugar comprises one or more groups selected from substituted silyl groups, RNA-cleaving groups, reporter groups, fluorescent labels, intercalators, groups for improving the pharmacokinetic properties of nucleic acids, groups for improving the pharmacodynamic properties of nucleic acids, or other substituents having similar properties. in some embodiments, the modification is made at one or more of the 2', 3', 4', or 5' positions, including the 3 'position of the sugar on the 3' -terminal nucleoside or the 5 'position of the 5' -terminal nucleoside.

In some embodiments, the 2' -OH of the ribose is replaced with a group selected from the group consisting of: -H, -F; -CF ₃、-CN、-N₃、-NO、-NO₂, -OR ', -SR', OR-N (R ') ₂, wherein each R' is independently described in the present disclosure; -O- (C ₁-C₁₀ alkyl), -S- (C ₁-C₁₀ alkyl), -NH- (C ₁-C₁₀ alkyl), or-N (C ₁-C₁₀ alkyl) ₂;-O-(C₂-C₁₀ alkenyl), -S- (C ₂-C₁₀ alkenyl), -NH- (C ₂-C₁₀ alkenyl), or-N (C ₂-C₁₀ alkenyl) ₂;-O-(C₂-C₁₀ alkynyl), -S- (C ₂-C₁₀ alkynyl), -NH- (C ₂-C₁₀ alkynyl), or-N (C ₂-C₁₀ alkynyl) ₂; or-O- (C ₁-C₁₀ alkylene) -O- (C ₁-C₁₀ alkyl), -O- (C ₁-C₁₀ alkylene) -NH- (C ₁-C₁₀ alkyl) or-O- (C ₁-C₁₀ alkylene) -NH (C ₁-C₁₀ alkyl) ₂、-NH-(C₁-C₁₀ alkylene) -O- (C ₁-C₁₀ alkyl), or-N (C ₁-C₁₀ alkyl) - (C ₁-C₁₀ alkylene) -O- (C ₁-C₁₀ alkyl), wherein alkyl, alkylene, alkenyl and alkynyl are each independently and optionally substituted. In some embodiments, the 2' -OH is replaced with-H (deoxyribose). In some embodiments, 2' -OH is replaced with-F. In some embodiments, the 2'-OH is replaced with-OR'. In some embodiments, the 2' -OH is replaced with-OMe. In some embodiments, 2' -OH is replaced with-OCH ₂CH₂ OMe.

In some embodiments, the sugar modification is a 2' -modification. Common 2 '-modifications include, but are not limited to, 2' -OR, where R is an optionally substituted C _1-6 aliphatic. In some embodiments, the modification is 2' or, wherein R is optionally substituted C _1-6 alkyl. In some embodiments, the modification is 2' -OMe. In some embodiments, the modification is a 2' -MOE. In some embodiments, the 2' -modification is S-cEt. In some embodiments, the modified sugar is an LNA sugar. In some embodiments, the 2' -modification is-F. In some embodiments, the 2' -modification is FANA. In some embodiments, the 2' -modification is FRNA. In some embodiments, the sugar modification is a5 '-modification, e.g., 5' -Me. In some embodiments, the sugar modification alters the size of the sugar ring. In some embodiments, the sugar modification is a sugar moiety in FHNA.

In some embodiments, the sugar modification replaces the sugar moiety with another cyclic or acyclic moiety. Examples of such moieties are widely known in the art and include, but are not limited to, those used in morpholino (optionally with its phosphorodiamidate linkages), diol nucleic acids, and the like.

In some embodiments, one or more sugars of the ds oligonucleotide targeting HSD17B13 are modified. In some embodiments, each sugar of the oligonucleotide is independently modified. In some embodiments, the modified sugar comprises a 2' -modification. In some embodiments, each modified sugar independently comprises a 2' -modification. In some embodiments, the 2 '-modification is 2' or, wherein R is an optionally substituted C _1-6 aliphatic. In some embodiments, the 2 '-modification is 2' -OMe. In some embodiments, the 2 '-modification is a 2' -MOE. In some embodiments, the 2' -modification is an LNA sugar modification. In some embodiments, the 2 '-modification is 2' -F. In some embodiments, each sugar modification is independently a 2' -modification. In some embodiments, each sugar modification is independently 2' -OR. In some embodiments, each sugar modification is independently 2' or, wherein R is optionally substituted C _1-6 alkyl. In some embodiments, each sugar modification is 2' -OMe. In some embodiments, each sugar modification is a 2' -MOE. In some embodiments, each sugar modification is independently a 2'-OMe or a 2' -MOE. In some embodiments, each sugar modification is independently a 2'-OMe, 2' -MOE, or LNA sugar.

In some embodiments, the modified sugar is an optionally substituted ENA sugar. In some embodiments, the sugar is a sugar described in the following: for example, seth et al, J Am Chem Soc. [ journal of American society of chemistry ]2010, 10 months, 27 days; 132 (42): 14942-14950. In some embodiments, the modified sugar is a sugar in XNA (heterologous nucleic acid (xenonucleic acid)), such as arabinose, anhydrohexitol, threose, 2' fluoroarabinose, or cyclohexene.

The modified sugar includes a cyclobutyl or cyclopentyl moiety in place of the pentofuranosyl sugar. Representative examples of such modified sugars include those described in US 4,981,957, US 5,118,800, US 5,319,080 or US 5,359,044. In some embodiments, the oxygen atom within the ribose ring is replaced with nitrogen, sulfur, selenium, or carbon. In some embodiments, -O-is replaced by-N (R '), -S-, -Se-, or-C (R') ₂ -. In some embodiments, the modified sugar is a modified ribose, wherein the oxygen atoms within the ribose ring are replaced with nitrogen, and wherein the nitrogen is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc.).

In some embodiments, the sugar is linked by internucleotide linkages (in some embodiments, modified internucleotide linkages). In some embodiments, the internucleotide linkages do not comprise a linkage phosphorus. In some embodiments, the internucleotide linkage is-L-. In some embodiments, the internucleotide linkage is-OP (O) (-c≡ch) O-, -OP (O) (R) O- (e.g., R is -CH₃)、3'-NHP(O)(OH)O-5'、3'-OP(O)(CH₃)OCH₂-5'、3'-CH₂C(O)NHCH₂-5'、3'-SCH₂OCH₂-5'、3'-OCH₂OCH₂-5'、3'-CH₂NR'CH₂-5'、3'-CH₂N(Me)OCH₂-5'、3'-NHC(O)CH₂CH₂-5'、3'-NR'C(O)CH₂CH₂-5'、3'-CH₂CH₂NR'-5'、3'-CH₂CH₂NH-5'、 or 3'-OCH ₂CH₂ N (R') -5 'in some embodiments, the 5' carbon may be optionally substituted with =o.

In some embodiments, the modified sugar is an optionally substituted pentose or hexose. In some embodiments, the modified sugar is an optionally substituted pentose. In some embodiments, the modified sugar is an optionally substituted hexose. In some embodiments, the modified sugar is an optionally substituted ribose or hexitol. In some embodiments, the modified sugar is an optionally substituted ribose. In some embodiments, the modified sugar is an optionally substituted hexitol.

In some embodiments, the sugar modification is a 5' -vinyl (R or S), a 5' -methyl (R or S), a 2' -SH, a 2' -F, a 2' -OCH ₃、2′-OCH₂CH₃、2′-OCH₂CH₂ F, or a 2' -O (CH ₂)₂₀CH₃. In some embodiments, the substituents at the 2' position, e.g., the 2' -modification are allyl, amino, azido, thio, O-allyl, O-C ₁-C₁₀ alkyl 、OCF₃、OCH₂F、O(CH₂)₂SCH₃、O(CH₂)₂-O-N(R_m)(R_n)、O-CH₂-C(＝O)-N(R_m)(R_n), and O-CH ₂-C(＝O)-N(R₁)-(CH₂)₂-N(R_m)(R_n), wherein each allyl, amino, and alkyl is optionally substituted, and R ₁、R_m and R _n are each independently R ' as described in the present disclosure. In some embodiments, R ₁、R_m and R _n are each independently-H or optionally substituted C ₁-C₁₀ alkyl.

In some embodiments, the saccharide is a tetrahydropyran or THP saccharide. In some embodiments, the modified nucleoside is a tetrahydropyran nucleoside or a THP nucleoside (which is a nucleoside that replaces the pentofuranosyl residue in a typical natural nucleoside with a six-membered tetrahydropyran sugar). THP sugars and/or nucleosides include those for Hexitol Nucleic Acid (HNA), aniytone Nucleic Acid (ANA), mannitol Nucleic Acid (MNA) (e.g., leumann, bioorg. Med. Chem. [ bioorganic chemistry and medicinal chemistry ],2002, 10, 841-854) or fluorohna (F-HNA).

In some embodiments, the sugar comprises a ring having more than 5 atoms and/or more than one heteroatom, such as morpholino sugar.

Modifications of sugar, nucleobases, internucleotide linkages, etc. may and often are used in combination with oligonucleotides (e.g., see the various oligonucleotides in table 1), as will be appreciated by those skilled in the art. For example, the combination of sugar modification and nucleobase modification is a 2' -F (sugar) 5-methyl (nucleobase) modified nucleoside. In some embodiments, the combination is substitution of the ribosyl epoxy atom with S and substitution at the 2' -position.

In some embodiments, the 2 '-modified sugar is a furanosyl sugar modified at the 2' position. In some embodiments, the 2' -modification is halogen, -R ' (where R ' is not-H), -OR ' (where R ' is not-H), -SR ', -N (R ') ₂, optionally substituted-CH ₂-CH＝CH₂, optionally substituted alkenyl, OR optionally substituted alkynyl. In some embodiments, the 2 '-modification is selected from -O[(CH₂)_nO]_mCH₃、-O(CH₂)_nNH₂、-O(CH₂)_nCH₃、-O(CH₂)_nF、-O(CH₂)_nONH₂、-OCH₂C(＝O)N(H)CH₃ and-O (CH ₂)_nON[(CH₂)_nCH₃]₂), wherein each n and m is independently 1 to about 10, in some embodiments, the 2' -modification is an optionally substituted C ₁-C₁₂ alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, an optionally substituted alkylaryl group, an optionally substituted arylalkyl group, an optionally substituted-O-alkylaryl group, an optionally substituted-O-arylalkyl 、-SH、-SCH₃、-OCN、-Cl、-Br、-CN、-F、-CF₃、-OCF₃、-SOCH₃、-SO₂CH₃、-ONO₂、-NO₂、-N₃、-NH₂、 optionally substituted heterocycloalkyl group, an optionally substituted aminoalkylamino group, an optionally substituted polyalkylamino group, a substituted silyl group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, a group for improving pharmacodynamic properties, and other substituents.

In some embodiments, a 2' -modified or 2' -substituted sugar or nucleoside is a sugar or nucleoside that contains a substituent other than-H (not typically considered a substituent) or-OH at the 2' position of the sugar. In some embodiments, the 2 '-modified sugar is a bicyclic sugar comprising a bridge connecting two carbon atoms of the sugar ring (one of which is the 2' carbon). In some embodiments, the 2' -modification is non-bridging, e.g., allyl, amino, azido, thio, optionally substituted-O-allyl, optionally substituted-O-C ₁-C₁₀ alkyl 、-OCF₃、-O(CH₂)₂OCH₃、2′-O(CH₂)₂SCH₃、-O(CH₂)₂ON(R_m)(R_n), or-OCH ₂C(＝O)N(R_m)(R_n), wherein each R _m and R _n is independently-H or optionally substituted C ₁-Cx₀ alkyl.

In some embodiments, the sugar is a sugar of N-methanol carba (N-methanocarba), LNA, cmOEBNA, cEt BNA, alpha-L-LNA, or a related analog, HNA, me-ANA, MOE-ANA, ara-FHNA, R-6'-Me-FHNA, S-6' -Me-FHNA, ENA, or c-ANA. In some embodiments, the modified internucleotide linkages are C3-amide (e.g., with an amide-modified sugar attached to C3', mutisya et al 2014Nucleic Acids Res [ nucleic acids research ]2014, month 6, 1; 42 (10): 6542-6551), methylal, thiomethylal, MMI [ e.g., methylene (methylimino), peoc' h et al 2006Nucleosides and Nucleotides [ nucleoside with nucleotide ]16 (7-9) ], PMO (phosphorodiamidate-attached morpholino) linkages, which attach two sugars, or PNA (peptide nucleic acid) linkages.

In some embodiments, the sugar is a sugar described in US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252, each of which is incorporated herein by reference.

A variety of additional sugars that can be used to prepare oligonucleotides or analogs thereof are known in the art and can be used in accordance with the present disclosure.

Nucleobases

According to the present disclosure, a variety of nucleobases can be used in the provided oligonucleotides. In some embodiments, the nucleobases are natural nucleobases, the most common natural nucleobases being A, T, C, G and U. In some embodiments, the nucleobase is a modified nucleobase because it is not A, T, C, G or U. In some embodiments, the nucleobase is an optionally substituted A, T, C, G or U, or a substituted tautomer of A, T, C, G or U. In some embodiments, the nucleobase is an optionally substituted A, T, C, G or U, e.g., 5mC, 5-hydroxymethyl C, etc. In some embodiments, the nucleobase is alkyl substituted A, T, C, G or U. In some embodiments, the nucleobase is a. In some embodiments, the nucleobase is T. In some embodiments, the nucleobase is C. In some embodiments, the nucleobase is G. In some embodiments, the nucleobase is U. In some embodiments, the nucleobase is 5mC. In some embodiments, the nucleobase is substituted A, T, C, G or U. In some embodiments, the nucleobase is a substituted tautomer of A, T, C, G or U. In some embodiments, substitution protects certain functional groups in the nucleobase to minimize unwanted reactions during oligonucleotide synthesis. Suitable techniques for nucleobase protection in oligonucleotide synthesis are well known in the art and may be used in accordance with the present disclosure. In some embodiments, the modified nucleobase improves the properties and/or activity of the oligonucleotide. For example, in many cases, 5mC may be used instead of C to modulate certain undesirable biological effects, such as immune responses. In some embodiments, when determining sequence identity, a substituted nucleobase with the same hydrogen bonding pattern is treated identically to an unsubstituted nucleobase, e.g., 5mC may be treated identically to C [ e.g., an oligonucleotide with 5mC instead of C (e.g., AT5 mCG) is considered to have the same base sequence as an oligonucleotide with C AT one or more corresponding positions (e.g., ATCG) ].

In some embodiments, the oligonucleotide comprises one or more A, T, C, G or U. In some embodiments, the oligonucleotide comprises one or more optionally substituted A, T, C, G or U. In some embodiments, the oligonucleotide comprises one or more 5-methylcytidine, 5-hydroxymethylcytosine, 5-formylcytosine, or 5-carboxycytosine. In some embodiments, the oligonucleotide comprises one or more 5-methylcytidine. In some embodiments, each nucleobase in the oligonucleotide is selected from the group consisting of optionally substituted A, T, C, G and U, and optionally substituted A, T, C, G and a tautomer of U. In some embodiments, each nucleobase in an oligonucleotide is optionally protected A, T, C, G and U. In some embodiments, each nucleobase in an oligonucleotide is optionally substituted A, T, C, G or U. In some embodiments, each nucleobase in an oligonucleotide is selected from the group consisting of A, T, C, G, U and 5 mC.

In some embodiments, the nucleobase is an optionally substituted 2AP or DAP. In some embodiments, the nucleobase is an optionally substituted 2AP. In some embodiments, the nucleobase is an optionally substituted DAP. In some embodiments, the nucleobase is 2AP. In some embodiments, the nucleobase is DAP.

In some embodiments, the nucleobase is a natural nucleobase or a modified nucleobase derived from a natural nucleobase. Examples include uracil, thymine, adenine, cytosine and guanine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2, 6-diaminopurine, azacytosine, pyrimidine analogs (e.g., pseudoisocytosine and pseudouracil), and other modified nucleobases (e.g., 8-substituted purines, xanthines, or hypoxanthines, the latter two being natural degradation products), optionally with their respective amino groups protected by an acyl protecting group. Some examples of modified nucleobases are disclosed in Chiu and Rana, RNA,2003,9, 1034-1048; limbach et al Nucleic ACIDS RESEARCH [ Nucleic acids research ],1994, 22, 2183-2196; and Revankar and Rao, comprehensive Natural Products Chemistry [ natural products integrated chemistry ], volume 7, 313. In some embodiments, the modified nucleobase is a substituted uracil, thymine, adenine, cytosine, or guanine. In some embodiments, the modified nucleobase is a functional replacement of uracil, thymine, adenine, cytosine, or guanine, for example, in terms of hydrogen bonding and/or base pairing. In some embodiments, the nucleobase is an optionally substituted uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine. In some embodiments, the nucleobase is uracil, thymine, adenine, cytosine, 5-methylcytosine, or guanine.

In some embodiments, provided oligonucleotides comprise one or more 5-methylcytosines. In some embodiments, the present disclosure provides oligonucleotides whose base sequences are disclosed herein, for example, in table 1, wherein each T can be independently replaced by U and vice versa, and each cytosine is optionally and independently replaced by 5-methylcytosine, or vice versa. As will be appreciated by those of skill in the art, in some embodiments, 5mC can be considered as C-such oligonucleotides comprising nucleobase modifications at the C-position in terms of the base sequence of the oligonucleotide (e.g., see the various oligonucleotides in table 1). In the description of oligonucleotides, nucleobases, sugars and internucleotide linkages are generally unmodified unless otherwise indicated.

In some embodiments, the modified base is an optionally substituted adenine, cytosine, guanine, thymine, or uracil or tautomer thereof. In some embodiments, the modified nucleobase is a modified adenine, cytosine, guanine, thymine, or uracil modified by one or more modifications by:

(1) The nucleobases are modified with one or more optionally substituted groups independently selected from: acyl, halogen, amino, azido, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl, heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin, substituted silyl, and combinations thereof;

(2) One or more atoms of the nucleobase are independently replaced by a different atom selected from carbon, nitrogen and sulfur;

(3) One or more double bonds in the nucleobase are independently hydrogenated; or alternatively

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

In some embodiments, the modified nucleobase is a modified nucleobase known in the art (e.g., WO 2017/210647). In some embodiments, the modified nucleobase is an enlarged size nucleobase in which one or more aryl and/or heteroaryl rings (e.g., benzene rings) have been added.

In some embodiments, the modified nucleobase is selected from the group consisting of 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6, and O-6 substituted purines. In some embodiments of the present invention, in some embodiments, the modified nucleobase is selected from the group consisting of 2-aminopropyl adenine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-C.ident.C-CH ₃) uracil, 5-propynyl cytosine, 6-azauracil, 6-azacytosine, 6-azathymine, 5-ribosyl uracil (pseudouracil), 4-thiouracil, 8-halopurine, 8-aminopurine, 8-mercaptopurine, 8-thioalkyl purine 8-hydroxy-, 8-aza-and other 8-substituted purines, 5-halogeno, in particular 5-bromo, 5-trifluoromethyl, 5-halogeno-uracil and 5-halogenocytidine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, hybrid bases, size-expanded bases, and fluorinated bases. In some embodiments, the modified nucleobase is a tricyclic pyrimidine, such as l, 3-diazaphenoxazin-2-one, l, 3-diazaphenothiazin-2-one, or 9- (2-aminoethoxy) -1, 3-diazaphenoxazin-2-one (G-clamp). In some embodiments, modified nucleobases are those in which a purine or pyrimidine base is replaced by another heterocycle, e.g., 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, or 2-pyridone.

In some embodiments, the modified nucleobase is substituted. In some embodiments, the modified nucleobase is substituted such that it contains a heteroatom, alkyl group, or linking moiety, for example, attached to a fluorescent moiety, biotin or avidin moiety, or other protein or peptide. In some embodiments, the modified nucleobase is a "universal base" that is not the most classical nucleobase, but functions similarly to a nucleobase. An example of a universal base is 3-nitropyrrole.

In some embodiments, nucleosides useful in the provided technology include modified nucleobases and/or modified sugars, such as 4-acetylcytidine; 5- (carboxyhydroxymethyl) uridine; 2' -O-methylcytidine; 5-carboxymethyl aminomethyl-2-thiouridine; 5-carboxymethyl aminomethyluridine; dihydrouridine; 2' -O-methyl pseudouridine; beta, D-galactosyl Q nucleoside; 2' -O-methylguanosine; n ⁶ -isopentenyl adenosine; 1-methyl adenosine; 1-methyl pseudouridine; 1-methylguanosine; 1-methyl inosine; 2, 2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; n ⁷ -methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytosine; 5-formyl cytosine; 5-carboxycytosine; n ⁶ -methyladenosine; 7-methylguanosine; 5-methylaminoethyl uridine; 5-methoxyaminomethyl-2-thiouridine; beta, D-mannosyl braided glycoside (mannosylqueosine); 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N ⁶ -isopentenyl adenosine; n- ((9- β, D-ribofuranosyl-2-methylthiopurin-6-yl) carbamoyl) threonine; n- ((9- β, D-ribofuranosylpurine-6-yl) -N-methylcarbamoyl) threonine; uridine-5-oxyacetic acid methyl ester; uridine-5-oxyacetic acid (v); pseudouridine; a Q nucleoside; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2' -O-methyl-5-methyluridine; 2' -O-methyluridine.

In some embodiments, nucleobases, e.g., modified nucleobases, comprise one or more biomolecule-binding moieties, such as, for example, antibodies, antibody fragments, biotin, avidin, streptavidin, receptor ligands, or chelating moieties. In other embodiments, the nucleobase is 5-bromouracil, 5-iodouracil, or 2, 6-diaminopurine. In some embodiments, the nucleobase comprises substitution by a fluorescent or biomolecular binding moiety. In some embodiments, the substituent is a fluorescent moiety. In some embodiments, the substituent is biotin or avidin.

In some embodiments, the nucleobases are those described in US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252, the respective nucleobases of which are incorporated herein by reference.

Additional chemical moieties

In some embodiments, the ds oligonucleotide that targets HSD17B13 comprises one or more additional chemical moieties. A variety of additional chemical moieties, e.g., targeting moieties, carbohydrate moieties, lipid moieties, etc., are known in the art and can be used in accordance with the present disclosure to modulate properties and/or activity, e.g., stability, half-life, activity, delivery, pharmacodynamic properties, pharmacokinetic properties, etc., of a ds oligonucleotide targeting HSD17B 13. In some embodiments, certain additional chemical moieties facilitate delivery of the oligonucleotides to desired cells, tissues and/or organs, including but not limited to cells of the central nervous system. In some embodiments, certain additional chemical moieties facilitate internalization of the oligonucleotide. In some embodiments, certain additional chemical moieties improve oligonucleotide stability. In some embodiments, the present disclosure provides techniques for incorporating various additional chemical moieties into an oligonucleotide.

In certain embodiments, ds oligonucleotides comprising additional chemical moieties exhibit increased delivery into and/or activity in tissue as compared to a reference oligonucleotide, e.g., a reference oligonucleotide that does not have additional chemical moieties but is otherwise identical.

In certain embodiments, non-limiting examples of additional chemical moieties include carbohydrate moieties, targeting moieties, and the like, which can improve one or more properties when incorporated into an oligonucleotide. In certain embodiments, the additional chemical moiety is selected from the group consisting of: glucose, gluNAc (N-acetylglucosamine) and an anisoamide moiety. In certain embodiments, a provided ds oligonucleotide may comprise two or more additional chemical moieties, wherein the additional chemical moieties are the same or different, or belong to the same class (e.g., carbohydrate moiety, sugar moiety, targeting moiety, etc.) or do not belong to the same class.

In certain embodiments, the additional chemical moiety is a targeting moiety. In certain embodiments, the additional chemical moiety is or comprises a carbohydrate moiety. In certain embodiments, the additional chemical moiety is or comprises a lipid moiety. In certain embodiments, the additional chemical moiety is or comprises a ligand moiety, e.g., a cellular receptor (such as sigma receptor, asialoglycoprotein receptor, etc.). In certain embodiments, the ligand moiety is or comprises an anisoamide moiety, which may be a ligand moiety of sigma receptor. In certain embodiments, the ligand moiety is or comprises a GalNAc moiety, which may be a ligand moiety of an asialoglycoprotein receptor. In certain embodiments, the additional chemical moiety facilitates delivery to the liver.

In certain embodiments, provided ds oligonucleotides may comprise one or more linkers and additional chemical moieties (e.g., targeting moieties), and/or may be chirally or achirally controlled, and/or have a base sequence and/or one or more modifications and/or forms described herein.

A variety of linkers, carbohydrate moieties, and targeting moieties (including many known in the art) may be used in accordance with the present disclosure. In certain embodiments, the carbohydrate moiety is a targeting moiety. In certain embodiments, the targeting moiety is a carbohydrate moiety. In certain embodiments, provided ds oligonucleotides comprise additional chemical moieties suitable for delivery, such as glucose, gluNAc (N-acetylglucosamine), anisoamide, or a structure selected from the group consisting of:

In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4. In certain embodiments, n is 5. In certain embodiments, n is 6. In certain embodiments, n is 7. In certain embodiments, n is 8.

In certain embodiments, the additional chemical moiety is any of the chemical moieties described in the examples (including examples of multiple additional chemical moieties incorporated into multiple ds oligonucleotides).

In certain embodiments, the additional chemical moiety conjugated to the ds oligonucleotide is capable of targeting the ds oligonucleotide to a cell in the central nervous system.

In certain embodiments, the additional chemical moiety comprises or is a cell receptor ligand. In certain embodiments, the additional chemical moiety comprises or is a protein binding agent, e.g., a protein binding agent that binds to a cell surface protein. These moieties are particularly useful for targeted delivery of ds oligonucleotides to cells expressing the corresponding receptor or protein. In certain embodiments, additional chemical moieties of the provided ds oligonucleotides comprise anisoamide or derivatives or analogs thereof, and are capable of targeting the ds oligonucleotides to cells expressing a particular receptor (such as the sigma 1 receptor).

In certain embodiments, the provided ds oligonucleotides are formulated for administration to body cells and/or tissues expressing their targets. In certain embodiments, additional chemical moieties conjugated to the ds oligonucleotides are capable of targeting the oligonucleotides to cells.

In certain embodiments, the additional chemical moiety is selected from optionally substituted phenyl, Wherein n' is 1,2, 3, 4,5, 6,7, 8,9, or 10, and each other variable is as described in the disclosure. In certain embodiments, R ^s is F. In certain embodiments, R ^s is OMc. In certain embodiments, R ^s is OH. In certain embodiments, R ^s is NHAc. In certain embodiments, R ^s is NHCOCF ₃. In certain embodiments, R' is H. In certain embodiments, R is H. In certain embodiments, R ^2s is NHAc and R ^5s is OH. In certain embodiments, R ^2s is p-anisoyl and R ^5s is OH. In certain embodiments, R ^2s is NHAc and R ^5s is p-anisoyl. In certain embodiments, R ^2s is OH and R ^5s is p-anisoyl. In certain embodiments, the additional chemical moiety is selected from In certain embodiments, n' is 1. In certain embodiments, n' is 0. In certain embodiments, n "is 1. In certain embodiments, n "is 2.

In certain embodiments, the additional chemical moiety is or comprises an asialoglycoprotein receptor (ASGPR) ligand.

Without wishing to be bound by any particular theory, the present disclosure states that ASGPR1 expression in the hippocampus and/or cerebellum purkinje cell layers of mice has also been reported. http: brake-map. Org/expert/show/2048

A variety of other ASGPR ligands are known in the art and may be used in accordance with the present disclosure. In certain embodiments, the ASGPR ligand is a carbohydrate. In certain embodiments, the ASGPR ligand is GalNac or a derivative or analog thereof. In certain embodiments, the ASGPR ligand is one described in Sanhueza et al, j.am.chem.soc. [ journal of american society of chemistry ],2017, 139 (9), pages 3528-3536. In certain embodiments, the ASGPR ligand is a ligand described in MAMIDYALA et al, j.am.chem.soc. [ journal of american chemistry ],2012, 134, pages 1978-1981. In certain embodiments, the ASGPR ligand is an ASGPR ligand described in US 20160207953. In certain embodiments, the ASGPR ligand is a substituted 6, 8-dioxabicyclo [3.2.1] octane-2, 3-diol derivative such as disclosed in US 20160207953. In certain embodiments, the ASGPR ligand is an ASGPR ligand such as described in US 20150329555. In certain embodiments, the ASGPR ligand is a substituted 6, 8-dioxabicyclo [3.2.1] octane-2, 3-diol derivative such as disclosed in US 20150329555. In certain embodiments, the ASGPR ligand is an ASGPR ligand described in US 8877917, US 20160376585, US 10086081, or US 8106022. ASGPR ligands described in these documents are incorporated herein by reference. Those skilled in the art will appreciate that a variety of techniques, including those described in this document, are known for assessing binding of chemical moieties to ASGPR and may be utilized in accordance with the present disclosure. In certain embodiments, the provided ds oligonucleotides are conjugated to ASGPR ligands. In certain embodiments, the provided ds oligonucleotides comprise ASGPR ligands. In certain embodiments, the additional chemical moiety comprises an ASGPR ligand that is Wherein each variable is independently as described in the present disclosure. In certain embodiments, R is-H. In certain embodiments, R' is-C (O) R.

In certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprises an optionally substitutedIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprisesIn certain embodiments, the additional chemical moiety is or comprises

In certain embodiments, the additional chemical moiety comprises one or more moieties that can bind to, for example, an oligonucleotide target cell. For example, in certain embodiments, the additional chemical moiety comprises one or more protein ligand moieties, e.g., in certain embodiments, the additional chemical moiety comprises a plurality of moieties, each of which is independently an ASGPR ligand. In certain embodiments, as in Mod 001, mod083, mod071, mod153, and Mod155, the additional chemical moiety comprises three such ligands.

Mod001：

Mod083：

Mod071

Mod077

Mod102：

Mod105：

Mod152 (in some embodiments, -C (O) -is connected to a linker-NH-), such as Mod 153:

Mod153

Mod154 (in some embodiments, -C (O) -is connected to a linker-NH-), such as Mod 155:

Mod155

In some embodiments, the oligonucleotide comprises In some embodiments, the-CH ₂ -attachment site is used as the C5 attachment site in the saccharide. In some embodiments, the attachment site on the loop serves as a C3 attachment site in the saccharide. Such moieties may be introduced using, for example, phosphoramidite (those skilled in the art understand that one or more groups, such as protecting groups for-OH, -NH ₂-、-N(i-Pr)₂、-OCH₂CH₂ CN, etc., may alternatively be used, and the protecting groups may be removed under a variety of suitable conditions, sometimes during oligonucleotide deprotection and/or cleavage steps). In some embodiments, the oligonucleotides comprise 2, 3, or more (e.g., 3 and no more than 3). In some embodiments, the oligonucleotides comprise 2, 3, or more (e.g., 3 and no more than 3). In some embodiments, copies of such moieties are linked by internucleotide linkages (e.g., natural phosphate linkages) as described herein. In some embodiments, the-CH ₂ -attachment site is bonded to the-OH when at the 5' terminus. In some embodiments, each-OR 'is-OAc, and-N (R') ₂ is-NHAc.

In certain embodiments, the additional chemical moiety is a Mod group as described herein, e.g., in table 1.

In certain embodiments, the additional chemical moiety is Mod001. In certain embodiments, the additional chemical moiety is Mod083. In certain embodiments, an additional chemical moiety (e.g., mod group) is conjugated (e.g., without a linker) directly to the remainder of the ds oligonucleotide. In certain embodiments, the additional chemical moiety is conjugated to the remainder of the ds oligonucleotide via a linker. In certain embodiments, additional chemical moieties (e.g., mod groups) can be directly linked and/or linked via a linker to nucleobases, sugars, and/or internucleotide linkages of the ds oligonucleotide. In certain embodiments, the Mod group is attached to the sugar directly or via a linker. In certain embodiments, the Mod group is attached to the 5' terminal sugar directly or via a linker. In certain embodiments, the Mod group is attached to the 5 'terminal sugar through a 5' carbon, either directly or via a linker. See table 1 for examples of various ds oligonucleotides. In certain embodiments, the Mod group is attached to the 3' terminal sugar directly or via a linker. In certain embodiments, the Mod group is attached to the 3 'terminal sugar through the 3' carbon directly or via a linker. In certain embodiments, the Mod group is attached to the nucleobase directly or via a linker. In certain embodiments, the Mod group is attached to the internucleotide linkage directly or via a linker. In certain embodiments, provided oligonucleotides comprise Mod001 attached to the 5' end of an oligonucleotide strand by L001.

As will be appreciated by those of skill in the art, additional chemical moieties may be attached to the ds oligonucleotide chain at various positions, such as the 5 'end, the 3' end, or intermediate positions (e.g., on sugars, bases, internucleotide linkages, etc.). In certain embodiments, it is attached at the 5' end. In certain embodiments, it is attached at the 3' terminus. In certain embodiments, it is attached at an intermediate nucleotide.

Certain additional chemical moieties (e.g., lipid moieties, targeting moieties, carbohydrate moieties), including but not limited to Mod012, mod039, mod062, mod085, mod086, and Mod094, and various linkers for attaching additional chemical moieties to the ds oligonucleotide chain, including but not limited to L001, L003, L004, L008, L009, and L010, described in WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/022473、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019032612、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/03612, each of which is independently incorporated herein by reference, and may be used in accordance with the present disclosure. In certain embodiments, the additional chemical moiety is digitoxin or biotin or a derivative thereof.

In certain embodiments, the ds oligonucleotide comprises a linker, e.g., L001, L004, L008, and/or additional chemical moieties, e.g., mod012, mod039, mod062, mod085, mod086, or Mod094. In certain embodiments, a linker, e.g., L001, L003, L004, L008, L009, L110, etc., is connected to Mod, e.g., mod012, mod039, mod062, mod085, mod086, mod094, mod152, mod153, mod154, mod155, etc. L001: -NH- (CH ₂)₆ -linker (also referred to as C6 linker, C6 amine linker or C6 amino linker) linked to Mod (if any) by-NH-, and linked to the 5 'end or 3' end of the ds oligonucleotide chain by a phosphate linkage (-O-P (O) (OH) -O-, which may be present in salt form and may be denoted O or PO) or a phosphorothioate linkage (-O-P (O) (SH) -O-, which may be present in salt form and may be denoted as os (if phosphorothioate is not chiral-controlled), or os S, S or Sp (if phosphorothioate is chiral-controlled and has Sp configuration)), or os R, R or Rp (if phosphorothioate is chiral-controlled and has Rp configuration)), if no Mod is present;

L003： And (3) a joint. In certain embodiments, it is linked by its amino group to Mod (if any) (if Mod is not present), and is linked to the 5 'end or the 3' end of the oligonucleotide strand, for example via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be either not chirally controlled or chirally controlled (Sp or Rp))); L004: a linker having the structure-NH (CH ₂)₄CH(CH₂OH)CH₂ -, wherein-NH-is attached to Mod (via-C (O) -) or-H, and the-CH ₂ -attachment site is linked to the oligonucleotide strand by a linkage (e.g., at the 3' end), which is, for example, a phosphodiester linkage (-O-P (O) (OH) -O-, which may be present in salt form, and may be represented as O or PO), phosphorothioate linkages (-O-P (O) (SH) -O-, which may exist in salt form and may be represented as onium (if phosphorothioate is not chirally controlled); Or S, S or Sp (if the phosphorothioate is chirally controlled and has the Sp configuration); or (R, R) or Rp (if the phosphorothioate is chirally controlled and has the Rp configuration)), or phosphorodithioate linkages (-O-P (S) (SH) -O-, which may be present in salt form and may be represented as PS2 or: or D). For example, an immediately preceding L004 with an asterisk (e.g., L004) indicates that the linkage is a phosphorothioate linkage, while an immediately preceding L004 without an asterisk indicates that the linkage is a phosphodiester linkage. For example, in an oligonucleotide terminating at mAL, linker L004 is linked to the 3' position of the 3' terminal sugar (which is 2' -OMe modified and linked to nucleobase a) by phosphodiester linkage (via the-CH ₂ -site), and the L004 linker is linked to-H via-NH-. Similarly, in one or more oligonucleotides, the L004 linker is linked to the 3 'position of the 3' terminal sugar by phosphodiester linkage (via-CH ₂ -site), and L004 is linked via-NH-to, for example, mod012, mod085, mod086, etc.; L008: a linker having a structure of-C (O) - (CH ₂)₉) -, wherein-C (O) -is attached to Mod (via-NH-) or-OH (if Mod is not indicated), and the-CH ₂ -attachment site is linked to the oligonucleotide strand by a linkage (e.g., at the 5' end), for example, a phosphodiester linkage (-O-P (O) (OH) -O-, it may exist in salt form and may be represented as O or PO), phosphorothioate linkages (-O-P (O) (SH) -O-, it may exist in salt form and may be represented as onium (if phosphorothioate is not chirally controlled); Or S, S or Sp (if the phosphorothioate is chirally controlled and has the Sp configuration); or (R, R) or Rp (if the phosphorothioate is chirally controlled and has the Rp configuration)), or phosphorodithioate linkages (-O-P (S) (SH) -O-, which may be present in salt form and may be represented as PS2 or: or D). For example, the number of the cells to be processed, in the presence of 5' -L008mN N exemplary oligonucleotides of sequence mN-3' and having stereochemistry/linkage of OXXXXXXXXX XXXXXXXX (where N is a base, wherein O is a natural phosphate internucleotide linkage, and wherein X is a stereorandom phosphorothioate), L008 is attached to-OH via-C (O) -and to the 5' end of the oligonucleotide chain via a phosphate linkage (denoted "O" in "stereochemistry/linkage"); As another example of the use of a catalyst, in a system having 5' -Mod062L008mN N in an exemplary oligonucleotide of mN-3' sequence and having a stereochemistry/linkage of OXXXXXXXXX XXXXXXXX (where N is a base), L008 is linked to Mod062 by-C (O) -and to the 5' -end of the oligonucleotide chain by phosphate linkage (denoted as "O" in "stereochemistry/linkage");

l009: -CH ₂CH₂CH₂ -. In certain embodiments, when L009 is present at the 5 'end of an oligonucleotide without Mod, one end of L009 is attached to-OH and the other end is attached to the 5' -carbon of the oligonucleotide chain, for example via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be either not chiral controlled or chiral controlled (Sp or Rp))); l010: In certain embodiments, when L010 is present at the 5 'end of the oligonucleotide without Mod, the 5' -carbon of L010 is attached to-OH and the 3 '-carbon is attached to the 5' -carbon of the oligonucleotide strand via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be either not chiral controlled or chiral controlled (Sp or Rp))); mod012 (in some embodiments, -C (O) -attached to a linker such as-NH- > of L001, L004, L008, etc.):

l010 is utilized with n001R to form L010n001R having the structure Wherein the configuration of the phosphorus linkage is Rp. In some embodiments, multiple L010n001R may be used. For example, L023L010n001RL010n001RL010n001R, which has the structure as follows (bonded to the 5 '-carbon at the 5' -end of the oligonucleotide chain, each bonded phosphorus is independently Rp):

l023 is utilized with n001 to form L023n001 having the structure

L023 is utilized with n009 to form L023n009, as in WV-42644, having the structure

In some embodiments, L023n001L009n001 may be utilized. For example, L023n001L009n001 in WV-42643

In some embodiments, L023n009L009n009 may be utilized. For example, as in WV-42646

In some embodiments, L023n009L009n009 may be utilized. For example, as in WV-42648

In some embodiments, L025 may be utilized; as in the case of WV-41390,

Wherein the —ch ₂ -attachment site serves as a C5 attachment site for a sugar (e.g., a DNA sugar) and is attached to another unit (e.g., 3 'of the sugar), and the attachment site on the loop serves as a C3 attachment site and is attached to another unit (e.g., 5' -carbon of the carbon), each independently attached, for example, via a linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may not be chiral controlled or chiral controlled (Sp or Rp)). When L025 is at the 5' end without any modification, its-CH ₂ -attachment site is bonded to-OH. For example, L025L025L 025-of the various oligonucleotidesThe structure of (may exist in various salt forms) and is linked to the 5' -carbon of the oligonucleotide chain via an indicated linkage (e.g., a phosphate linkage (O or PO) or a phosphorothioate linkage (which may be either not chiral controlled or chiral controlled (Sp or Rp)));

in some embodiments, L026 may be utilized; as in the case of WV-44444,

In some embodiments, L027 may be utilized; as in the case of WV-44445,

In some embodiments, mU may be utilized; as in the case of WV-42079,

FU may be utilized in some embodiments; as in the case of WV-44433,

In some embodiments, dT may be utilized; as in the case of WV-44434,

In some embodiments POdT or PO4-dT may be utilized; as in the case of WV-44435,

In some embodiments, PO5MRdT may be utilized; as in the case of WV-44436,

In some embodiments, PO5MSdT may be utilized; as in the case of WV-44437,

In some embodiments VPdT may be utilized; as in the case of WV-44438,

In some embodiments, 5mvpdT may be utilized; as in the case of WV-44439,

In some embodiments, 5mrpdT may be utilized; as in the case of WV-44440,

In some embodiments, 5mspdT may be utilized; as in the case of WV-44441,

PNdT may be utilized in some embodiments; as in the case of WV-44442,

In some embodiments SPNdT may be utilized; as in the case of WV-44443,

In some embodiments, 5ptzdT may be utilized; as in the case of WV-44446,

Mod039 (in certain embodiments, -C (O) -is attached to a linker such as-NH-), L001, L003, L004, L008, L009, L110, etc.:

mod062 (in some embodiments, -C (O) -is connected to-NH-) of a linker such as L001, L003, L004, L008, L009, L110, etc.):

Mod085 (in certain embodiments, -C (O) -is attached to a linker such as-NH-), L001, L003, L004, L008, L009, L110, etc.:

mod086 (in certain embodiments, -C (O) -is attached to a linker such as-NH-), L001, L003, L004, L008, L009, L110, etc.:

Mod094 (in certain embodiments, linked to internucleotide linkages, or to the 5 'or 3' ends of oligonucleotides via linkages such as phosphate linkages, phosphorothioate linkages (which are optionally chirally controlled), and the like, e.g., in an exemplary oligonucleotide having a sequence of 5'-mN N Mod094 is linked to the 3' -end of the oligonucleotide chain (3 '-carbon of the 3' -terminal sugar) via a phosphate group (the phosphate group is not shown below and may exist in salt form; and denoted as "O" in "stereochemistry/linkage" (.. XXXXO):

In certain embodiments, the additional chemical moiety is a chemical moiety described in WO 2012/030683. In certain embodiments, provided ds oligonucleotides comprise the chemical structures described in WO 2012/030683 (e.g., linkers, lipids, solubilizing groups, and/or targeting ligands).

In certain embodiments, provided ds oligonucleotides comprise additional chemical moieties and/or modifications (e.g., modifications of nucleobases, sugars, internucleotide linkages, etc.) described in the following documents: U.S. patent nos. 5,688,941;6,294,664;6,320,017;6,576,752;5,258,506;5,591,584;4,958,013;5,082,830;5,118,802;5,138,045;6,783,931;5,254,469;5,414,077;5,486,603;5,112,963;5,599,928;6,900,297;5,214,136;5,109,124;5,512,439;4,667,025;5,525,465;5,514,785;5,565,552;5,541,313;5,545,730;4,835,263;4,876,335;5,578,717;5,580,731;5,451,463;5,510,475;4,904,582;5,082,830;4,762,779;4,789,737;4,824,941;4,828,979;5,595,726;5,214,136;5,245,022;5,317,098;5,371,241;5,391,723;4,948,882;5,218,105;5,112,963;5,567,810;5,574,142;5,578,718;5,608,046;4,587,044;4,605,735;5,585,481;5,292,873;5,552,538;5,512,667;5,597,696;5,599,923;7,037,646;5,587,371;5,416,203;5,262,536;5,272,250; or 8,106,022.

In certain embodiments, additional chemical moieties (e.g., mod) are attached via a linker. Various linkers are available in the art and may be used in accordance with the present disclosure, for example, those for conjugating various moieties to proteins (e.g., to antibodies to form antibody-drug conjugates), nucleic acids, and the like. Some useful linkers are described in US 9982257、US 20170037399、US 20180216108、US 20180216107、US 9598458、WO 2017/062862、WO 2018/067973、WO 2017/160741、WO 2017/192679、WO 2017/210647、WO 2018/098264、WO 2018/223056、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、 and/or WO 2019/032612, the respective linker portions of which are incorporated herein by reference in their own right. In certain embodiments, the linker is L001, L004, L009, or L010, as non-limiting examples. In certain embodiments, the oligonucleotide comprises a linker, but no additional chemical moiety other than a linker. In certain embodiments, the ds oligonucleotide comprises a linker, but no additional chemical moiety other than a linker, wherein the linker is L001, L004, L009, or L010.

As demonstrated herein, in certain embodiments, the provided techniques can provide high levels of activity and/or desirable properties without utilizing specific structural elements (e.g., modifications, linkage configurations and/or patterns, etc.) reported to be desirable and/or necessary (e.g., those reported in WO 2019/219581), but some such structural elements can be incorporated into ds oligonucleotides in combination with a variety of other structural elements according to the present disclosure. For example, in certain embodiments, a ds oligonucleotide of the disclosure has fewer nucleosides 3 'of the nucleoside opposite the target glycoside (e.g., target adenosine), comprises one or more phosphorothioate internucleotide linkages at one or more positions where phosphorothioate internucleotide linkages are reportedly undesirable or disallowed, comprises one or more Sp phosphorothioate internucleotide linkages at one or more positions where Sp phosphorothioate internucleotide linkages are reportedly undesirable or disallowed, comprises one or more Sp phosphorothioate internucleotide linkages at one or more positions where Rp phosphorothioate internucleotide linkages are reportedly undesirable or disallowed, and/or comprises different modifications (e.g., reportedly internucleotide linkages, sugar modifications, etc.) and/or stereochemistry (e.g., sp phosphorothioate linkages are reportedly favored and/or are present at certain oligonucleotide linkages, other than those modifications and/or stereochemically desired at certain oligonucleotide linkages, at certain phosphorothioate linkages at certain positions, other than those positions where Rp phosphorothioate linkages are reportedly favored and/or desired; as demonstrated herein, the provided techniques can provide desirable properties and/or high activity without utilizing a 2' -MOE, without avoiding phosphorothioate linkages at one or more such specific positions, without avoiding Sp phosphorothioate linkages at one or more such specific positions, and/or without avoiding Rp phosphorothioate linkages at one or more of such specific positions). Additionally or alternatively, provided ds oligonucleotides comprise previously unrecognized structural elements, such as with certain modifications (e.g., base modifications, sugar modifications (e.g., 2' -F), linkage modifications (e.g., non-negatively charged internucleotide linkages), additional moieties, and the like), as well as levels, patterns, and combinations thereof.

For example, in certain embodiments, as described herein, provided oligonucleotides comprise no more than 5, 6, 7, 8, 9, 10, 11, or 12 nucleosides 3' of a nucleoside opposite a target nucleoside (e.g., target adenosine).

Alternatively or additionally, as described herein (e.g., as displayed in certain examples), for structural elements 3' of a nucleoside opposite a target glycoside (e.g., target adenosine), in certain embodiments, about 50% -100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) of internucleotide linkages of the nucleoside opposite the target glycoside (e.g., target adenosine) are each independently modified internucleotide linkages, which are optionally chirally controlled. In certain embodiments, no more than 1, 2, or 3 internucleotide linkages at the nucleoside opposite the target glycoside are natural phosphate linkages. In certain embodiments, no such internucleotide linkages are natural phosphate linkages. In certain embodiments, no more than 1 such internucleotide linkage is a natural phosphate linkage. In certain embodiments, no more than 2 such internucleotide linkages are natural phosphate linkages. In certain embodiments, no more than 3 such internucleotide linkages are natural phosphate linkages. In certain embodiments, each modified internucleotide linkage is independently a phosphorothioate or a non-negatively charged internucleotide linkage (e.g., n 001). In certain embodiments, each phosphorothioate internucleotide linkage is chiral controlled. In certain embodiments, no more than 1, 2, or 3 internucleotide linkages at the nucleoside opposite the target glycoside are Rp phosphorothioate internucleotide linkages.

Alternatively or additionally, as described herein (e.g., as shown in certain examples), in certain embodiments, about 50% -100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the internucleotide linkages at the 5' nucleoside opposite the target glycoside (e.g., target adenosine) are each independently modified internucleotide linkages, optionally under chiral control. In certain embodiments, no internucleotide linkages or no more than 1,2, or 3 internucleotide linkages are not modified internucleotide linkages at the 5' of the nucleoside opposite the target glycoside (e.g., target adenosine). In certain embodiments, no internucleotide linkages or no more than 1,2, or 3 internucleotide linkages are not phosphorothioate internucleotide linkages at the 5' of the nucleoside opposite the target glycoside (e.g., target adenosine). In certain embodiments, no internucleotide linkages or no more than 1,2, or 3 internucleotide linkages other than Sp phosphorothioate internucleotide linkages are 5' of the nucleoside opposite the target glycoside (e.g., target adenosine). In certain embodiments, no more than 1,2, or 3 internucleotide linkages at the nucleoside opposite the target glycoside (e.g., target adenosine) are natural phosphate linkages. In certain embodiments, no such internucleotide linkages are natural phosphate linkages. in certain embodiments, no more than 1 such internucleotide linkage is a natural phosphate linkage. In certain embodiments, no more than 2 such internucleotide linkages are natural phosphate linkages. In certain embodiments, no more than 3 such internucleotide linkages are natural phosphate linkages. In certain embodiments, each modified internucleotide linkage is independently a phosphorothioate or a non-negatively charged internucleotide linkage (e.g., n 001). In certain embodiments, there are no 2, 3, or 4 consecutive internucleotide linkages 5' of the nucleoside opposite the target glycoside, each of which is not a phosphorothioate internucleotide linkage. In certain embodiments, there are no 2, 3, or 4 consecutive internucleotide linkages 5' of the nucleoside opposite the target glycoside, each of which is chirally controlled and is not an Sp phosphorothioate internucleotide linkage. In certain embodiments, no or no more than 1, 2, 3, 4, or 5 internucleotide linkages at the 5' opposite nucleoside from the target glycoside (e.g., target adenosine) are Rp phosphorothioate internucleotide linkages. In certain embodiments, at least about 1,2,3,4, 5, 6,7, 8, 9, or 10, or about 1,2,3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, or about 50% -100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the internucleotide linkages at the 5' opposite nucleoside from the target glycoside (e.g., target adenosine) are each independently chirally controlled, and is Sp internucleotide linkage. In certain embodiments, at least about 1,2, 3, 4, 5, 6, 7, 8, 9, or 10, or about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32, or about 50% -100% (e.g., about or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of phosphorothioate internucleotide linkages at the 5' nucleoside opposite the target glycoside (e.g., target adenosine) are each independently chirally controlled, And is Sp. In certain embodiments, each phosphorothioate internucleotide linkage at the 5' of the nucleoside opposite the target glycoside (e.g., target adenosine) is chirally controlled. In certain embodiments, each phosphorothioate internucleotide linkage at the 5' of the nucleoside opposite the target glycoside (e.g., target adenosine) is Sp.

Oligonucleotide and composition production

A variety of methods can be used to generate oligonucleotides and compositions, and can be used in accordance with the present disclosure. For example, traditional phosphoramidite chemistry can be used to prepare stereorandom oligonucleotides and compositions, and certain reagents and techniques of chirality control can be used to prepare chirality-controlled oligonucleotide compositions, e.g., ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252, each of which is incorporated herein by reference.

In some embodiments, the chiral controlled/stereoselective preparation of oligonucleotides and compositions thereof includes the use of chiral auxiliary reagents, for example, as part of monomeric phosphoramidites. Examples of such chiral auxiliary reagents and phosphoramidites are described in ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252 below, each of which is incorporated herein by reference independently. In some embodiments, the chiral auxiliary is(DPSE chiral auxiliary). In some embodiments, the chiral auxiliary isIn some embodiments, the chiral auxiliary is In some embodiments, the chiral auxiliary comprises-SO ₂R^AU, wherein R ^AU is an optionally substituted group selected from the group consisting of: c _1-20 aliphatic, C _1-20 heteroaliphatic having 1-10 heteroatoms, C _6-20 aryl, C _6-20 arylaliphatic, C _6-20 arylaliphatic having 1-10 heteroatoms, 5-20 membered heteroaryl having 1-10 heteroatoms, and 3-20 membered heterocyclyl having 1-10 heteroatoms. In some embodiments, the chiral auxiliary is In some embodiments, R ^AU is optionally substituted aryl. In some embodiments, R ^AU is optionally substituted phenyl. In some embodiments, R ^AU is an optionally substituted C _1-6 aliphatic. In some embodiments, the chiral auxiliary is (PSM chiral auxiliary). In some embodiments, the use, protection, removal, etc. of such chiral auxiliary (e.g., formulation), phosphoramidite comprising such chiral auxiliary, intermediate oligonucleotide comprising such auxiliary (these auxiliary are typically bonded to the phosphorus linkage through-O-of-OH, while-NH-is optionally capped (e.g., through-C (O) R)), are described in US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612、 and/or WO 2020/191252, and incorporated herein by reference.

In some embodiments, chiral controlled preparation techniques (including oligonucleotide synthesis cycles, reagents and conditions) are described below in ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252, the respective oligonucleotide synthesis methods, cycles, reagents and conditions of which are independently incorporated herein by reference.

Once synthesized, ds oligonucleotides and compositions targeting HSD17B13 will typically be further purified. Suitable purification techniques are well known and practiced by those skilled in the art, including but not limited to those ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252 described below, the respective purification techniques of which are individually incorporated herein by reference.

In some embodiments, cycling includes or consists of coupling, capping, modifying and deblocking. In some embodiments, cycling includes or consists of coupling, capping, modifying, capping, and deblocking. The steps are typically performed in the order in which they are listed, but in some embodiments the order of certain steps, such as capping and modification, may be altered as understood by those skilled in the art. If desired, one or more steps may be repeated to increase conversion, yield and/or purity, as is commonly done in syntheses by those skilled in the art. For example, in some embodiments, the coupling may be repeated; in some embodiments, the modification (e.g., oxidation to install = O, sulfidation to install = S, etc.) may be repeated; in some embodiments, the coupling is repeated after modification, which may convert the P (III) linkage to a P (V) linkage that may be more stable in some cases, and the coupling is typically followed by modification to convert the newly formed P (III) linkage to a P (V) linkage. In some embodiments, different conditions (e.g., concentration, temperature, reagents, time, etc.) may be employed when repeating the steps.

In some embodiments, the oligonucleotide is attached to a solid support. In some embodiments, the solid support is a support for oligonucleotide synthesis. In some embodiments, the solid support comprises glass. In some embodiments, the solid support is CPG (controlled pore glass). In some embodiments, the solid support is a polymer. In some embodiments, the solid support is polystyrene. In some embodiments, the solid support is Highly Crosslinked Polystyrene (HCP). In some embodiments, the solid support is a hybrid support of Controlled Pore Glass (CPG) and Highly Crosslinked Polystyrene (HCP). In some embodiments, the solid support is a metal foam. In some embodiments, the solid support is a resin. In some embodiments, the oligonucleotides are cleaved from the solid support.

Techniques for formulating the provided oligonucleotides and/or preparing pharmaceutical compositions, e.g., techniques for administration to a subject via various routes, are readily available in the art and may be used in accordance with the present disclosure, e.g., those ：US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612 and/or WO 2020/191252 described below.

Metabolic products and shortened forms of oligonucleotides

In some embodiments, the ds oligonucleotide that targets HSD17B13 corresponds to a metabolite produced by cleavage of a longer oligonucleotide, e.g., a longer ds oligonucleotide that targets HSD17B13 (e.g., by enzymatic cleavage of a nuclease). In some embodiments, the disclosure relates to a ds oligonucleotide targeting HSD17B13 corresponding to a portion or fragment of a ds oligonucleotide targeting HSD17B13 disclosed herein.

In some embodiments, the disclosure relates to oligonucleotides corresponding to metabolites of the ds oligonucleotides disclosed herein that target HSD17B 13. In some embodiments, the disclosure relates to oligonucleotides that are 1, 2,3,4, 5, 6, 7, 8, 9,10, 11, 12, 13 or more bases shorter than the oligonucleotides disclosed herein. In some embodiments, the disclosure relates to oligonucleotides having a base sequence that is 1, 2,3,4, 5, 6, 7, 8, 9,10, 11, 12, 13 or more bases shorter than the base sequence of the oligonucleotides disclosed herein.

In some embodiments, the metabolite is named 3'-N- # or 5' N- # where # denotes the number of bases removed and 3 'or 5' denote from which end of the molecule the base is deleted. For example, 3'-N-1 represents a fragment or metabolite in which 1 base is removed from the 3' -end.

In some embodiments, the disclosure relates to oligonucleotides corresponding to fragments or metabolites of the oligonucleotides disclosed herein, wherein the fragments or metabolites may be described as corresponding to 3'-N-1、3'-N-2、3'-N-3、3'-N-4、3'-N-5、3'-N-6、3'-N-7、3'-N-8、3'-N-9、3'-N-10、3'-N-11、3'-N-12、5'-N-1、5'-N-2、5'-N-3、5'-N-4、5'-N-5、5'-N-6、5'-N-7、5'-N-8、5'-N-9、5'-N-10、5'-N-11、 or 5' -N-12 of the oligonucleotides described herein, wherein each T may be independently replaced by U, and vice versa.

In some embodiments, the disclosure relates to oligonucleotides corresponding to a metabolite of the oligonucleotides, wherein the metabolite is truncated at the 5 'and/or 3' ends relative to the oligonucleotides disclosed herein, wherein each T may be independently replaced by U, and vice versa. In some embodiments, the disclosure relates to oligonucleotides corresponding to a metabolite of the oligonucleotides, wherein the metabolite is truncated at both the 5 'and 3' ends relative to the oligonucleotides disclosed herein, wherein each T can be independently replaced by U, and vice versa. In some embodiments, the disclosure relates to oligonucleotides that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more total bases shorter than the oligonucleotides disclosed herein at the 5 'end and/or the 3' end. In some embodiments, the disclosure relates to oligonucleotides having a base sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more total bases shorter at the 5 'end and/or the 3' end than the base sequence of the oligonucleotides disclosed herein, wherein each T can be independently replaced by U, and vice versa.

In some embodiments, the disclosure relates to oligonucleotides that are cleavage products of the oligonucleotides disclosed herein that cleave at natural phosphate linkages. In some embodiments, the disclosure relates to oligonucleotides that are cleavage products of the oligonucleotides disclosed herein that cleave at Rp phosphorothioate internucleotide linkages.

In accordance with the present disclosure, metabolites and/or shortened ds oligonucleotides targeting HSD17B13 may be identified, characterized, and/or assessed using a variety of techniques, such as those described in US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0249173、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2019/032607、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612、 and/or WO 2020/191252.

Characterization and evaluation

In some embodiments, the properties and/or activity of ds oligonucleotides and compositions thereof that target HSD17B13 can be characterized and/or assessed using various techniques available to those of skill in the art, such as biochemical assays (e.g., rnase H assays), cell-based assays, animal models, clinical trials, and the like.

In some embodiments, a method of identifying and/or characterizing an oligonucleotide composition, e.g., a ds oligonucleotide composition that targets HSD17B13, comprises the steps of:

Providing at least one composition comprising a plurality of oligonucleotides; and

Delivery was assessed relative to a reference composition.

In some embodiments, the disclosure provides methods of identifying and/or characterizing an oligonucleotide composition, e.g., a ds oligonucleotide composition that targets HSD17B13, comprising the steps of:

Cellular uptake was assessed relative to a reference composition.

The reduction of transcripts of the target gene and/or products encoded thereby relative to a reference composition is assessed.

Reduction of tau levels was assessed, which was aggregated and/or spread relative to the reference composition.

In some embodiments, the identity and/or activity of an oligonucleotide, e.g., a ds oligonucleotide targeting HSD17B13 and compositions thereof, is compared to a reference oligonucleotide and compositions thereof, respectively.

In some embodiments, the reference oligonucleotide composition is a stereorandom oligonucleotide composition. In some embodiments, the reference oligonucleotide composition is a stereorandom composition of oligonucleotides in which all internucleotide linkages are phosphorothioate. In some embodiments, the reference oligonucleotide composition is a DNA oligonucleotide composition having all phosphate linkages. In some embodiments, the reference oligonucleotide composition is otherwise identical to the provided chirally controlled oligonucleotide composition except that it is not chirally controlled. In some embodiments, the reference oligonucleotide composition is otherwise identical to the provided chirally controlled oligonucleotide composition except that it has a different stereochemical pattern. In some embodiments, the reference oligonucleotide composition is similar to the provided oligonucleotide composition except that it has different modifications or different modes of modification to one or more sugars, bases, and/or internucleotide linkages. In some embodiments, the oligonucleotide compositions are stereorandom, while the reference oligonucleotide compositions are also stereorandom, but they differ in one or more modifications of sugar and/or base or pattern thereof.

In some embodiments, the reference composition is a composition of oligonucleotides having the same base sequence and the same chemical modification. In some embodiments, the reference composition is a composition of oligonucleotides having the same base sequence and the same pattern of chemical modification. In some embodiments, the reference composition is an achiral controlled (or stereorandom) composition of oligonucleotides having the same base sequence and chemical modification. In some embodiments, the reference composition is an achiral controlled (or stereorandom) composition of oligonucleotides of the same composition as the provided chiral controlled oligonucleotide composition.

In some embodiments, the reference oligonucleotide composition is an oligonucleotide having a different base sequence. In some embodiments, the reference oligonucleotide composition has an oligonucleotide that does not target HSD17B13 (e.g., as a negative control for certain assays).

In some embodiments, the reference composition is a composition of oligonucleotides having the same base sequence but different chemical modifications (including but not limited to the chemical modifications described herein). In some embodiments, the reference composition is a composition of stereochemically and/or chemically modified oligonucleotides having the same base sequence but different modes of internucleotide linkage and/or internucleotide linkages.

Various methods are known in the art for detecting gene products whose expression, level and/or activity may be altered following the introduction or administration of the provided oligonucleotides. For example, qPCR can be used to detect and quantify transcripts and their knockdown, and protein levels can be determined by western blotting.

In some embodiments, the assessment of oligonucleotide efficacy may be performed in a biochemical assay or in vitro in a cell. In some embodiments, the ds oligonucleotide targeting HSD17B13 can be introduced into a cell by various methods available to those skilled in the art, e.g., naked (gymnotic) delivery, transfection, lipofection, etc.

In some embodiments, putative ds oligonucleotides targeting HSD17B13 may be tested for efficacy in vitro.

In some embodiments, the efficacy of a putative ds oligonucleotide targeting HSD17B13 may be tested in vitro using any known method of testing the expression, level and/or activity of the HSD17B13 gene or gene product thereof.

In some embodiments, the safety and/or efficacy of animal models administered ds oligonucleotides targeting HSD17B13 can be assessed.

In some embodiments, one or more effects of administering the oligonucleotide to an animal can be assessed, including any effects on behavior, inflammation, and toxicity. In some embodiments, after administration, signs of toxicity can be observed in the animal, including disturbing grooming behavior, lack of food consumption, and any other signs of somnolence.

In some embodiments, after administration of the ds oligonucleotide targeting HSD17B13 to an animal, the animal may be sacrificed and analysis of the tissues or cells may be performed to determine alterations of HSD17B13 or other biochemical or other alterations. In some embodiments, after necropsy, liver, heart, lung, kidney and spleen can be collected, fixed and processed for histopathological assessment (standard optical microscopy of hematoxylin and eosin stained tissue slides).

In some embodiments, behavioral changes can be monitored or assessed following administration of a ds oligonucleotide targeting HSD17B13 to an animal. In some embodiments, such assessment may be performed using techniques described in the scientific literature.

The various effects tested in the animals described herein can also be monitored in human subjects or patients following administration of ds oligonucleotides targeting HSD17B 13.

In addition, the efficacy of ds oligonucleotides targeting HSD17B13 in human subjects can be measured by assessing any of a variety of parameters known in the art after administration of the oligonucleotide, including, but not limited to, reduction of the symptoms of the disease.

In some embodiments, the cells and/or tissues are collected for analysis after treatment of the human with the oligonucleotide, or after contacting the cells or tissues with the oligonucleotide in vitro.

In some embodiments, in various cells and/or tissues, target nucleic acid levels can be quantified by methods available in the art (many of which can be accomplished with commercially available kits and materials). Such methods include, for example, northern blot analysis, competitive Polymerase Chain Reaction (PCR), quantitative real-time PCR, and the like. RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. Probes and primers are designed to hybridize to the nucleic acid to be detected. Methods for designing real-time PCR probes and primers are well known in the art and are widely practiced. For example, to detect and quantify HSD17B13 RNA, one exemplary method comprises isolating total RNA (e.g., including mRNA) from cells or animals treated with an oligonucleotide or composition and subjecting the RNA to reverse transcription and/or quantitative real-time PCR, such as described herein or Moon et al 2012Cell meta 15: 240-246.

In some embodiments, protein levels can be assessed or quantified using various methods known in the art, such as, for example, enzyme-linked immunosorbent assay (ELISA), western blot analysis (immunoblot), immunocytochemistry, fluorescence Activated Cell Sorting (FACS), immunohistochemistry, immunoprecipitation, protein activity assays (e.g., caspase activity assays), and quantitative protein assays. Antibodies useful for detection of mouse, rat, monkey and human proteins are commercially available and can be produced when desired. For example, various HSD17B13 antibodies have been reported.

Various techniques for detecting the level of an oligonucleotide or other nucleic acid are available and/or known in the art. Such techniques can be used to detect ds oligonucleotides targeting HSD17B13 when administered to assess, for example, delivery, cellular uptake, stability, distribution, and the like.

In some embodiments, selection criteria are used to evaluate the data obtained from various assays and to select specific desirable oligonucleotides having certain properties and activities, such as the desired ds oligonucleotide targeting HSD17B 13. In some embodiments, the selection criteria includes an IC ₅₀ of less than about 10nM, less than about 5nM, or less than about 1 nM. In some embodiments, the selection criteria for stability analysis include at least 50% stability on day 1 [ at least 50% of the oligonucleotides remain remaining and/or detectable ]. In some embodiments, the selection criteria for stability analysis includes at least 50% stability on day 2. In some embodiments, the selection criteria for stability analysis includes at least 50% stability on day 3. In some embodiments, the selection criteria for stability analysis includes at least 50% stability on day 4. In some embodiments, the selection criteria for stability analysis includes at least 50% stability on day 5. In some embodiments, the selection criteria for stability analysis includes at least 80% [ at least 80% of oligonucleotides remaining ] on day 5.

In some embodiments, the efficacy of a ds oligonucleotide targeting HSD17B13 is assessed directly or indirectly by monitoring, measuring or detecting a condition, disorder or disease associated with HSD17B13 or a change in a biological pathway.

In some embodiments, the efficacy of a ds oligonucleotide targeting HSD17B13 is assessed directly or indirectly by monitoring, measuring, or detecting a change in response affected by the knockdown of HSD17B 13.

In some embodiments, the provided oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) can be analyzed by sequence analysis to determine which other genes [ e.g., genes that are not target genes (e.g., HSD17B 13) ] have a sequence that is complementary to the base sequence of the provided oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) or has 0, 1, 2, or more mismatches to the base sequence of the provided oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13). Knockdown (if any) by these potentially off-target oligonucleotides can be determined to assess the potential off-target effect of the oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13). In some embodiments, off-target effects are also referred to as unintended effects and/or are associated with hybridization of bystander (non-target) sequences or genes.

Oligonucleotides that knock down HSD17B13 have been evaluated and tested for efficacy have a variety of uses, for example, in the treatment or prevention of HSD17B 13-related conditions, disorders or diseases or symptoms thereof.

In some embodiments, ds oligonucleotides targeted to HSD17B13 that have been evaluated and tested for their ability to provide a particular biological effect (e.g., reduce the level, expression, and/or activity of an HSD17B13 target gene or gene product thereof) are useful in treating, ameliorating, and/or preventing a HSD17B 13-related condition, disorder, or disease.

Treatment of HSD17B 13-related conditions, disorders or diseases

In some embodiments, the disclosure provides ds oligonucleotides targeting HSD17B13 that target HSD17B13 and direct target-specific knockdown of HSD17B 13. In some embodiments, the disclosure provides methods for preventing and/or treating HSD17B 13-related conditions, disorders, or diseases using the provided ds oligonucleotides that target HSD17B13 and compositions thereof. In some embodiments, the disclosure provides oligonucleotides and compositions thereof for use as a medicament, e.g., for use in HSD17B 13-related conditions, disorders or diseases. In some embodiments, the disclosure provides oligonucleotides and compositions thereof for use in treating HSD17B 13-related conditions, disorders or diseases. In some embodiments, the disclosure provides oligonucleotides and compositions thereof for use in the manufacture of a medicament for the treatment of HSD17B 13-related conditions, disorders or diseases.

In some embodiments, the disclosure provides methods for treating and/or ameliorating one or more symptoms associated with a HSD17B 13-associated condition, disorder or disease in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a ds oligonucleotide or composition thereof that targets HSD17B 13. In some embodiments, the disclosure provides a method for reducing the susceptibility of a mammal in need thereof to an HSD17B 13-related condition, disorder or disease, the method comprising: administering to the mammal a therapeutically effective amount of a ds oligonucleotide or a composition thereof that targets HSD17B 13. In some embodiments, the disclosure provides a method for preventing or delaying the onset of an HSD17B 13-related condition, disorder or disease in a mammal in need thereof, the method comprising: administering to the mammal a therapeutically effective amount of a ds oligonucleotide or a composition thereof that targets HSD17B 13. In some embodiments, the present disclosure provides a method for treating and/or ameliorating one or more symptoms associated with a HSD17B 13-associated condition, disorder or disease in a mammal in need thereof, the method comprising: administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising a ds oligonucleotide that targets HSD17B 13. In some embodiments, the disclosure provides a method for reducing the susceptibility of a mammal in need thereof to an HSD17B 13-related condition, disorder or disease, the method comprising: administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising a ds oligonucleotide that targets HSD17B 13. In some embodiments, the disclosure provides a method for preventing or delaying the onset of an HSD17B 13-related condition, disorder or disease in a mammal in need thereof, the method comprising: administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising a ds oligonucleotide that targets HSD17B 13. In some embodiments, the mammal is a human. In some embodiments, the mammal is susceptible to, suffering from, and/or suffering from a HSD17B 13-related condition, disorder, or disease. In some embodiments, the HSD17B 13-associated condition, disorder or disease is NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis.

In some embodiments, the provided oligonucleotides and compositions are useful for preventing and/or treating neurodegenerative diseases, such as NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis, etc. In some embodiments, the disclosure provides methods for preventing and/or treating a degenerative disease, e.g., NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, pharmaceutical liver injury, or hepatocyte necrosis, etc., comprising administering to a subject susceptible to or suffering from the above disease a therapeutically effective amount of a ds oligonucleotide or composition targeting HSD17B13 as described herein. In some embodiments, the disclosure provides methods for treating a neurodegenerative disease comprising administering to a subject having the neurodegenerative disease a therapeutically effective amount of a ds oligonucleotide or composition as described herein that targets HSD17B 13. In some embodiments, the disclosure provides methods for preventing and/or treating tauopathies comprising administering to a subject susceptible to or suffering from the tauopathies a therapeutically effective amount of a ds oligonucleotide or composition that targets HSD17B13 as described herein. In some embodiments, the disclosure provides methods for treating tauopathies comprising administering to a subject suffering from the tauopathies a therapeutically effective amount of a ds oligonucleotide or composition as described herein that targets HSD17B 13. In some embodiments, the disclosure provides methods for preventing and/or treating NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury, or hepatocyte necrosis, comprising administering to a subject susceptible to or suffering from the above disease a therapeutically effective amount of a ds oligonucleotide or composition as described herein that targets HSD17B 13. In some embodiments, the disclosure provides methods for treating NAFLD, NASH, ASH, alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, pharmaceutical liver injury, or hepatocyte necrosis, comprising administering to a subject having the above disease a therapeutically effective amount of a ds oligonucleotide or composition targeting HSD17B13 as described herein. In some embodiments, the subject has increased expression of HSD17B13 and/or increased levels of one or more HSD17B13 products (e.g., transcripts, proteins, etc.) as compared to, for example, a healthy subject (or population thereof), a subject (or population thereof) that is not susceptible to and/or does not have a HSD17B 13-related disease, etc.

In some embodiments, the provided oligonucleotides and compositions may optionally be used in combination with one or more other therapeutic agents.

Administration of oligonucleotides and compositions

In accordance with the present disclosure, a number of delivery methods, protocols, etc. can be used to administer the provided ds oligonucleotides and compositions thereof (typically pharmaceutical compositions for therapeutic purposes), including various techniques known in the art.

In some embodiments, the oligonucleotide composition (e.g., a ds oligonucleotide composition that targets HSD17B 13) is administered at a lower dose and/or frequency than an otherwise comparable reference oligonucleotide composition, and has a comparable or improved effect. In some embodiments, the chirally controlled oligonucleotide composition is administered at a lower dose and/or frequency than a comparable, otherwise identical, stereorandomized reference oligonucleotide composition and has a comparable or improved effect, e.g., in improving knockdown of a target transcript.

In some embodiments, the present disclosure recognizes that properties and activities of oligonucleotides and compositions thereof, such as knockdown activity, stability, toxicity, etc., can be modulated and optimized by chemical modification and/or stereochemistry. In some embodiments, the disclosure provides methods for optimizing oligonucleotide properties and/or activity via chemical modification and/or stereochemistry. In some embodiments, the present disclosure provides oligonucleotides and compositions thereof with improved properties and/or activity. Without wishing to be bound by any theory, e.g., due to its better activity, stability, delivery, distribution, toxicity, pharmacokinetics, pharmacodynamics, and/or efficacy profile, applicants note that the provided oligonucleotides and compositions thereof may be administered in lower doses and/or with reduced frequency in some embodiments to achieve comparable or better efficacy, and in some embodiments at higher doses and/or with increased frequency to provide enhanced effects.

In some embodiments, the present disclosure provides an improvement in a method of administering an oligonucleotide composition comprising a plurality of oligonucleotides sharing a common base sequence, the improvement comprising administering an oligonucleotide comprising a plurality of oligonucleotides characterized by improved delivery relative to a reference oligonucleotide composition having the same common base sequence.

In some embodiments, the provided oligonucleotides, compositions, and methods provide improved delivery. In some embodiments, the provided oligonucleotides, compositions, and methods provide improved cytoplasmic delivery. In some embodiments, the improved delivery is into a cell population. In some embodiments, the improved delivery is into tissue. In some embodiments, the improved delivery is into an organ. In some embodiments, the improved delivery is into an organism (e.g., a patient or subject). Exemplary structural elements (e.g., chemical modifications, stereochemistry, combinations thereof, etc.), oligonucleotides, compositions, and methods that provide improved delivery are detailed in the present disclosure.

Various dosing regimens may be used to administer the oligonucleotides and compositions of the disclosure. In some embodiments, multiple unit doses are administered at intervals of time. In some embodiments, the specified composition has a recommended dosing regimen, which may involve one or more administrations. In some embodiments, the dosing regimen comprises multiple doses, each of which is spaced apart from each other by a period of the same length; in some embodiments, the dosing regimen includes multiple doses and at least two different periods of time separated by individual doses. In some embodiments, all administrations within a dosage regimen have the same unit dosage. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose of a first dose followed by another dose or doses of a second dose different from the first dose. In some embodiments, the dosing regimen comprises a first dose administered followed by another dose administered in one or more times a second (or subsequent) dose that is the same as or different from the first dose (or another previous dose). In some embodiments, the chirally controlled oligonucleotide composition is administered according to a dosing regimen that is different from the dosing regimen for an achiral controlled (e.g., stereorandom) oligonucleotide composition of the same sequence and/or the dosing regimen for a different chirally controlled oligonucleotide composition of the same sequence. In some embodiments, the chirally controlled oligonucleotide composition is administered according to a dosing regimen that is reduced compared to a dosing regimen of an achiral controlled (e.g., stereorandom) oligonucleotide composition of the same sequence that achieves a lower level of total exposure per given unit time, involves one or more lower unit doses, and/or includes fewer dosing times per given unit time. In some embodiments, the achiral controlled oligonucleotide is administered according to a dosing regimen that lasts for a longer period of time than a dosing regimen of an achiral controlled (e.g., stereorandom) oligonucleotide composition of the same sequence. Without wishing to be bound by theory, applicants note that in some embodiments, shorter dosing regimens and/or longer periods between doses may be based on improved stability, bioavailability, and/or efficacy of the chirally controlled oligonucleotide composition. In some embodiments, with improved delivery (and other characteristics), the provided compositions can be administered at lower doses and/or at lower frequencies to achieve biological effects, such as clinical efficacy.

Pharmaceutical composition

When used as a therapeutic agent, the provided ds oligonucleotides (e.g., ds oligonucleotides targeting HSD17B 13) or oligonucleotide compositions thereof are typically administered as a pharmaceutical composition. In some embodiments, the present disclosure provides pharmaceutical compositions comprising a provided compound (e.g., an oligonucleotide) or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier. In some embodiments, the oligonucleotides of the present disclosure are provided as pharmaceutical compositions for therapeutic and clinical purposes. As will be appreciated by those of skill in the art, the oligonucleotides of the present disclosure may be provided in their acid, base or salt forms. In some embodiments, the oligonucleotide may be in the acid form, e.g., for natural phosphate linkages, -OP (O) (OH) O-; for phosphorothioate internucleotide linkages, -OP (O) (SH) O-forms; etc. In some embodiments, the ds oligonucleotide targeting HSD17B13 may be in the form of a salt, e.g., in the form of the sodium salt of-OP (O) (ONa) O-for natural phosphate linkages; for phosphorothioate internucleotide linkages, the sodium salt form-OP (O) (SNa) O-; etc. Unless otherwise indicated, the oligonucleotides of the present disclosure may be present in acid, base and/or salt form.

In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is provided by dissolving the solid oligonucleotide composition or diluting the concentrated oligonucleotide composition with a suitable solvent (e.g., water or a pharmaceutically acceptable buffer). In some embodiments, the liquid composition comprises an anionic form of the provided oligonucleotides and one or more cations. In some embodiments, the liquid composition has a pH in the weakly acidic, about neutral, or alkaline range. In some embodiments, the pH of the liquid composition is about physiological pH, for example about 7.4.

In some embodiments, the provided oligonucleotides are formulated for administration to and/or contact with body cells and/or tissues expressing their targets. For example, in some embodiments, the provided ds oligonucleotides targeting HSD17B13 are formulated for administration to body cells and/or tissues expressing HSD17B 13. In some embodiments, such body cells and/or tissues are neurons or cells and/or tissues of the central nervous system. In some embodiments, the broad distribution of oligonucleotides and compositions may be achieved by intraparenchymal administration, intrathecal administration, or intraventricular administration.

In some embodiments, the pharmaceutical composition is formulated for intravenous injection, oral administration, inhalation, nasal administration, topical administration, ocular administration, or otic administration. In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops.

In some embodiments, the present disclosure provides pharmaceutical compositions comprising a chirally controlled oligonucleotide or a composition thereof admixed with a pharmaceutically acceptable inactive ingredient (e.g., a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, etc.). In some embodiments, the present disclosure provides pharmaceutical compositions that deliver a chirally controlled oligonucleotide or a composition thereof admixed with a pharmaceutically acceptable inactive ingredient (e.g., a pharmaceutically acceptable excipient, a pharmaceutically acceptable carrier, etc.). Those of skill in the art will recognize that pharmaceutical compositions include provided oligonucleotides or pharmaceutically acceptable salts of the compositions. In some embodiments, the pharmaceutical composition is a chirally controlled oligonucleotide composition. In some embodiments, the pharmaceutical composition is a stereopure oligonucleotide composition.

In some embodiments, the disclosure provides salts of oligonucleotides and pharmaceutical compositions thereof. In some embodiments, the salt is a pharmaceutically acceptable salt. In some embodiments, the pharmaceutical composition comprises an oligonucleotide, optionally in the form of a salt thereof, and a sodium salt. In some embodiments, the pharmaceutical composition comprises an oligonucleotide, optionally in the form of a salt thereof, and sodium chloride. In some embodiments, each hydrogen ion that can be given to a base (e.g., under conditions of aqueous solution, pharmaceutical composition, etc.) of the oligonucleotide is replaced with a non-H ⁺ cation. For example, in some embodiments, the pharmaceutically acceptable salt of an oligonucleotide is an all-metal ion salt, wherein each hydrogen ion (e.g., -OH, -SH, etc.) of each internucleotide linkage (e.g., natural phosphate linkage, phosphorothioate internucleotide linkage, etc.) is replaced with a metal ion. Various suitable metal salts for pharmaceutical compositions are well known in the art and may be used in accordance with the present disclosure. In some embodiments, the pharmaceutically acceptable salt is a sodium salt. In some embodiments, the pharmaceutically acceptable salt is a magnesium salt. In some embodiments, the pharmaceutically acceptable salt is a calcium salt. In some embodiments, the pharmaceutically acceptable salt is a potassium salt. In some embodiments, the pharmaceutically acceptable salt is an ammonium salt (cation N (R) ₄ ⁺). In some embodiments, the pharmaceutically acceptable salt comprises one and no more than one type of cation. In some embodiments, the pharmaceutically acceptable salt comprises two or more types of cations. In some embodiments, the cation is Li ⁺、Na⁺、K⁺、Mg²⁺ or Ca ²⁺. In some embodiments, the pharmaceutically acceptable salt is the sodium salt. In some embodiments, the pharmaceutically acceptable salt is the holodium salt, wherein each internucleotide linkage being a natural phosphate linkage (acid form-O-P (O) (OH) -O-) (if present) exists in its sodium salt form (-O-P (O) (ONa) -O-), and each internucleotide linkage being a phosphorothioate internucleotide linkage (acid form-O-P (O) (SH) -O-) (if present) exists in its sodium salt form (O-P (O) (SNa) -O-).

In accordance with the present disclosure, various techniques known in the art for delivering nucleic acids and/or oligonucleotides may be utilized. For example, a variety of supramolecular nanocarriers may be used to deliver nucleic acids. Exemplary nanocarriers include, but are not limited to, liposomes, cationic polymer complexes, and various polymer compounds. Complexing nucleic acids with various polycations is another method for intracellular delivery; this includes the use of pegylated polycations, polyvinylamine (PEI) complexes, cationic block copolymers, and dendrimers. Several cationic nanocarriers (including PEI and polyamide dendrimers) help to release the contents from the endosomes. Other methods include the use of polymeric nanoparticles, microspheres, liposomes, dendrimers, biodegradable polymers, conjugates, prodrugs, inorganic colloids such as sulfur or iron, antibodies, implants, biodegradable microspheres, permeation-controlled implants, lipid nanoparticles, emulsions, oily solutions, aqueous solutions, biodegradable polymers, poly (lactic-co-glycolic acid)), poly (lactic acid), liquid reservoirs, polymer micelles, quantum dots, and lipid complexes. In some embodiments, the oligonucleotide is conjugated to another molecule.

In therapeutic and/or diagnostic applications, compounds of the present disclosure, e.g., oligonucleotides, may be formulated for a variety of modes of administration, including systemic and topical (localized) administration. Techniques and formulations can generally be found in Remington, THE SCIENCE AND PRACTICE of Pharmacy [ pharmaceutical science and practice ] (20 th 2000).

Pharmaceutically acceptable salts of the basic moiety are generally well known to those of ordinary skill in the art and may include, for example, acetate, benzenesulfonate (benzenesulfonate), benzenesulfonate (besylate), benzoate, bicarbonate, bitartrate, bromide, ethylenediamine tetraacetate, taurine, carbonate, citrate, ethylenediamine tetraacetate, ethanedisulfonate, propionate laurylsulfate (estolate), phenolsulfoethylamine (esylate), fumarate, gluconate (gluceptate), gluconate (gluconate), glutamate, glycolamidophenylarsonate (glycollylarsanilate), hexylresorcinol (hexylresorcinate), hydramine (hydrobromide), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactose aldehyde, malate, maleate, mandelate, methanesulfonate, muciate, naphthalene sulfonate, nitrate, pamoate/embonate), pantothenate, phosphate/hydrogen phosphate, polygalacturonate, salicylate, stearate, basic acetate (subacetate), succinate, tannin, tartrate, tea, or tartrate. Other pharmaceutically acceptable salts may be found, for example, in Remington, THE SCIENCE AND PRACTICE of Pharmacy [ leimington: pharmaceutical science and practice ] (20 th edition 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, naphthalene sulfonate, pamoate (pamoate, embonate), phosphate, salicylate, succinate, sulfate or tartrate.

In some embodiments, the ds oligonucleotide targeting HSD17B13 is formulated in a pharmaceutical composition described in WO 2005/060697, WO 2011/076807, or WO 2014/136086.

Depending on the particular condition, disorder or disease being treated, the provided agents, e.g., oligonucleotides, may be formulated in liquid or solid dosage forms and administered systemically or locally. As known to those skilled in the art, the provided oligonucleotides may be delivered, for example, in a timed or sustained low release form. Techniques for formulation and administration can be found in Remington, THE SCIENCE AND PRACTICE of Pharmacy [ leimington: pharmaceutical science and practice ] (20 th edition 2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections, or other means of delivery.

For injection, the provided reagents, e.g., oligonucleotides, may be formulated and diluted in an aqueous solution, e.g., in a physiologically compatible buffer, e.g., hank's solution, ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier penetration are used in the formulation. Such penetrants are well known in the art, and may be used in accordance with the present disclosure.

The use of pharmaceutically acceptable carriers for practicing the present disclosure for formulating compounds (e.g., provided oligonucleotides) into dosages suitable for various modes of administration is well known in the art. By appropriate choice of carrier and appropriate manufacturing methods, the compositions of the present disclosure, e.g., formulated as solutions, may be administered by various routes, e.g., parenterally, e.g., by intravenous injection.

In some embodiments, the composition comprising a ds oligonucleotide that targets HSD17B13 further comprises any or all of: calcium chloride dihydrate, magnesium chloride hexahydrate, potassium chloride, sodium chloride, anhydrous disodium phosphate, sodium phosphate, monobasic dihydrate, and/or water for injection. In some embodiments, the composition further comprises any or all of the following: calcium chloride dihydrate (0.21 mg) USP, magnesium chloride hexahydrate (0.16 mg) USP, potassium chloride (0.22 mg) USP, sodium chloride (8.77 mg) USP, anhydrous disodium hydrogen phosphate (0.10 mg) USP, sodium dihydrogen phosphate dihydrate (0.05 mg) USP and water for injection USP.

In some embodiments, the composition comprising the oligonucleotide further comprises any or all of the following: cholesterol, (6 z,9z,28z,31 z) -thirty-seven-6, 9, 28, 31-tetraenyl-19-yl-4- (dimethylamino) butanoate (DLin-MC 3-DMA), 1, 2-distearoyl-sn. glycerol-3-phosphorylcholine (DSPC), α - (3' - { [1, 2-bis (myristyloxy) propoxy ] carbonylamino } propyl) - ω -methoxy, polyoxyethylene (PEG 2000-C-DMG), anhydrous potassium dihydrogen phosphate NF, sodium chloride, disodium hydrogen phosphate heptahydrate, and water for injection. In some embodiments, the pH of the composition comprising a ds oligonucleotide that targets HSD17B13 is about 7.0. In some embodiments, the composition comprising the oligonucleotide further comprises any or all of the following: in a total volume of about 1mL, 6.2mg cholesterol USP, 13.0mg (6Z, 9Z,28Z, 31Z) -thirty-seven-6, 9, 28, 31-tetraenyl-19-yl-4- (dimethylamino) butyrate (DLin-MC 3-DMA), 3.3mg 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1.6mg α - (3' - { [1, 2-bis (myristyloxy) propoxy ] carbonylamino } propyl) - ω -methoxy, polyoxyethylene (PEG 2000-C-DMG), 0.2mg anhydrous potassium dihydrogen phosphate NF, 8.8mg sodium chloride USP, 2.3mg disodium hydrogen phosphate heptahydrate USP, and water for injection USP.

The provided compounds (e.g., oligonucleotides) can be readily formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. In some embodiments, such carriers enable the provided oligonucleotides to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for example, for oral ingestion by a subject (e.g., a patient) to be treated.

For nasal or inhalation delivery, the provided compounds, e.g., oligonucleotides, can be formulated by methods known to those skilled in the art, and can include, for example, examples of solubilizing, diluting or dispersing substances (e.g., saline, preservatives (e.g., benzyl alcohol), absorption promoters, and fluorocarbons).

In certain embodiments, parenteral administration is by injection, e.g., by syringe, pump, etc. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or site, such as the striatum, caudate nucleus, cortex, hippocampus, and/or cerebellum.

In certain embodiments, methods of specifically locating a provided compound (e.g., an oligonucleotide), such as by bolus injection, can reduce the median effective concentration (EC 50) by a factor of 20, 25, 30, 35, 40, 45, or 50. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments, the targeted tissue is striatal tissue. In certain embodiments, reducing the EC50 is desirable because it reduces the dosage required to achieve a pharmacological result in a patient in need thereof.

In certain embodiments, the provided oligonucleotides are delivered monthly, bi-monthly, 90-day, 3-month, 6-month, twice a year, or once a year by injection or infusion.

Pharmaceutical compositions suitable for use in the present disclosure include compositions comprising an effective amount of an active ingredient, such as an oligonucleotide, to achieve its intended purpose. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the specific disclosure provided herein.

In addition to the active ingredient, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers (including excipients and auxiliaries), which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Formulations formulated for oral administration may be in the form of tablets, dragees, capsules or solutions.

In some embodiments, the pharmaceutical composition for oral use can be obtained by the following method: the active compound is combined with solid excipients, the resulting mixture is optionally ground, and the particulate mixture is processed (after addition of suitable auxiliaries, if desired) to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose (CMC), and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (such as sodium alginate).

In some embodiments, the dragee cores are provided with a suitable coating. For this purpose concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol, polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablet or dragee coating for identifying or characterizing different combinations of active compound doses.

Pharmaceutical formulations for oral use include plug-in capsules (push-fit capsules) made of gelatin and sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The plug-in capsules may contain the active ingredient, for example an oligonucleotide, in admixture with fillers (e.g. lactose), binders (e.g. starches) and/or lubricants (e.g. talc or magnesium stearate) and, optionally, stabilizers. In soft capsules, the active compounds, e.g., oligonucleotides, may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol (PEG). In addition, stabilizers may be added.

In some embodiments, provided compositions comprise lipids. In some embodiments, the lipid is conjugated to an active compound, such as an oligonucleotide. In some embodiments, the lipid is not conjugated to an active compound. In some embodiments, the lipid comprises a C ₁₀-C₄₀ straight saturated or partially unsaturated aliphatic chain. In some embodiments, the lipid comprises a C ₁₀-C₄₀ straight saturated or partially unsaturated aliphatic chain optionally substituted with one or more C _1-4 aliphatic groups. In some embodiments, the lipid is selected from the group consisting of: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), alginic acid and linoleyl alcohol. In some embodiments, the active compound is a provided oligonucleotide. In some embodiments, the composition comprises a lipid and an active compound, and further comprises another component that is another lipid or targeting compound or moiety. In some embodiments, the lipid is an amino lipid; an amphiphilic lipid; anionic lipids; apolipoproteins; cationic lipids; low molecular weight cationic lipids; cationic lipids such as CLinDMA and DLinDMA; an ionizable cationic lipid; a masking component; auxiliary lipids; a lipopeptide; neutral lipids; neutral zwitterionic lipids; a hydrophobic small molecule; a hydrophobic vitamin; PEG-lipid; an uncharged lipid modified by one or more hydrophilic polymers; a phospholipid; phospholipids such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; stealth lipids; sterols; cholesterol; targeting lipids; or another lipid described herein or reported in the art as suitable for pharmaceutical use. In some embodiments, the composition comprises a lipid and a portion of another lipid capable of mediating at least one function of the other lipid. In some embodiments, the targeting compound or moiety is capable of targeting a compound (e.g., an oligonucleotide) to a particular cell or tissue or subset of cells or tissues. In some embodiments, the targeting moiety is designed for cell-specific or tissue-specific expression with a particular target, receptor, protein, or another subcellular component. In some embodiments, the targeting moiety is a ligand (e.g., a small molecule, an antibody, a peptide, a protein, a carbohydrate, an aptamer, etc.) that targets the composition to a cell or tissue and/or binds to a target, receptor, protein, or another subcellular component.

Certain exemplary lipids for delivering active compounds, such as oligonucleotides, allow (e.g., do not prevent or interfere with) the function of the active compound. In some embodiments, the lipid is lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), alginic acid, or linoleyl alcohol.

Lipid conjugation (e.g., to fatty acids) can improve one or more properties of the oligonucleotides, as described in the present disclosure.

In some embodiments, the composition for delivering an active compound, e.g., an oligonucleotide, is capable of targeting the active compound to a particular cell or tissue as desired. In some embodiments, the composition for delivering the active compound is capable of targeting the active compound to muscle cells or tissue. In some embodiments, the present disclosure provides compositions and methods related to the delivery of an active compound, wherein the compositions comprise the active compound and a lipid. In various embodiments of muscle cells or tissues, the lipid is selected from lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid (cis-DHA), alginic acid, and linoleyl alcohol.

In some embodiments, the ds oligonucleotide that targets HSD17B13 is delivered via a composition comprising or involving the use of any one or more of the following delivery methods: nanoparticles targeting transferrin receptor; cationic liposome-based delivery strategies; cationic liposomes; polymerizing the nanoparticles; a viral carrier; a retrovirus; adeno-associated virus; stabilized nucleic acid lipid particles; a polymer; a cell penetrating peptide; a lipid; a dendrimer; neutral lipids; cholesterol; a lipid molecule; fusion lipids; a hydrophilic molecule; polyethylene glycol (PEG) or derivatives thereof; shielding lipids; polyethylene glycol lipid; PEG-C-DMSO; PEG-C-DMSA; DSPC; ionizing the lipid; guanidine-based cholesterol derivatives; ion-coated nanoparticles; metal ion coated nanoparticles; manganese ion coated nanoparticles; angubindin-1; a nanogel; incorporating a ds oligonucleotide targeting HSD17B13 into the branched nucleic acid structure; and/or incorporating a ds oligonucleotide targeting HSD17B13 into a branched nucleic acid structure comprising 2, 3, 4 or more oligonucleotides.

In some embodiments, the composition comprising the oligonucleotide is lyophilized. In some embodiments, the composition comprising the oligonucleotide is lyophilized and the lyophilized oligonucleotide is placed in a vial. In some embodiments, the vial is backfilled with nitrogen. In some embodiments, the lyophilized oligonucleotide composition is reconstituted prior to administration. In some embodiments, the lyophilized oligonucleotide composition is reconstituted with a sodium chloride solution prior to administration. In some embodiments, the lyophilized oligonucleotide composition is reconstituted with a 0.9% sodium chloride solution prior to administration. In some embodiments, reconstitution is performed at a clinical site for administration. In some embodiments, in the lyophilized composition, the oligonucleotide composition is chirally controlled or comprises at least one chirally controlled internucleotide linkage and/or ds oligonucleotide targeting HSD17B13.

Transgenic HSD17B13 non-human animals

In some embodiments, the disclosure provides engineered animals and cells thereof, wherein the animals are engineered to comprise or express HSD17B13 polypeptides or characteristic portions thereof, and/or polynucleotides encoding such HSD17B13 polypeptides or characteristic portions thereof. In some embodiments, the HSD17B13 polypeptide or a characteristic portion thereof is or comprises a sequence sharing about 80% -100%, e.g., about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with a primate, e.g., human HSD17B13 or a characteristic portion thereof. In some embodiments, the HSD17B13 polypeptide or a characteristic portion thereof is or comprises a sequence sharing about 80% -100%, e.g., about or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity with one or more domains of human HSD17B13 (e.g., HSD17B13 lipid droplet targeting domain (comprising an N-terminal hydrophobic domain, PAT-like domain, and putative α -helix/β -sheet/α -helix domain) and HSD17B13 enzyme activity domain (comprising a catalytic site, a substrate binding site, and a homodimer interaction site).

Exemplary HSD17B13 sequence example

In certain embodiments, the HSD17B13 polynucleotide or human HSD17B13 gene incorporated into a non-human animal (e.g., a rodent, such as a rat or mouse) is represented by or comprises the following: a sequence encoding human HSD17B13 or a genomic locus or a characteristic portion thereof.

As used herein, the term "characteristic moiety" refers in the broadest sense to the following substance moiety: its presence (or absence) is related to the presence (or absence) of a particular feature, attribute or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion found in the substance and in related substances that share a particular feature, property, or activity, but not in those substances that do not share the particular feature, property, or activity. In certain embodiments, the characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion of a contiguous segment or collection of contiguous segments of amino acids that together are characteristic of the protein or polypeptide. In some embodiments, each such continuous segment generally comprises at least 2, 5, 10, 15, 20, 50, or more amino acids. Typically, a characteristic portion of a substance (e.g., a characteristic portion of a protein, antibody, etc.) is a portion that shares at least one functional property with the relevant intact substance in addition to the sequence and/or structural identity specified above. In some embodiments, the characteristic moiety may be bioactive.

Exemplary HSD17B13 coding sequences are described above.

Exemplary promoters

In some embodiments, the construct (e.g., a construct comprising the human HSD17B13 gene) comprises a promoter. The term "promoter" refers to a DNA sequence recognized by an enzyme/protein that can promote and/or initiate transcription of an operably linked gene (e.g., the human HSD17B13 gene). For example, a promoter generally refers to a nucleotide sequence from which, for example, RNA polymerase and/or any related factors bind and from which transcription can be initiated. Thus, in some embodiments, the construct (e.g., a targeting construct and/or vector comprising the human HSD17B13 gene) comprises a promoter operably linked to one of the non-limiting exemplary promoters described herein.

In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue specific promoter, an insertion site endogenous promoter, or any other type of promoter known in the art. In some embodiments, the promoter is an RNA polymerase II promoter, e.g., a mammalian RNA polymerase II promoter. In some embodiments, the promoter is an RNA polymerase III promoter, including but not limited to a HI promoter, a human U6 promoter, a mouse U6 promoter, or a porcine U6 promoter. The promoter will typically be one that is capable of promoting transcription in inner ear cells. In some embodiments, the promoter is a cochlear specific promoter or a cochlear-directed promoter. In some embodiments, the promoter is a hair cell-specific promoter or a supporting cell-specific promoter.

A variety of promoters are known in the art and may be used herein. Non-limiting examples of promoters useful herein include: human EFla, human Cytomegalovirus (CMV) (U.S. Pat. No. 5,168,062), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1, polyomaadenovirus, simian virus 40 (SV 40), beta-globin, beta-actin, alpha fetoprotein, gamma-globin, beta-interferon, gamma-glutamyl transferase, mouse Mammary Tumor Virus (MMTV), rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g., human T cell leukemia Virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5E2, stromelysin, murine MX gene, glucose regulatory proteins (GRP 78 and GRP 94), alpha-2-macroglobulin, vimentin, MHC class I gene H-2 _K B, HSP70, proliferative protein, tumor necrosis factor, thyroid stimulating hormone gene, immunoglobulin light chain, T cell receptor, HLA DQa and DQ, interleukin-2 receptor, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (thyroxine transporter), elastase I, albumin gene, C-los, C-HA-ras, neural Cell Adhesion Molecule (NCAM), H2B (TH 2B) histone, rat growth hormone, human serum amyloid protein (SAA), troponin I (TNI), dunaliella muscular dystrophy, human immunodeficiency virus, and gibbon leukemia virus (GALV) promoters. Other examples of promoters are known in the art. See, e.g., loish, molecular Cell Biology [ molecular cell biology ], FREEMAN AND Company [ Frieman, N.Y. 2007.

In some embodiments, the promoter is a CMV immediate early promoter. In some embodiments, the promoter is a CAG promoter or a CAG/CBA promoter. In certain embodiments, the promoter comprises a CMV/CBA enhancer/promoter construct. In certain embodiments, the promoter comprises a CAG promoter or a CMV/CBA/SV-40 enhancer/promoter construct.

The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., a pendrin protein), results in transcription of RNA from the nucleic acid in a cell under most or all physiological conditions.

Examples of constitutive promoters include, but are not limited to, the retrovirus Rous Sarcoma Virus (RSV) LTR promoter, the Cytomegalovirus (CMV) promoter (see, e.g., bosharp et al, cell [ Cell ]41:521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EFI-alpha promoter (Invitrogen).

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature or the presence of specific physiological states such as the acute phase, specific differentiation states of cells, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to, england, cloning technology (Clontech), and Ariad. Other examples of inducible promoters are known in the art.

Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep Metallothionein (MT) promoter, dexamethasone (Dex) inducible Mouse Mammary Tumor Virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); ecdysone insect promoters (No. et al, proc.Natl.Acad Sci.US.A. [ Proc.Natl.Acad.Sci.Sci.U.S. 1996 ], tetracycline inhibition systems (Gossen et al, proc.Natl.Acad.Sci.US.A. [ Proc.Acad.Sci.U.S. Sci.A. 89:5547-5551, 1992), tetracycline induction systems (Gossen et al, science [ Science ],268:1766-1769, 1995, see also Harvey et al, curr.Opin.chem.Bio1.[ New Biol.2:512-518, 1998), RU486 inducible systems (Wang et al, natl.Biotech. [ Nature Biotech ] 15:239-239, 1997 and Wang et al, gene therapy [ 4:432-441, 1997), and rapamycin inducible systems (Magari et al J. Invin. J. Clinical research [ 2865:2865, 2872).

The term "tissue-specific" promoter refers to a promoter that is active only in certain specific cell types and/or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcriptional regulator and/or control proteins that bind to the tissue-specific promoter).

In some embodiments, the regulatory sequences and/or control sequences confer tissue specific gene expression. In some cases, the tissue-specific regulatory sequences and/or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

In some embodiments, the tissue-specific promoter is a Central Nervous System (CNS) -specific promoter. Non-limiting examples of CNS-specific promoters include, but are not limited to, promoters of the following genes or functional portions ：Aldh1l1、CaMIIα、Dlx1、Dlx5/6、Gad2、GFAP、Grik4、Lepr、Nes、nNOS、Pdgfrα、PLP1、Pv(Pvalb)、Slc17a6、Sst、Vip、Pcp2、Slc6a3(DAT)、ePet(Fev)、Npy2r、Cdh3 and/or Htr6 thereof; see, e.g., kim et al, "Mouse Cre-LoxP system: GENERAL PRINCIPLES to determine tissue-specific roles of TARGET GENES [ mouse Cre-LoxP system: general principle for determining tissue-specific effects of target genes ] "Laboratory ANIMAL RESEARCH [ Laboratory animal research ] (2018) 34 (4), 147-159. In certain embodiments, the CNS-specific promoter comprises or consists of: a nucleotide sequence that is identical to a promoter of a gene or has at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% homology ：Aldh1l1、CaMIIα、Dlx1、Dlx5/6、Gad2、GFAP、Grik4、Lepr、Nes、nNOS、Pdgfra、PLP1、Pv(Pvalb)、Slc17a6、Sst、Vip、Pcp2、Slc6a3(DAT)、ePet(Fev)、Npy2r、Cdh3 and/or Htr6.

In some embodiments, the tissue-specific promoter is an eye cell-specific promoter. Non-limiting examples of eye cell specific promoters include, but are not limited to, promoters of the following genes or functional portions thereof: EFS, GRK1, CRX, NRL, and/or RCVRN. In certain embodiments, the ocular system specific promoter comprises or consists of: a nucleotide sequence that is identical to or has at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% homology to the promoter of a gene of: EFS, GRK1, CRX, NRL, and/or RCVRN.

In some embodiments, the tissue-specific promoter is a liver system-specific promoter. Non-limiting examples of liver system specific promoters include, but are not limited to, promoters of the following genes or functional portions thereof: EFS, EF-la, MSCV, PGK, CAG, ALB, and/or SERPINA1. In certain embodiments, the liver system-specific promoter comprises or consists of: a nucleotide sequence that is identical to or has at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% homology to the promoter of a gene of: EFS, EF-la, MSCV, PGK, CAG, ALB, and/or SERPINA1.

In some embodiments, the nucleic acid constructs provided comprise a promoter sequence selected from CAG, CBA, CMV or CB7 promoters. In certain embodiments, the promoter comprises or consists of: a nucleotide sequence that is identical to CAG, CBA, CMV or CB7 promoter or has at least 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% homology.

In some embodiments of any of the nucleic acid constructs described herein, the first or unique nucleic acid construct further comprises at least one promoter sequence selected from CNS, eye and/or hepatocyte specific promoters, or a functional portion thereof.

Exemplary enhancers

In some cases, the construct may include an enhancer sequence. In some embodiments, the term "enhancer" refers to a nucleotide sequence that can increase the level of transcription of a nucleic acid encoding a protein of interest (e.g., a human and/or NHP HSD17B13 protein). Enhancer sequences (typically 50-1500bp in length) generally increase transcription levels by providing additional binding sites for transcription related proteins (e.g., transcription factors). In some embodiments, the enhancer sequence is found in the intron sequence. Unlike a promoter sequence, an enhancer sequence may function farther from the transcription initiation site (e.g., compared to the promoter). Non-limiting examples of enhancers include the RSV enhancer, the CMV enhancer, and/or the SV40 enhancer. In some embodiments, the construct comprises a CMV enhancer. In some embodiments, the SV-40-derived enhancer is an SV-40T intron sequence. In some embodiments, the enhancer sequence is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Exemplary coding sequences flanking untranslated regions

In some embodiments, any of the constructs described herein can include an untranslated region (UTR), such as a 5'UTR or a 3' UTR. The UTR of the gene is transcribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR starts immediately with the stop codon and continues until the transcription termination signal. The regulatory and/or control features of UTRs may be incorporated into any construct, composition, kit or method as described herein to enhance or otherwise modulate expression of HSD17B13 protein.

The native 5' utr includes sequences that play a role in translation initiation. In some embodiments, the 5' utr may comprise a sequence, such as a kozak sequence, which is generally known to be involved in the process of ribosome initiation of many gene translations. The kozak sequence has the consensus sequence CCR (a/G) CCAUGG, where R is a purine (a or G) three bases upstream of the start codon (AUG), followed by another "G". In certain embodiments, the kozak sequence is GCCACC. It is also known that the 5' UTR can also form secondary structures involved in elongation factor binding.

In some embodiments, the 5' utr is comprised in any of the constructs described herein. Non-limiting examples of 5' utrs include those from the following genes: albumin, serum amyloid a, apolipoprotein a/B/E, transferrin, alpha fetoprotein, erythropoietin, factor VIII and HSD17B13 may be used to enhance expression of nucleic acid molecules such as mRNA.

In some embodiments, the 5' utr from mRNA transcribed from cells in the CNS may be included in any of the constructs, compositions, kits, and methods described herein. In some embodiments, the 5' utr is derived from an endogenous HSD17B13 locus.

The 3'UTR is immediately 3' to the stop codon of the gene of interest. In some embodiments, the 3' utr from mRNA transcribed from cells in the CNS may be included in any of the constructs, compositions, kits, and methods described herein. In some embodiments, the 3' utr is derived from the endogenous HSD17B13 locus and may include all or part of the endogenous sequence.

It is known that 3' UTR has embedded therein a stretch of adenosine and uridine (in RNA form) or thymidine (in DNA form). These AU-rich features are particularly prevalent in high turnover genes. AU-rich elements (ARE) can be divided into three classes based on their sequence features and functional properties (Chen et al, mal cell.bios1. [ molecular cell biology ]15:5777-5788, 1995; chen et al, mal cell Biol. [ molecular cell biology ]15:2010-2018, 1995): class I ARE contain several discrete copies of the AUUUA motif within the U-rich region. For example, c-Myc and MyoD mRNA contains class I AREs. Class II AREs have two or more overlapping UUAUUUA (U/A) (U/A) nonamers. GM-CSF and TNF- α mRNA ARE examples containing class II AREs. Class III ARE less well defined. These U-rich regions do not contain the AUUUA motif, two well-studied examples of which are c-Jun and myogenin mRNA.

Most proteins that bind ARE known to disrupt messenger stability, whereas members of the ELAV family, particularly HuR, have been described as increasing mRNA stability. HuR binds to ARE of all three categories. Engineering a HuR specific binding site into the 3' utr of a nucleic acid molecule will result in HuR binding, thereby stabilizing the message in vivo.

In some embodiments, the introduction, removal, or modification of a 3' utr ARE useful for modulating the stability of mRNA encoding HSD17B13 protein. In other embodiments, AREs may be removed or mutated to increase intracellular stability and thus increase translation and production of HSD17B13 protein.

In other embodiments, non-ARE sequences may incorporate 5 'or 3' utrs. In some embodiments, an intron or a portion of an intron sequence may be incorporated into flanking regions of a polynucleotide in any of the constructs, compositions, kits, and methods provided herein. Incorporation of an intron sequence may increase protein production and mRNA levels.

Exemplary Internal Ribosome Entry Site (IRES)

In some embodiments, the construct encoding the HSD17B13 protein may include an Internal Ribosome Entry Site (IRES). IRES forms a complex secondary structure that allows translation to begin from any position of the mRNA immediately downstream of the position where IRES is located (see, e.g., pelletier and Sonenberg, mal.cell.biol. [ molecular cell biology ]8 (3): 1103-1112, 1988).

Several IRES sequences are known to those skilled in the art, including those from the following: such as Foot and Mouth Disease Virus (FMDV), encephalomyocarditis virus (EMCV), human Rhinovirus (HRV), cricket paralysis virus, human Immunodeficiency Virus (HIV), hepatitis A Virus (HAV), hepatitis C Virus (HCV), and Poliovirus (PV). See, e.g., alberts, molecular Biology of the Cell [ cell molecular biology ], GARLAND SCIENCE [ galanthe ],2002; and Hellen et al, genes Dev [ gene development ]15 (13): 1593-612, 2001.

In some embodiments, the IRES sequence incorporated into the construct encoding the HSD17B13 protein is a Foot and Mouth Disease Virus (FMDV) 2A sequence. Foot-and-mouth disease virus 2A sequence is a small peptide (about 18 amino acids in length) that has been shown to mediate cleavage of multiple proteins (Ryan, MD et al, EMBO 4:928-933, 1994; station et al, J Virology [ J Virology ]70:8124-8127, 1996; furler et al, GENE THERAPY [ Gene therapy ]8:864-873, 2001; and Halpin et al, plant Journal [ J. Plant ]4:453-459, 1999). Cleavage activity of the 2A sequence has been previously demonstrated in artificial systems, including plasmids and gene therapy constructs (AAV and retroviruses) (Ryau et al, EMBO 4:928-933, 1994; station et al, J Virology [ Journal of Virology ]70:8124-8127, 1996; furler et al, GENETHERAPY [ gene therapy ]8:864-873, 2001; and Halpin et al, plant Journal [ Journal of plants ]4:453-459, 1999; de Felipe et al, GENE THERAPY [ gene therapy ]6:198-208, 1999; de Felipe et al, human GENE THERAPY [ Human gene therapy ] II:1921-1931, 2000; and Klump et al, GENE THERAPY [ gene therapy ]8:811-817, 2001).

IRES can be used with any of the constructs described herein. In some embodiments, the IRES may be part of a composition comprising more than one construct. In some embodiments, IRES is used to produce more than one polypeptide from a single gene transcript.

Exemplary splice sites

In some embodiments, any of the constructs provided herein can include splice donor and/or splice acceptor sequences that are functional during RNA processing that occurs during transcription. In some embodiments, the splice site is involved in trans-splicing.

Exemplary polyadenylation sequences

In some embodiments, constructs provided herein can include a polyadenylation (poly (a)) signal sequence. Most nascent eukaryotic mRNAs have poly (A) tails at their 3' ends that are added in a complex process involving cleavage of the primary transcript and coupled polyadenylation reactions driven by poly (A) signal sequences (see, e.g., proudfoot et al, cell [ Cell ]108:501-512, 2002). The poly (A) tail confers mRNA stability and transferability (Molecular Biology of the Cell [ cell molecular biology ], B.Alberts et al, 3 rd edition, garland Publishing [ Garland Press ], 1994). In some embodiments, the poly (a) signal sequence is located 3' of the coding sequence.

As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylation moiety or modified variant thereof to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylation at the 3' end. The 3' poly (a) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA by the action of an enzyme (polya polymerase). In some embodiments, the poly (a) tail is added to a transcript comprising a specific sequence (e.g., poly (a) signal). The poly (a) tail and related proteins help protect mRNA from exonuclease degradation. Polyadenylation also plays a role in transcription termination, mRNA export from the nucleus and translation. Polyadenylation usually occurs in the nucleus immediately after transcription of DNA into RNA, but may also occur later in the cytoplasm. After termination of transcription, the mRNA strand is cleaved by the action of an endonuclease complex associated with the RNA polymerase. The cleavage site is generally characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end at the cleavage site.

As used herein, a "poly (a) signal sequence" or "polyadenylation signal sequence" is a sequence that triggers an endonuclease to cleave mRNA and add a series of adenosines to the 3' end of the cleaved mRNA.

There are several poly (a) signal sequences available, including those derived from: bovine growth hormone (bGH) (Woychik et al, proc.Natl. Acad Sci.US.A. [ Proc. Natl. Acad. Sci.U.S. Sci ]81 (13) 3944-3948, 1984; U.S. Pat. No. 5,122,458), mouse- β -globin, mouse- α -globin (Orkin et al, EMBO J [ European journal of molecular biology ]4 (2): 453-456, 1985; thein et al, blood [ Blood ]71 (2): 313-319, 1988), human collagen, polyomavirus (Batt et al, mal. Cell Biol. [ molecular cell biology ]15 (9): 4783-4790, 1995), herpes simplex virus thymidine kinase gene (HSV TK), igG heavy chain gene polyadenylation signal (US 2006/0040354), human growth hormone (hGH) (Szymanski et al, mal. Therapy [ molecular therapy ]15 (7): 1340-1347, 2007), a group consisting of SV40 poly (A) sites, such as SV40 late and early poly (A) sites (Schek et al, mal. Cell Biol. [ molecular cell biology ]12 (535386-533, 1992).

The poly (a) signal sequence may be AATAAA. The AATAAA sequence may be substituted with other hexanucleotide sequences which are homologous to AATAAA and capable of signaling polyadenylation, including ATTAAA、AGTAAA、CATAAA、TATAAA、GATAAA、ACTAAA、AATATA、AAGAAA、AATAAT、AAAAAA、AATGAA、AATCAA、AACAAA、AATCAA、AATAAC、AATAGA、AATTAA、 or AATAAG (see, e.g., WO 06/12414).

In some embodiments, the poly (A) signal sequence may be a synthetic polyadenylation site (see, e.g., the pCl-neo expression construct based on Levitt et al, genes Dev. [ Gene and development ]3 (7): 1019-1025, 1989, promega). In some embodiments, the poly (A) signal sequence is a polyadenylation signal (AAATAAAATACGAAATG) of soluble neuropilin-1 (sNRP) (see, e.g., WO 05/073384). In some embodiments, the poly (a) signal sequence comprises or consists of bGHpA. In some embodiments, the poly (a) signal sequence comprises or consists of the SV40 poly (a) site. Other examples of poly (a) signal sequences are known in the art.

Exemplary destabilizing Domains

In some embodiments, any of the constructs provided herein can optionally include a sequence encoding a destabilizing domain for time control of protein expression ("destabilizing sequence"). Non-limiting examples of destabilizing sequences include sequences encoding FK506 sequences, dihydrofolate reductase (DHFR) sequences, or other exemplary destabilizing sequences.

In the absence of stabilizing ligands, protein sequences operably linked to destabilizing sequences are degraded by ubiquitination. Conversely, protein degradation is inhibited in the presence of the stabilizing ligand, thereby allowing active expression of the protein sequence operably linked to the destabilizing sequence. As a positive control for stabilizing protein expression, protein expression may be detected by conventional means, including enzymatic assays, radiographic assays, colorimetric assays, fluorescent assays, or other spectroscopic assays; fluorescence Activated Cell Sorting (FACS) assay; immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

Additional examples of destabilizing sequences are known in the art. In some embodiments, the destabilizing sequences are FK506 and rapamycin binding protein (FKBP 12) sequences and the stabilizing ligand is Shield-1 (Shield 1) (Banaszynski et al (2012) Cell [ Cell ]126 (5): 995-1004). In some embodiments, the destabilizing sequence is a DHFR sequence and the stabilizing ligand is Trimethoprim (TMP) (Iwamoto et al (2010) Chem Biol [ chemical and biological ] 17:981-988).

In some embodiments, the destabilizing sequence is an FKBP12 sequence and the presence of the nucleic acid construct carrying the FKBP12 gene is detected in a test cell (e.g., a rodent cell, e.g., a rat or mouse cell) by western blotting. In some embodiments, the destabilizing sequences can be used to verify the time-specific activity of any of the nucleic acid constructs described herein.

Exemplary report sequences or elements

In some embodiments, the constructs provided herein may optionally include sequences encoding a reporter polypeptide and/or protein ("reporter sequences"). Non-limiting examples of reporter sequences include DNA sequences encoding: beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green Fluorescent Protein (GFP), red fluorescent protein, mCherry fluorescent protein, yellow fluorescent protein, chloramphenicol Acetyl Transferase (CAT), and luciferase. Additional examples of reporting sequences are known in the art. When associated with a control element that drives its expression, the reporter sequence may provide a signal that is detectable by conventional means, including enzymatic assays, radiographic assays, colorimetric assays, fluorometric assays or other spectroscopic assays; fluorescence Activated Cell Sorting (FACS) assay; immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

In some embodiments, the reporter sequence is a LacZ gene and the presence of a construct carrying the LacZ gene is detected in a non-human cell (e.g., a rodent cell, e.g., a rat or mouse cell) as determined by β -galactosidase activity. When the reporter gene is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a construct carrying the fluorescent protein or luciferase in a non-human cell (e.g., a rodent cell, e.g., a rat or mouse) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or light generation in a photometer (e.g., a spectrophotometer or IVIS imaging instrument). In some embodiments, the reporter sequence can be used to verify tissue-specific targeting ability and tissue-specific promoter regulatory activity and/or control activity of any of the constructs described herein.

In some embodiments, the reporter sequence is a FLAG tag (e.g., a 3xFLAG tag), and the presence of a construct carrying the FLAG tag in a non-human cell (e.g., a rodent cell, e.g., a rat or mouse) is detected by a protein binding or detection assay (e.g., western blot, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry).

Targeting vectors

Targeting vectors can be used to introduce a nucleic acid construct into a target genomic locus. The targeting vector may comprise a nucleic acid construct and a homology arm flanking the nucleic acid construct; those skilled in the art will recognize a variety of choices and features that are generally applicable to the design, structure, and/or use of a targeting vector. For example, the targeting vectors may be in linear or circular form, and they may be single-stranded or double-stranded. The targeting vector may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). For ease of reference, homology arms are referred to herein as 5 'and 3' (i.e., upstream and downstream, i.e., left and right) homology arms. The term relates to the relative position of the homology arm and the targeting vector within the nucleic acid construct. The 5 'and 3' homology arms correspond to regions within the targeted locus or within another targeting vector, which are referred to herein as "5 'target sequence" and "3' target sequence", respectively. In some embodiments, the homology arms may also function as 5 'or 3' target sequences. In some embodiments, the disclosure provides targeting vectors comprising the provided technology whose sequences encode HSD17B13 polypeptides or characteristic portions thereof as described herein.

In some embodiments, the methods described herein provide for the production of traditional transgenic non-human animals. In such embodiments, a vector comprising the exogenous HSD17B13 gene is injected into a fertilized egg and randomly integrated into the genome. In some embodiments, such random insertion sites may be within the protein coding region and may result in altered function of endogenous proteins and/or genes. In some such embodiments, the exogenous HSD17B13 gene may be incorporated as a separate coding region, as a coding region comprising a protein tag, as a coding region having an operably linked promoter, as a coding region comprising a poly (a) site, as a coding region comprising any additional regulatory region, or any combination thereof.

In some embodiments, the methods described herein provide for the production of traditional transgenic non-human animals utilizing the Tol2 transposon subsystem. In such embodiments, a vector comprising the exogenous HSD17B13 gene is injected into a fertilized egg and randomly integrated into the a/T rich region of the genome. In some embodiments, such random insertion sites may be within the protein coding region and may result in altered function of endogenous proteins and/or genes. In some such embodiments, the exogenous HSD17B13 gene may be incorporated as a separate coding region, as a coding region comprising a protein tag, as a coding region having an operably linked promoter, as a coding region comprising a poly (a) site, as a coding region comprising any additional regulatory region, or any combination thereof.

In some embodiments, the methods described herein that provide for the production of traditional transgenic non-human animals may utilize large genomic fragments (e.g., 1mb, 10mb, 100mb, and/or 1000 mb). In some embodiments, a traditional transgenic non-human animal may comprise transgenic regions including promoters, introns, exons, and/or additional genomic regulatory regions. In some embodiments, the production of a traditional transgenic non-human animal may utilize a Yeast Artificial Chromosome (YAC), a Bacterial Artificial Chromosome (BAC), a human artificial chromosome, a P1-derived artificial chromosome (PAC), or any other engineered region that may be contained in a suitable host cell.

In some embodiments, the methods described herein employ two, three, or more targeting vectors that are capable of recombining with each other. In some embodiments, the first, second, and third targeting vectors each comprise 5 'and 3' homology arms. The 3 'homology arm of the first targeting vector comprises a sequence that overlaps (i.e., overlaps) with the 5' homology arm of the second targeting vector, which allows for homologous recombination between the first and second vectors.

In some embodiments of the two-component targeting method, the 5 'homology arm of the first targeting vector and the 3' homology arm of the second targeting vector may resemble respective segments within the target genomic locus (i.e., target sequence), which may facilitate homologous recombination of the first and second targeting vectors with the respective genomic segments and modify the target genomic locus.

In some embodiments of the three-component targeting method, the 3 'homology arm of the second targeting vector may comprise a sequence that overlaps (i.e., overlaps) with the 5' homology arm of the third targeting vector, which may allow for homologous recombination between the second and third targeting vectors. The 5 'homology arm of the first targeting vector and the 3' homology arm of the third targeting vector may resemble corresponding segments within the target genomic locus (i.e., target sequence), which may facilitate homologous recombination of the first and third targeting vectors with the corresponding genomic segments and modify the target genomic locus.

In some embodiments, the homology arm and the target sequence or both homology arms share a sufficient level of sequence identity with each other in both regions such that they can "correspond" or "correspond" to each other when they serve as substrates for homologous recombination reactions. The sequence identity between a given target sequence and the corresponding homology arms found on the targeting vector (i.e., overlapping sequences) or between two homology arms may be any degree of sequence identity that allows homologous recombination to occur. To name just one example, the amount of sequence identity shared by the homology arm of a targeting vector (or fragment thereof) with a target sequence of another targeting vector or a target sequence of a target genomic locus (or fragment thereof) may be, for example, but not limited to, at least 50％、55％、60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％ or 100% sequence identity, such that the sequences undergo homologous recombination.

Furthermore, the respective region of similarity (e.g., identity) between the homology arms and the respective target sequences may be of any length sufficient to promote homologous recombination at the target genomic locus. For example, a given homology arm and/or corresponding target sequence may comprise corresponding similar regions of a length of, but not limited to, about 0.2-0.5kb, 0.2-1kb, 0.2-1.5kb, 0.2-2kb, 0.2-2.5kb, 0.2-3kb, 0.2-3.5kb, 0.2-4kb, 0.2-4.5kb, or 0.2-5kb, such that the homology arms have sufficient similarity to homologous recombine with one or more corresponding target sequences within the target genomic locus of the cell or within another targeting vector. In some embodiments, a given homology arm and/or corresponding target sequence may comprise corresponding similar regions, such as, but not limited to, about 5-10kb、5-15kb、5-20kb、5-25kb、5-30kb、5-35kb、5-40kb、5-45kb、5-50kb、5-55kb、5-60kb、5-65kb、5-70kb、5-75kb、5-80kb、5-85kb、5-90kb、5-95kb、5-100kb、100-200kb or 200-300kb in length (as described elsewhere herein), such that the homology arm has sufficient similarity to homologous recombine with one or more corresponding target sequences within the target genomic locus of a cell or within another targeting vector. In some embodiments, a given homology arm and/or corresponding target sequence comprises corresponding similar regions, such as, but not limited to, about 10-100kb、15-100kb、20-100kb、25-100kb、30-100kb、35-100kb、40-100kb、45-100kb、50-100kb、55-100kb、60-100kb、65-100kb、70-100kb、75-100kb、80-100kb、85-100kb、90-100kb、 or 95-100kb in length (as described elsewhere herein), such that the homology arm has sufficient similarity to homologous recombine with one or more corresponding target sequences within the target genomic locus of the cell or within another targeting vector.

In some embodiments, the overlapping sequence of the 3 'homology arm of the first targeting vector and the 5' homology arm of the second targeting vector or the 3 'homology arm of the second targeting vector and the 5' homology arm of the third targeting vector may be of any length sufficient to promote homologous recombination between the targeting vectors. For example, a given homology arm and/or corresponding target sequence may comprise corresponding similar regions of length such as, but not limited to, about 0.2-0.5kb, 0.2-1kb, 0.2-1.5kb, 0.2-2kb, 0.2-2.5kb, 0.2-3kb, 0.2-3.5kb, 0.2-4kb, 0.2-4.5kb, or 0.2-5kb, such that the homology arms have sufficient similarity to homologous recombine with one or more corresponding target sequences within the target genomic locus of the cell or within another targeting vector. In some embodiments, a given overlapping sequence of homology arms may comprise corresponding overlapping regions that are about 1-5kb、5-10kb、5-15kb、5-20kb、5-25kb、5-30kb、5-35kb、5-40kb、5-45kb、5-50kb、5-55kb、5-60kb、5-65kb、5-70kb、5-75kb、5-80kb、5-85kb、5-90kb、5-95kb、5-100kb、100-200kb or 200-300kb in length, such that the overlapping sequences of the homology arms have sufficient similarity to perform homologous recombination with corresponding overlapping sequences in another targeting vector. In some embodiments, a given overlapping sequence of homology arms comprises an overlapping region that is about 1-100kb、5-100kb、10-100kb、15-100kb、20-100kb、25-100kb、30-100kb、35-100kb、40-100kb、45-100kb、50-100kb、55-100kb、60-100kb、65-100kb、70-100kb、75-100kb、80-100kb、85-100kb、90-100kb or 95-100kb in length, such that the overlapping sequences of the homology arms have sufficient similarity to homologous recombine with corresponding overlapping sequences in another targeting vector. In some embodiments, the overlapping sequences are 1-5kb, inclusive. In some embodiments, the overlapping sequences are from about 1kb to about 70kb, inclusive. In some embodiments, the overlapping sequences are from about 10kb to about 70kb, inclusive. In some embodiments, the overlapping sequences are from about 10kb to about 50kb, inclusive. In some embodiments, the overlapping sequence is at least 10kb. In some embodiments, the overlapping sequence is at least 20kb. For example, overlapping sequences may be from about 1kb to about 5kb (inclusive), from about 5kb to about 10kb (inclusive), from about 10kb to about 15kb (inclusive), from about 15kb to about 20kb (inclusive), from about 20kb to about 25kb (inclusive), from about 25kb to about 30kb (inclusive), from about 30kb to about 35kb (inclusive), from about 35kb to about 40kb (inclusive), from about 40kb to about 45kb (inclusive), from about 45kb to about 50kb (inclusive), from about 50kb to about 60kb (inclusive), from about 60kb to about 70kb (inclusive), from about, About 70kb to about 80kb (inclusive), about 80kb to about 90kb (inclusive), about 90kb to about 100kb (inclusive), about 100kb to about 120kb (inclusive), about 120kb to about 140kb (inclusive), about 140kb to about 160kb (inclusive), about 160kb to about 180kb (inclusive), about 180kb to about 200kb (inclusive), about 200kb to about 220kb (inclusive), about 220kb to about 240kb (inclusive), about 240kb to about 260kb (inclusive), About 260kb to about 280kb (inclusive) or about 280kb to about 300kb (inclusive). By way of example only, the overlapping sequences may be from about 20kb to about 60kb, inclusive. Alternatively, the overlapping sequence may be at least 1kb, at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, at least 45kb, at least 50kb, at least 60kb, at least 70kb, at least 80kb, at least 90kb, at least 100kb, at least 120kb, at least 140kb, at least 160kb, at least 180kb, at least 200kb, at least 220kb, at least 240kb, at least 260kb, at least 280kb, or at least 300kb. In some embodiments, overlapping sequences may be up to 400kb, up to 350kb, up to 300kb, up to 280kb, up to 260kb, up to 240kb, up to 220kb, up to 200kb, up to 180kb, up to 160kb, up to 140kb, up to 120kb, up to 100kb, up to 90kb, up to 80kb, up to 70kb, up to 60kb, or up to 50kb.

Exemplary sites for genome incorporation

In some embodiments, the homology arms may correspond to loci native to the cell (e.g., targeted loci), or alternatively they may correspond to regions of heterologous or exogenous DNA segments integrated into the cell genome, including, for example, heterologous or exogenous regions of transgene, expression cassette, or DNA. In some embodiments, the homology arm may correspond to a region on a targeting vector in a cell. In some embodiments, the homology arm of the targeting vector may correspond to a region of a Yeast Artificial Chromosome (YAC), a Bacterial Artificial Chromosome (BAC), a human artificial chromosome, a P1-derived artificial chromosome (PAC), or any other engineered region contained in a suitable host cell. Still further, the homology arm of the targeting vector may correspond to or be derived from a region of a BAC library, cosmid library, or P1 phage library. In some particular embodiments, the homology arm of the targeting vector corresponds to a natural, heterologous, or exogenous locus of a prokaryote, yeast, avian (e.g., chicken), non-human mammal, rodent, human, rat, mouse, hamster, rabbit, pig, cow, deer, sheep, goat, cat, dog, ferret, primate (e.g., marmoset, rhesus), domesticated mammal, agricultural mammal, or any other organism of interest. In some embodiments, the homology arms correspond to loci of cells that exhibit limited sensitivity to targeting using conventional methods or exhibit relatively low levels of successful integration and/or significant levels of off-target integration at the targeted site in the absence of nicks or double strand breaks induced by nuclease agents (e.g., cas protein, zinc finger nuclease protein, and/or TALEN protein). In some embodiments, the homology arm is designed to include engineered DNAo

In some embodiments, the 5 'and 3' homology arms of one or more targeting vectors correspond to the targeted genome. Alternatively, the homology arms correspond to the relevant genome. For example, the targeted genome is a mouse genome of a first strain, and the targeting arm corresponds to a mouse genome of a second strain, wherein the first strain and the second strain are different. In certain embodiments, the homology arms correspond to the genome of the same animal or from the same strain, e.g., the targeted genome is the mouse genome of the first strain, and the targeted arms correspond to the mouse genome from the same mouse or same strain.

The homology arm of the targeting vector may be of any length sufficient to promote homologous recombination events with the corresponding target sequence, including, for example, a length of 0.2-1kb (inclusive), 1-5kb (inclusive), 5-10kb (inclusive), 5-15kb (inclusive), 5-20kb (inclusive), 5-25kb (inclusive), 5-30kb (inclusive), 5-35kb (inclusive), 5-40kb (inclusive), 5-45kb (inclusive), 5-50kb (inclusive), 5-55kb (inclusive), 5-60kb (inclusive), 5-65kb (inclusive), 5-70kb (inclusive), 5-75kb (inclusive), 5-80kb (inclusive), 5-85kb (inclusive), 5-90kb (inclusive), 5-95kb (inclusive), 5-100kb (inclusive), 100-200kb (inclusive), or 200-300kb (inclusive). In some embodiments, the homology arms of the targeting vector have a length sufficient to promote homologous recombination events with the corresponding target sequence of 0.2-100kb (inclusive), 1-100kb (inclusive), 5-100kb (inclusive), 10-100kb (inclusive), 15-100kb (inclusive), 20-100kb (inclusive), 25-100kb (inclusive), 30-100kb (inclusive), 35-100kb (inclusive), 40-100kb (inclusive), 45-100kb (inclusive), 50-100kb (inclusive), 55-100kb (inclusive), 60-100kb (inclusive), 65-100kb (inclusive), 70-100kb (inclusive), 75-100kb (inclusive), 80-100kb (inclusive), 85-100kb (inclusive), 90-100kb (inclusive), or 95-100kb (inclusive). As described herein, a larger targeting vector may use a longer targeting arm.

In certain embodiments, the HSD17B13 polynucleotide or the exogenous HSD17B13 locus is incorporated into an endogenous locus of a non-human animal (e.g., a rodent, such as a rat or mouse). In some cases, the endogenous locus is the HSD17B13 locus. In some such cases, the endogenous HSD17B13 locus may be replaced with the exogenous HSD17B13 gene. In some embodiments, the substitution may be partial or may be complete. In some embodiments, the HSD17B13 polynucleotide or the exogenous HSD17B13 gene is incorporated into the endogenous HSD17B13 locus and is operably linked to the endogenous HSD17B13 promoter.

In certain embodiments, the HSD17B13 polynucleotide or the exogenous HSD17B13 locus is incorporated into an endogenous locus of a non-human animal (e.g., a rodent, such as a rat or mouse). In some cases, the endogenous locus is a locus driven by a constitutive promoter. In some embodiments, the endogenous locus is a locus driven by a tissue specific promoter.

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated into a non-human animal (e.g., a rodent, such as a rat or mouse) at a site susceptible to Cre/LoxP manipulation. In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 is incorporated into or located within a site within a targeting vector flanking the LoxP recombination site. In certain embodiments, a non-human animal (e.g., a rodent, such as a rat or mouse) having an exogenous HSD17B13 gene comprising or incorporating a site flanking the LoxP site can be further hybridized to an animal expressing Cre recombinase under the control of one or more tissue-specific, time-specific, and/or inducible promoters.

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated into a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse) cell or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue at a locus that is amenable to manipulation using Cre-Lox P and/or Flp-FRT; see, e.g., kim et al, "Mouse Cre-LoxP system: GENERAL PRINCIPLES to determine tissue-specific roles of TARGET GENES [ mouse Cre-LoxP system: general principle for determining tissue-specific effects of target genes ] "Laboratory ANIMAL RESEARCH [ Laboratory animal research ] (2018) 34 (4), 147-159.

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated into a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse) cell, or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue at the Cre/LoxP termination or inducible Cre/LoxP site. In some such embodiments, the locus can produce tissue-specific exogenous HSD17B13 expression in a transgenic animal when hybridized to a mouse having Cre under a tissue-specific promoter.

In some embodiments of non-human (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cells or non-human (e.g., rodent, e.g., rat or mouse) tissue as described herein, the non-human, non-human cells or non-human tissue are homozygous or heterozygous for the exogenous HSD17B13 gene integrated at a site operably linked to an inducible promoter (e.g., a tetracycline responsive element, an estrogen receptor targeting motif, and/or under the control of tamoxifen).

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated at a locus of a non-human animal (e.g., a rodent, e.g., a rat or mouse), a non-human (e.g., a rodent, e.g., a rat or mouse) cell or a non-human (e.g., a rodent, e.g., a rat or mouse) tissue known to function as a transcription hotspot and/or transcription safety harbor (as is abundant and well known in the art).

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated at a ROSA26 locus of a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse), or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue.

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated at an H11 locus of a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse) cell, or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue.

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated at the TIGRE locus of a non-human animal (e.g., a rodent, e.g., a rat or a mouse), a non-human (e.g., a rodent, e.g., a rat or a mouse) cell, or a non-human (e.g., a rodent, e.g., a rat or a mouse) tissue.

In certain embodiments, the HSD17B13 polynucleotide or exogenous HSD17B13 gene is incorporated at a MYH9 locus of a non-human animal (e.g., a rodent, e.g., a rat or mouse), a non-human (e.g., a rodent, e.g., a rat or mouse) cell, or a non-human (e.g., a rodent, e.g., a rat or mouse) tissue.

In some embodiments of non-human (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cells or non-human (e.g., rodent, e.g., rat or mouse) tissue as described herein, the non-human, non-human cells or non-human tissue are homozygous or heterozygous for the HSD17B13 polynucleotide or exogenous HSD17B13 gene integrated at a site operably linked to a ubiquitously expressed promoter (e.g., CMV, SV40, elongation factor 1a, CBA/CAGG, ubiquitin C and/or phosphoglycerate kinase 1).

Nuclease agents facilitating targeted wearer integration

In some embodiments, nuclease agents (e.g., CRISPR/Cas systems, zinc finger nucleases, and/or TALENs) can be used in combination with targeting vectors to facilitate modification of a target locus (e.g., modification of the HSD17B13 locus and/or modification of a locus targeted for foreign protein insertion). Such nuclease agents and their use are well known in the art and can promote homologous recombination between the targeting vector and the target locus. When the nuclease agent is used in combination with a targeting vector, the targeting vector can comprise 5 'and 3' homology arms corresponding to 5 'and 3' target sequences sufficiently close to the nuclease cleavage site to promote homologous recombination events between the target sequences and homology arms upon nicking or double strand breaks at the nuclease cleavage site. In some embodiments, the term "nuclease cleavage site" includes a DNA sequence (e.g., cas9 cleavage site) at which a nick or double-strand break is created by a nuclease agent. Target sequences within the targeting gene locus corresponding to the 5 'and 3' homology arms of the targeting vector are "located sufficiently close" to the nuclease cleavage site if they are, for example, sufficiently distant to facilitate the occurrence of homologous recombination events between the 5 'and 3' target sequences and the homology arms following nicking or double strand breaks at the recognition site. Thus, in certain embodiments, the target sequence corresponding to the 5 'and/or 3' homology arm of the targeting vector is within at least one nucleotide of a given recognition site or within at least 10 nucleotides to about 14kb of a given recognition site. In some embodiments, the nuclease cleavage site is immediately adjacent to at least one, two, three, four, and/or more target sequences.

The spatial relationship of the target sequence and nuclease cleavage site corresponding to the homology arm of the targeting vector can vary. For example, the target sequence may be located 5 'to the nuclease cleavage site, the target sequence may be located 3' to the recognition site, or the target sequence may flank the nuclease cleavage site.

The combined use of a targeting vector with a nuclease agent can result in increased targeting efficiency compared to the use of the targeting vector alone. For example, when the targeting vector is used in combination with a nuclease agent, the targeting efficiency of the targeting vector may be increased by at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or within the range formed by these integers, e.g., 2-10 times, as compared to using the targeting vector alone.

In some embodiments, the targeting vector comprises homology arms that correspond to and are derived from nucleic acid sequences that are larger than those typically used by other methods intended for homologous recombination in cells. In some embodiments, the targeting vector comprises homology arms that correspond to and are derived from nucleic acid sequences that are shorter than those typically used by other methods intended for homologous recombination in cells. In some embodiments, the homology arms are at least 10kb in length, or the sum of the 5 'homology arm and the 3' homology arm can be, for example, at least 10kb. In some embodiments, the homology arms are less than 10kb in length, or the sum of the 5 'homology arm and the 3' homology arm may be, for example, less than 10kb.

In some embodiments, the targeting vector comprising the nucleic acid construct is larger than those typically used by other methods intended for homologous recombination in a cell. For example, in some embodiments, large loci that traditionally cannot be accommodated by plasmid-based targeting vectors due to their size limitations can still be used by using large targeting vectors. For example, the targeted loci can be (i.e., the 5 'and 3' homology arms can correspond to) cellular loci that are not targetable using conventional methods, or cellular loci that can only be targeted erroneously or with significantly lower efficiency in the absence of nicks or double strand breaks induced by nuclease agents (e.g., cas proteins). In some embodiments, the large targeting vector may include a vector derived from a Bacterial Artificial Chromosome (BAC), a human artificial chromosome, or a Yeast Artificial Chromosome (YAC). The large targeting vector may be in linear or circular form. Examples of large targeting vectors and methods for their preparation are described in the following: such as Macdonald (2014), U.S. Pat. nos. 6,586,251, 6,596,541, and 7,105,348; and International patent application publication No. WO 2002/036789.

Method for preparing the provided non-human animals

Those of skill in the art will appreciate that various techniques may be utilized to prepare engineered cells, tissues, animals, etc. according to the present disclosure. Provided herein are compositions and methods for preparing non-human animals (e.g., rodents, such as mice) whose germline genomes comprise an engineered human HSD17B13 gene.

In some embodiments, the non-human HSD17B13 locus may be a locus for insertion of the human HSD17B13 gene. In some embodiments, any suitable integration locus may be a site for insertion of the human HSD17B13 coding sequence.

In some embodiments, the human HSD17B13 gene may be under the control of one or more heterologous protein enhancers and/or promoters. In some embodiments, the methods described herein comprise inserting a single human HSD17B13 gene encoding a human HSD17B13 protein. In some embodiments, the methods described herein comprise inserting more than one human HSD17B13 gene encoding more than one human HSD17B13 polypeptide.

Provided herein are compositions and methods for making non-human animals (e.g., rodents, such as mice) whose germline genome comprises an engineered non-human primate (NHP) HSD17B13 locus comprising one or more functional HSD17B13 domains (e.g., a lipid droplet targeting domain comprising an N-terminal hydrophobic domain, a PAT-like domain, and a putative alpha-helix/beta-sheet/alpha-helix domain) and an enzyme activity domain comprising a catalytic site, a substrate binding site, and a homodimer interaction site).

In some embodiments, the non-human HSD17B13 locus may be a site for insertion of the NHPHSD B13 gene. In some embodiments, any suitable integration locus may be a site for insertion of the NHP HSD17B13 coding sequence.

In some embodiments, the NHP HSD17B13 gene may be under the control of one or more heterologous protein enhancers and/or promoters. In some embodiments, the methods described herein comprise inserting a single NHP HSD17B13 gene encoding a NHP HSD17B13 protein. In some embodiments, the methods described herein comprise inserting more than one NHP HSD17B13 gene encoding a NHP HSD17B13 polypeptide.

Embryo-in-cell modification for producing transgenic animals

In some embodiments, methods of making the provided non-human animals include inserting genetic material comprising the exogenous HSD17B13 gene into embryonic stem cells of a non-human animal (e.g., a rodent, such as a rat or mouse). In some embodiments, the method comprises multiple insertions in a single ES cell clone. In some embodiments, the method comprises sequential insertion in successive ES cell clones. In some embodiments, the method comprises a single insertion in an engineered ES cell clone.

In some embodiments, methods involving the preparation of non-human transgenic animals using embryonic stem cells may have targeting vectors and/or nucleic acid constructs introduced by any means known in the art. In some embodiments, the transgene is introduced into the embryonic stem cell by a method including, but not limited to: electroporation, lipid-based transfection, lipid-based nanoparticles, retroviral infection and/or lentiviral infection.

In some embodiments of the method of making a non-human animal (e.g., a rodent, such as a mouse), the DNA fragment is introduced into the non-human embryonic stem cells.

In some embodiments, methods involving the use of embryonic stem cell modifications to produce transgenic animals can utilize any of the molecular biology techniques or reagents described herein.

In some embodiments, a targeting vector comprising the HSD17B13 coding sequence is electroporated into a mouse ES cell using methods known in the art. Screening and/or selection of clones that have undergone homologous recombination yields modified ES cells for use in generating chimeric mice expressing huHSD B131. Positive ES cell clones were confirmed by PCR screening using primers and probes specific for huHSD B13 transgene. Primers and probes vary depending on the desired insertion locus. The targeted ES cells are used as donor ES cells and introduced into 8-cell stage mouse embryos using appropriate methods (e.g., byMethods (see, e.g., U.S. Pat. Nos. 7,294,754 and Poueymirou et al (2007).F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses[, which are essentially entirely derived from F0 mice that are targeted to ES cells by a donor gene that allows immediate phenotypic analysis), nature Biotech 25 (1): 91-99). Transgenic mice expressing huHSD B13 were identified by genotyping using methods known in the art. Mice were bred for stable heterozygous and/or homozygous transgene delivery at huHSD B13 insert loci.

Fertilized egg injection for producing transgenic animals

Where appropriate, the exogenous HSD17B13 gene (e.g., the human HSD17B13 gene encoding the human HSD17B13 protein) may be modified alone to include codons optimized for expression in a non-human animal (see, e.g., U.S. Pat. nos. 5,670,356 and 5,874,304). The codon-optimized sequence is an engineered sequence and preferably encodes the same polypeptide (or a biologically active fragment of a characteristic portion of a polypeptide having substantially the same activity as the full-length polypeptide) encoded by the non-codon-optimized parent polynucleotide. In some embodiments, the exogenous HSD17B13 gene encoding the exogenous HSD17B13 protein may include an altered sequence alone to optimize codon usage for a particular cell type (e.g., a rodent cell, such as a mouse cell). For example, as described herein, codons of each nucleotide sequence to be inserted into the genome of a non-human animal (e.g., a rodent, e.g., a mouse) can be optimized for expression in cells of the non-human animal. Such sequences may be described as codon optimized sequences.

In some embodiments, the insertion of the nucleotide sequence encoding the exogenous HSD17B13 gene employs minimal modification of the non-human animal germline genome as described herein and results in expression of the exogenous HSD17B13 gene (e.g., the human HSD17B13 gene or the NHP HSD17B13 gene). Methods for producing engineered non-human animals (e.g., rodents, such as rats or mice), including knockouts and knockins, are known in the art (see, e.g., GENE TARGETING: A PRACTICAL Approach [ Gene targeting: a practical method ], joyner, eds., oxford University Press, inc. [ oxford university Press ], 2000). For example, the generation of a genetically engineered rodent may optionally include disruption of a genetic locus of one or more endogenous rodent genes (or gene segments) and, in some embodiments, introduction of one or more heterologous genes (or gene segments or nucleotide sequences) into the rodent genome at the same location as the endogenous rodent genes (or gene segments). In some embodiments, the nucleotide sequence encoding the exogenous HSD17B13 gene (e.g., the human HSD17B13 gene or the NHP HSD17B13 gene) is randomly inserted into the rodent germline genome. In some embodiments, the nucleotide sequence encoding the exogenous HSD17B13 gene is introduced upstream of the non-human (e.g., rodent, e.g., rat or mouse) HSD17B13 locus in the rodent germline genome; in some particular embodiments, the endogenous HSD17B13 locus is altered, modified or engineered to comprise human and/or NHP HSD17B13 gene segments, wherein any combination of HSD17B13 gene segments derived from rodents, humans and/or NHPs may be utilized.

Once produced, the targeting vector can be injected into a rodent fertilized egg, or alternatively electroporated into a rodent Embryonic Stem (ES) cell to produce a rodent whose germline genome comprises the exogenous HSD17B13 gene. In some embodiments, confirmation of rodent ES cells comprising a targeting vector containing the exogenous HSD17B13 gene can be selected and/or screened using methods known in the art. As described in the examples section below, rodent fertilized eggs comprising an injected nucleic acid construct comprising a targeting vector comprising an exogenous HSD17B13 gene can be used to produce transgenic non-human animals comprising an integrated exogenous HSD17B13 gene, such animals can be screened from a population of viable injected fertilized eggs that are transplanted into a surrogate mother.

In some embodiments, the targeting vector is introduced into a non-human (e.g., rodent, e.g., mouse or rat) embryo cell (e.g., fertilized egg and/or stem cell) by electroporation such that the sequence contained in the targeting vector results in the ability of the non-human (e.g., rodent, e.g., rat or mouse) cell or non-human (e.g., rodent, e.g., rat or mouse) to express the exogenous HSD17B13 gene. As described herein, genetically engineered non-human animals are produced in which the exogenous HSD17B13 gene has been produced and/or incorporated into the germline genome of the non-human animal (e.g., at a defined locus, and/or at a random locus). In some embodiments, insertion and/or expression of the exogenous HSD17B13 gene is confirmed using methods known in the art (e.g., PCR, western blot, etc.). In some embodiments, the oligonucleotides as described herein are then characterized in vitro or in vivo using tissues, cells, and/or animals derived from non-human embryonic stem cells comprising the exogenous HSD17B13 gene.

In some embodiments, methods of making a genetically modified non-human animal (e.g., a rodent, such as a mouse) include engineering a human HSD17B13 gene in the germline genome of the non-human animal to comprise a sequence operably linked to a tissue-specific regulatory region.

In some embodiments, methods of making a genetically modified non-human animal (e.g., a rodent, such as a mouse) include engineering a human HSD17B13 gene in the germline genome of the non-human animal to comprise a sequence operably linked to a time-specific regulatory region.

In some embodiments, methods of making a genetically modified non-human animal (e.g., a rodent, such as a mouse) include engineering a human HSD17B13 gene in the germline genome of the non-human animal to comprise a sequence operably linked to a substrate-specific regulatory region.

In some embodiments, non-human animals (e.g., rodents, such as rats or mice) prepared, produced, obtained, or obtainable by a method as described herein are provided.

In some embodiments of the method of making a non-human animal (e.g., a rodent, such as a rat or mouse), the DNA fragment is introduced into a non-human embryonic stem cell and/or fertilized egg whose germline genome comprises the endogenous HSD17B13 locus. Alternatively and/or additionally, in some embodiments, the germline genome of a non-human animal (e.g., a rodent, such as a rat or mouse) as described herein further comprises a deleted, inactivated, functionally silent, or otherwise nonfunctional endogenous HSD17B13 locus. Genetic modifications to delete or disable a gene or genetic locus may be accomplished using the methods described herein and/or known in the art.

Genetically engineered, established non-human animals (e.g., rodents, such as rats or mice) can be identified based on the presence of the exogenous HSD17B13 gene in its germline genome and/or expression of the exogenous HSD17B13 protein in tissues or cells of the non-human animal as described herein. The genetically engineered, first-built non-human animals can then be used to reproduce additional non-human animals carrying the exogenous HSD17B13 gene, thereby producing a set of non-human animals, each carrying one or more copies of the exogenous HSD17B13 gene. In addition, genetically engineered non-human animals carrying the exogenous HSD17B13 gene may be further crossed as desired with other genetically engineered non-human animals carrying other transgenes (e.g., human disease genes) or other mutated endogenous loci.

Genetically engineered non-human animals (e.g., rodents, such as rats or mice) can also be produced to include selected systems that allow for regulated, targeted, inducible and/or cell type-specific expression of the transgene or one or more integrated sequences. For example, a non-human animal as described herein may be engineered to comprise one or more conditionally expressed sequences encoding the exogenous HSD17B13 gene (e.g., reviewed in Rajewski, K.et al, 1996, J. Clin. Invest. [ J. Clinical study ]98 (3): 600-3). Exemplary systems include the Cre/loxP recombinase system of phage P1 (see, e.g., lakso, M. Et al, 1992, proc. Natl. Acad. Sci. U.S.A. [ Proc. Natl. Acad. Sci. U.S.A. [ Proc. Natl. Acad. Sci. USA ] 89:6232-6) and the FLP/Frt recombinase system of Saccharomyces cerevisiae (O' Gorman, S. Et al, 1991, science [ science ] 251:1351-5). Such animals may be provided by: a "double" genetically engineered animal is constructed, for example, by mating two genetically engineered animals, one of which contains a transgene comprising a selected modification (e.g., the exogenous HSD17B13 gene as described herein) and the other of which contains a transgene encoding a recombinase (e.g., cre recombinase).

Non-human animals suitable for exogenous HSD17B13 gene expression

A non-human animal (e.g., a rodent, such as a rat or mouse) as described herein may be prepared as described above or using methods known in the art to include additional human, humanized or otherwise engineered genes, generally depending on the intended use of the non-human animal. Genetic material of such human, humanized or otherwise engineered genes may be introduced by: further altering the genome of the cell with the genetic modification or alteration as described above (e.g., embryonic stem cells, and/or injection of fertilized eggs derived from transgenic rodents comprising the exogenous HSD17B13 gene), or other genetically modified or engineered lines as desired by breeding techniques known in the art.

As will be appreciated by those of skill in the art, a variety of compatible mouse strains (e.g., WTs, containing one or more transgenes, containing one or more mutations in endogenous loci, etc.) can be bred into any of the engineered mice described herein to produce any number of genetically modified mouse strains expressing HSD17B13 (e.g., NHP HSD17B13, human HSD17B13, etc.) polypeptides or characteristic portions thereof and any other genetic characteristics (e.g., native mouse mutant loci, disease modeling endogenous mouse gene mutant loci, transgenic derived mutant animals expressing a human gene mutation of interest, etc.). Various techniques can be used to generate mice heterozygous or homozygous for a transgenic polynucleotide encoding an HSD17B13 polypeptide or a characteristic portion thereof (e.g., human HSD17B 13) as described herein. In some embodiments, huHSD B13 homozygous or heterozygous genetically modified mice (e.g., those described in the examples) are bred into mice that are homozygous or heterozygous for mutations (deletions, gain of function, loss of function, etc.) in the endogenous mouse gene of interest that may be associated with HSD17B13 function. The resulting offspring expressing the desired HSD17B13 or a characteristic portion thereof and heterozygous for the gene of interest are crossed to obtain mice homozygous and/or heterozygous for the HSD17B13 and/or the gene of interest. In some embodiments, breeding may be performed by commercial breeders, such as jackson laboratories (The Jackson Laboratory). In certain embodiments, mice heterozygous for the transgenic HSD17B13 insertion (e.g., as described herein) are crossed with a balanced line to maintain stable heterozygous transgenic HSD17B13 delivery. In some embodiments, closely linked phenotypically detectable markers are genetically engineered into transgenic HSD17B13 mice to aid hybridization and/or genotyping.

Although embodiments describing the construction of the exogenous HSD17B13 gene in a mouse (i.e., a mouse having the exogenous HSD17B13 gene integrated into its germline genome) are discussed extensively herein, other non-human animals comprising the exogenous HSD17B13 gene are also provided. Such non-human animals include any animal that can be genetically modified to express an exogenous HSD17B13 polypeptide and/or fragment thereof as described herein, including, for example, mammals, such as mice, rats, rabbits, pigs, cows (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus), and the like. For example, for non-human animals for which suitable genetically modifiable ES cells are not readily available, other methods are employed to prepare non-human animals comprising genetic modifications. Such methods include, for example, modifying a non-ES cell genome (e.g., a fibroblast or induced pluripotent cell) and transferring the genetically modified genome to a suitable cell, such as an enucleated oocyte, using Somatic Cell Nuclear Transfer (SCNT), and inoculating the modified cell (e.g., modified oocyte) in a non-human animal under suitable conditions to form an embryo.

Methods of modifying the genome of a non-human animal germline (e.g., porcine, bovine, rodent, chicken, etc.) include, for example, the use of Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or Cas proteins (i.e., CRISPR/Cas systems) to include the exogenous HSD17B13 gene. Guidelines for methods for modifying the germ line genome of a non-human animal can be found, for example, in U.S. patent No. 9,738,897 and U.S. patent application publication No. US 2016/0145646 (release of 2016 at 5-26) and US2016/0177339 (release of 2016 at 6-23).

In some embodiments, the non-human animal as described herein is a mammal. In some embodiments, the non-human animal as described herein is a small mammal, such as a small mammal of jerboa general family (Dipodoidea) or murine general family (Muroidea). In some embodiments, the genetically modified animal as described herein is a rodent. In some embodiments, the rodent as described herein is selected from a mouse, a rat, and a hamster. In some embodiments, the rodent as described herein is selected from the murine superfamily. In some embodiments, the genetically modified animal as described herein is from a family selected from the group consisting of: the hamster family (Calomyscidae) (e.g., hamster-like), hamster family (CRICETIDAE) (e.g., hamster, new World (New World) rats and mice, field mice), murine family (true mice and rats, gerbil, african spiny mice, coronaries), equine island murine family (Nesomyidae) (climbing, rock, white tail, motor gas rats and mice), sarcandidae family (Platacanthomyidae) (e.g., spiny sleeping mice (spiny dormice)) and mole murine family (SPALACIDAE) (e.g., mole, bamboo and zokor). In some specific embodiments, the genetically modified rodent as described herein is selected from a real mouse or rat (murine), a gerbil, a spiny mouse, and a coronamouse. In some specific embodiments, the genetically modified mouse as described herein is from a member of the murine family. In some embodiments, the non-human animal as described herein is a rodent. In some particular embodiments, the rodent as described herein is selected from a mouse and a rat. In some embodiments, the non-human animal as described herein is a mouse.

In some embodiments, the non-human animal as described herein is a rodent that is a mouse of the C57BL strain selected from C57BL/A、C57BL/An、C57BL/GrFa、C57BL/KaLwN、C57BL/6、C57BL/6J、C57BL/6ByJ、C57BL/6NJ、C57BL/10、C57BL/10ScSn、C57BL/10Cr and C57 BL/Ola. In some specific embodiments, the mouse as described herein is a 129-strain selected from the group consisting of: lines 129P1, 129P2, 129P3, 129X1, 129S1 (see, e.g., ,129S1/SV、129S1/SvIm)、129S2、129S4、129S5、129S9/SvEvH、129/SvJae、129S6(129/SvEvTac)、129S7、129S8、129T1、129T2(, e.g., festing et al, 1999,Mammalian Genome [ mammalian genome ]10:836; auerbach, W. Et al, 2000, biotechnology [ biotech ]29 (5): 1024-1028, 1030, 1032). In some specific embodiments, the genetically modified mouse as described herein is a mixture of the 129 strain described above and the C57BL/6 strain described above. In some specific embodiments, the mice as described herein are a mix of the 129 strain described above, or a mix of the BL/6 strain described above. In some particular embodiments, the 129 line of the mixture as described herein is a 129S6 (129/SvEvTac) line. In some embodiments, the mice as described herein are BALB strains, e.g., BALB/c strains. In some embodiments, the mouse as described herein is a mixture of a BALB strain and another of the foregoing strains.

In some embodiments, the non-human animal as described herein is a rat. In some specific embodiments, the rat as described herein is selected from the group consisting of Wistar rats, LEA strains, sprague Dawley strains, fischer strains, F344, F6, and Dark Agouti. In some specific embodiments, the rat strain as described herein is a mixture of two or more strains selected from the group consisting of Wistar, LEA, sprague Dawley, fischer, F344, F6, and Dark Agouti.

The rat pluripotent and/or totipotent cells may be from any rat strain including, for example, an ACI rat strain (an inbred strain originally derived from August and Copenhagen strains), darkAgouti (DA) rat strain, wistar rat strain, LEA rat strain, spragueDawley (SD) rat strain, or Fischer rat strain, such as Fisher F344 or FisherF6. Rat pluripotent and/or totipotent cells may also be obtained from lines derived from a mixture of two or more of the lines described above. For example, the pluripotent and/or totipotent cells of the rat may be from a DA strain or an ACI strain. ACI rat strains are characterized by having black spines with white abdomen and feet and RT1av1 haplotypes. Such lines are available from a variety of sources, including the halan laboratory (Harlan Laboratories). An example of a rat ES cell line from an ACI rat is aci.g1 rat ES cells. DA rat strains are characterized by having a spiny coat and RT1av1 haplotype. Such rats are available from a variety of sources, including the Charles river (CHARLES RIVER) and Ha Lan laboratories. Examples of rat ES cell lines from DA rats are the da.2b rat ES cell line and the da.2c rat ES cell line. In some embodiments, the rat pluripotent and/or totipotent cells are from a strain of inbred rats (see, e.g., U.S. patent application publication No. 2014-0235933 A1). Guidance for modification in the rat genome (e.g., in rat ES cells) using methods and/or constructs as described herein can be found, for example, in U.S. patent application publication nos. 2014-0310828 and 2017-0204430.

In some embodiments, useful techniques are described in, for example, US 10314297 and may be used in accordance with the present disclosure. As will be appreciated by those skilled in the art, many useful techniques are commercially available from various suppliers and/or service providers.

In some embodiments, the present disclosure provides methods for evaluating an agent (e.g., an oligonucleotide) or a composition thereof, comprising administering the agent or composition to an animal, cell, or tissue described herein. In some embodiments, the agent or composition is evaluated for use in preventing or treating a condition, disorder, or disease. In some embodiments, an animal, cell, tissue, e.g., as described in various embodiments herein, is an animal model or cell or tissue for a variety of conditions, disorders, or diseases (e.g., comprising mutations associated with a variety of conditions, disorders, or diseases, and/or cells, tissues, organs, etc. associated with a variety of conditions, disorders, or diseases, or cells, tissues, organs, etc. of a variety of conditions, disorders, or diseases), which animal model or cell or tissue is engineered to comprise and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, the animal may be provided by mating and breeding (e.g., IVF, natural breeding, etc.) an animal that is a model animal for various conditions, disorders, or diseases, but that is not engineered to contain and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof, with an animal that is engineered to contain and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, the cell or tissue may be provided by introducing into the cell or tissue a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, the disclosure provides methods for preventing or treating a condition, disorder, or disease, the methods comprising administering to a subject an effective amount of an agent or composition thereof, wherein the agent or composition is evaluated in an animal provided herein (e.g., an animal engineered to comprise an HSD17B13 polypeptide or a characteristic portion thereof, an animal engineered to comprise and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof, a model animal engineered to comprise an HSD17B13 polypeptide or a characteristic portion thereof for a condition, disorder, or disease, a model animal engineered to comprise and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof for a condition, disorder, or disease). In some embodiments, the present disclosure provides methods for preventing or treating a condition, disorder, or disease, the method comprising administering to a subject an effective amount of an agent or composition thereof, wherein the agent or composition is evaluated in a cell or tissue provided herein. In some embodiments, the animal is a non-human animal. In some embodiments, the cell is a non-human animal cell. In some embodiments, the tissue is non-human animal tissue. In some embodiments, the non-human animal is a rodent. In some embodiments, the non-human animal is a mouse. In some embodiments, the non-human animal is a rat. In some embodiments, the non-human animal is a non-human primate.

As will be appreciated by those of skill in the art, in some embodiments, the animal may be heterozygous in one or more or all of the sequences. In some embodiments, the animal is homozygous for one or more or all of the sequences. In some embodiments, the animal is semi-synthetic in terms of one or more or all of the engineered sequences. In some embodiments, the animal is homozygous for one or more sequences and heterozygous for one or more sequences. In some embodiments, the animal is heterozygous for a polynucleotide whose sequence encodes the HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, the animal is homozygous for the polynucleotide whose sequence encodes the HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, the animal is homozygous wild-type for the locus encoding the polynucleotide whose sequence encodes the HSD17B13 polypeptide or a characteristic portion thereof (e.g., does not express the exogenous HSD17B13 polypeptide or a characteristic portion thereof), and can serve as a relative control. In some embodiments, certain animals are heterozygous for one or more polynucleotide sequences associated with various conditions, disorders, or diseases, and are heterozygous for a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, certain animals are homozygous for one or more polynucleotide sequences associated with various conditions, disorders, or diseases, and heterozygous for a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, certain animals are heterozygous for one or more polynucleotide sequences associated with various conditions, disorders, or diseases, and are homozygous for a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, certain animals are homozygous for one or more polynucleotide sequences associated with various conditions, disorders, or diseases, and are homozygous for polynucleotides whose sequences encode HSD17B13 polypeptides or portions thereof. Cells or tissues are similarly heterozygous, hemizygous and/or homozygous for each sequence.

In some embodiments, the present disclosure provides methods comprising: 1) Assessing an agent or a composition thereof comprising contacting the agent or composition thereof with a provided cell or tissue associated with a condition, disorder or disease or with a cell or tissue of a condition, disorder or disease, and 2) administering an effective amount of the agent or composition thereof to a subject suffering from or susceptible to the condition, disorder or disease. In some embodiments, the present disclosure provides methods comprising: 1) Assessing an agent or a composition thereof comprising administering the agent or composition thereof to a provided animal that is an animal model of a condition, disorder or disease, and 2) administering an effective amount of the agent or composition thereof to a subject suffering from or susceptible to the condition, disorder or disease. In some embodiments, the cell, tissue, or animal is engineered to comprise an HSD17B13 polypeptide or a characteristic portion thereof, as described herein. In some embodiments, the cell, tissue or animal is engineered to comprise and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof. In some embodiments, the cell, tissue, or animal further comprises a nucleotide sequence (e.g., a mutation) associated with a condition, disorder, or disease. In some embodiments, the animal is a rodent, such as a mouse, rat, or the like. In some embodiments, the cell or tissue is a rodent (e.g., mouse, rat, etc.) cell or tissue. In some embodiments, the cell is a germ line cell. In some embodiments, a portion of a population of cells, tissue, or animal, but not all cells, e.g., cells of a particular cell type or tissue or location, comprises a nucleotide sequence (e.g., a mutation) associated with a condition, disorder, or disease, and the portion of the cells is engineered to comprise the HSD17B13 polypeptide or a characteristic portion thereof or is engineered to comprise and/or express a polynucleotide whose sequence encodes the HSD17B13 polypeptide or a characteristic portion thereof. Those of skill in the art understand that a variety of techniques may be used to optionally controllably introduce and/or express nucleotide sequences in a variety of cells, tissues or organs and may be utilized in accordance with the present disclosure. In some embodiments, the cell, tissue, or animal comprises in the genome, and in some embodiments in the germline genome, a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof, as described herein. In some embodiments, the cell, tissue, or animal comprises in the genome, in some embodiments a nucleotide sequence (e.g., a mutation) associated with a condition, disorder, or disease in the germline genome, as described herein.

Methods of using the provided non-human animals, cells or tissues

Non-human animals (e.g., rodents, such as rats or mice), non-human (e.g., rodents, such as rats or mice) cells and non-human (e.g., rodents, such as rats or mice) tissues can be used as platforms for developing therapeutic agents (e.g., oligonucleotides). In particular, non-human animals, non-human cells and non-human cellular tissues as described herein represent particularly advantageous platforms for identifying and characterizing agents, e.g., ds oligonucleotides targeted to HSD17B13 may be used to treat conditions, disorders or diseases associated with HSD17B13 expression, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic Steatohepatitis (ASH), alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug-induced liver injury or hepatocyte necrosis.

In some embodiments, the disclosure provides that non-human animals (e.g., rodents, such as rats or mice), non-human (e.g., rodents, such as rats or mice) cells, and non-human (e.g., rodents, such as rats or mice) tissues described herein can be used in methods of characterizing/evaluating various agents (e.g., ds oligonucleotides targeted to HSD17B 13) that can be used to treat a condition, disorder, or disease associated with HSD17B13 expression. In some embodiments, the composition is an oligonucleotide composition. In some embodiments, the oligonucleotides comprise various modifications, e.g., base, sugar, internucleotide linkage modifications, and the like. In some embodiments, the linkage phosphorus in the modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages) is chiral (as understood by those skilled in the art, natural phosphoester linkages common in natural DNA and RNA molecules are achiral). In some embodiments, for various biological or therapeutic uses, the oligonucleotides comprise a number of modifications, and in some cases, are free of natural RNA sugars for, e.g., improved stability. In some embodiments, the composition is a stereogenic oligonucleotide composition. In some embodiments, the composition is a chirally controlled oligonucleotide composition in which one or more or all of the chiral linked phosphites are independently chirally controlled.

In vivo agent assessment using non-human animals

In some embodiments, non-human (e.g., rodent, e.g., rat or mouse) cells and non-human (e.g., rodent, e.g., rat or mouse) tissues as described herein can be used to characterize oligonucleotides in vivo, wherein expression of the exogenous HSD17B13 gene in the non-human provides an improved characterization platform as compared to WT non-human (e.g., rodent, e.g., rat or mouse).

In some embodiments, a non-human animal (e.g., a genetically modified rodent, e.g., a genetically modified rat or mouse) as described herein is treated (e.g., injected) with a ds oligonucleotide of interest under conditions and for a time sufficient for the ds oligonucleotide to target exogenous HSD17B 13. The sequence of an RNA molecule (e.g., HSD17B13 sequence) is isolated and/or identified from a treated non-human animal (or one or more cells, e.g., one or more B cells) and characterized using various assays that measure, for example, affinity, specificity, edit level, transcript stability, translational efficiency, protein binding partner, nuclear localization, etc. In various embodiments, the oligonucleotides characterized using non-human animals, non-human cells, and/or non-human tissues as described herein comprise one or more regions that help target HSD17B13 targets.

In some embodiments, a non-human animal (e.g., a genetically modified rodent, e.g., a genetically modified rat or mouse) as described herein is treated with a ds oligonucleotide of interest and the effect of the ds oligonucleotide in a particular tissue is monitored and/or assessed.

In some embodiments, non-human (e.g., rodent, e.g., rat or mouse) cells as described herein comprising a transgenic HSD17B13 locus can be used to characterize a potentially therapeutically effective oligonucleotide method comprising characterizing in cells derived from HSD17B13 transgenic mice. In some embodiments, such cells may be of any desired cell lineage and/or desired type known in the art. In some embodiments, such cells may be, but are not limited to: primary mouse hepatocytes, epidermal cells, epithelial cells, cortical neurons, sensory neurons, effector neurons, hormone-secreting cells, exocrine epithelial cells, barrier cells, cardiomyocytes, leukocytes, lymphocytes, B cells, T cells, bone marrow cells, osteoblasts, chondrocytes, chondroblasts, adipocytes, cardiac myocytes, muscle cells, fibroblasts, germ cells, trophoblasts, renal cells, and/or induced stem cells derived from any of the foregoing or products thereof.

Non-human (e.g., rodent, e.g., rat or mouse), non-human (e.g., rodent, e.g., rat or mouse) cells and non-human (e.g., rodent, e.g., rat or mouse) tissues as described herein may be used to identify ds oligonucleotides targeting HSD17B13, which may be used to treat conditions, disorders or diseases associated with HSD17B13 expression, e.g., non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic Steatohepatitis (ASH), alcoholic liver disease, non-alcoholic liver disease, alcoholic liver cirrhosis, non-alcoholic liver cirrhosis, steatohepatitis, hepatic steatosis, hepatocellular carcinoma, HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, primary sclerosing cholangitis, drug liver injury or hepatocyte necrosis.

Non-human (e.g., rodent, such as rat or mouse) cells and non-human (e.g., rodent, such as rat or mouse) tissues as described herein provide improved in vivo systems and sources of biological material (e.g., cells, nucleotides, polypeptides, protein complexes) for the production and characterization of oligonucleotides and/or polynucleotides useful in various assays. In various embodiments, non-human animals, non-human cells, and non-human tissues as described herein are used to develop therapeutic agents that target RNA of interest (e.g., RNA molecules known to function in disease-related pathways) and/or modulate one or more activities associated with the RNA molecule of interest and/or modulate interactions of the RNA molecule of interest with other potential binding partners (e.g., any regulatory mechanism that can act on intracellular RNA molecules, such as proteins and/or RNA species involved in translation, proteins and/or RNA species involved in innate immunity, proteins and/or RNA species involved in RNA interference, etc.).

In various embodiments, non-human animals (e.g., rodents, e.g., rats or mice), non-human (e.g., rodents, e.g., rats or mice) cells and non-human (e.g., rodents, e.g., rats or mice) tissues as described herein are used to determine the pharmacokinetic profile of one or more candidate ds oligonucleotides. In various embodiments, one or more non-human animals, non-human cells, and non-human tissues as described herein, and one or more control or reference non-human animals, non-human cells, and non-human tissues are each exposed to one or more agents, e.g., various doses of ds oligonucleotides (e.g., less than 0.1mg/kg、0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、1mg/kg、2mg/kg、3mg/kg、4mg/kg、5mg/mg、7.5mg/kg、10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、40mg/kg or 50mg/kg or more). In some embodiments, the oligonucleotides may be administered to the non-human animal at a rate that varies with sex, e.g., in some embodiments, the male animal may receive a higher dose than the comparable female animal, while in other embodiments, the female animal may receive a higher dose than the comparable male animal. In some embodiments, the candidate therapeutic oligonucleotides can be administered to the non-human animal by any desired route of administration as described herein, including parenteral and non-parenteral routes of administration. Parenteral routes include, for example, intravenous, intra-arterial, portal, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intraventricular, intracranial, intrapleural, or other injection routes. In some embodiments, the administration may be non-parenteral, and in some embodiments, non-parenteral routes include, for example, oral, nasal, transdermal, pulmonary, rectal, buccal, vaginal, ocular. In some embodiments, administration may also be by continuous infusion, topical administration, sustained release from implants (gels, films, etc.), and/or intravenous injection. In some embodiments, biological tissue (e.g., organs, blood, cells, secretions, etc.) is isolated from non-human animals (humanized and controls) at different time points (e.g., 0 hours, 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or up to 30 days or more). Various assays can be performed using samples obtained from non-human animals, non-human cells, and non-human tissues described herein to determine the pharmacokinetic profile of the candidate therapeutic oligonucleotides administered, including but not limited to, edit levels, transcript levels, translation levels, and the like.

In various embodiments, non-human (e.g., rodent, e.g., rat or mouse) cells and non-human (e.g., rodent, e.g., rat or mouse) tissues as described herein are used to measure therapeutic effects that block or modulate the activity of an RNA molecule of interest and the effect on gene expression due to cellular changes thereof.

Cells from a provided non-human animal (e.g., a rodent, e.g., a rat or mouse) can be isolated and used as desired, or can be maintained in culture for multiple generations. In various embodiments, cells from the provided non-human animals are immortalized (e.g., by use of viruses) and maintained in culture indefinitely (e.g., in continuous culture).

In some embodiments, the non-human (e.g., rodent, e.g., rat or mouse) cells are non-human lymphocytes. In some embodiments, the non-human cells are selected from the group consisting of B cells, dendritic cells, macrophages, monocytes and T cells. In some embodiments, the non-human cells are immature B cells, mature naive B cells, activated B cells, memory B cells, and/or plasma cells.

In some embodiments, the non-human (e.g., rodent, e.g., rat or mouse) cells are non-human Embryonic Stem (ES) cells. In some embodiments, the non-human ES cells are rodent ES cells. In some particular embodiments, the rodent ES cells are mouse ES cells and are from line 129, C57BL, BALB/C or mixtures thereof. In some particular embodiments, the rodent embryonic stem cells are mouse embryonic stem cells and are a mixture of 129 and C57BL strains. In some specific embodiments, the rodent embryonic stem cells are mouse embryonic stem cells and are a mix of 129, C57BL and BALB/C strains.

In some embodiments, there is provided the use of non-human (e.g., rodent, e.g., rat or mouse) ES cells described herein to prepare a non-human animal. In some specific embodiments, the non-human ES cells are mouse ES cells and are used to prepare mice comprising exogenous HSD17B13 as described herein. In some specific embodiments, the non-human ES cells are rat ES cells and are used to prepare rats comprising exogenous HSD17B13 as described herein.

In some embodiments, the non-human (e.g., rodent, e.g., rat or mouse) tissue is selected from, but is not limited to, fat, bladder, brain, breast, bone marrow, eye, heart, intestine, kidney, liver, lung, lymph node, muscle, pancreas, plasma, serum, skin, spleen, stomach, thymus, testis, egg, and/or combinations thereof.

In some embodiments, immortalized cells prepared, generated, produced, or obtained from isolated non-human cells or tissues as described herein are provided.

In some embodiments, non-human (e.g., rodent, e.g., rat or mouse) embryos prepared, generated, produced, or obtained from non-human ES cells as described herein are provided. In some particular embodiments, the non-human embryo is a rodent embryo; in some embodiments, the mouse embryo; in some embodiments, the rat embryo.

In some embodiments, non-human (e.g., rodent, e.g., rat or mouse) cells or non-human (e.g., rodent, e.g., rat or mouse) tissues as described herein are provided for use in the manufacture and/or development of a medicament (e.g., ds oligonucleotide or fragment thereof) for therapy or diagnosis.

In some embodiments, non-human (e.g., rodent, e.g., rat or mouse) cells or non-human (e.g., rodent, e.g., rat or mouse) tissues as described herein are provided for use in the manufacture and/or development of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition.

In some embodiments, there is provided use of a non-human animal (e.g., a rodent, such as a rat or mouse), a non-human (e.g., a rodent, such as a rat or mouse), or a non-human (e.g., a rodent, such as a rat or mouse) cell or a non-human (e.g., a rodent, such as a rat or mouse) tissue as described herein in the manufacture and/or development of a medicament or vaccine for use in medicine, such as for use as a drug.

Kit for detecting a substance in a sample

The invention also provides a package or kit comprising one or more containers containing at least a non-human cell, a protein (single or complex (e.g., an antibody or fragment thereof)), a DNA fragment, a targeting vector, or any combination thereof, as described herein. The kit can be used in any suitable method (e.g., research methods). Optionally, associated with such one or more containers may be a notification in the form prescribed by a government agency that regulates the manufacture, use, or sale of the pharmaceutical or biological product, reflecting (a) approval of the human administration by the manufacture, use, or sale agency, (b) instructions for use, and/or (c) a contract to manage the transfer of material and/or biological product (e.g., non-human animal or non-human cells as described herein, and combinations thereof.

In some embodiments, kits are provided that comprise non-human cells, non-human tissue, immortalized cells, non-human ES cells, or non-human embryos as described herein. In some embodiments, kits are provided that comprise amino acids from non-human animals, non-human cells, non-human tissues, immortalized cells, non-human ES cells, or non-human embryos as described herein. In some embodiments, kits are provided that comprise nucleic acids (e.g., nucleic acids encoding the human HSD17B13 sequences described herein) from non-human animals, non-human cells, non-human tissues, immortalized cells, non-human ES cells, or non-human embryos as described herein. In some embodiments, kits are provided that comprise sequences (amino acid and/or nucleic acid sequences) identified from non-human animals, non-human cells, non-human tissues, immortalized cells, non-human ES cells, or non-human embryos as described herein.

In some embodiments, kits as described herein for manufacturing and/or developing a drug (e.g., an oligonucleotide) for therapy or diagnosis are provided.

In some embodiments, kits as described herein for the manufacture and/or development of a medicament (e.g., an oligonucleotide) for the treatment, prevention, or amelioration of a disease, disorder, or condition are provided.

Other features of certain embodiments will become apparent in the course of the following description of exemplary embodiments, which are given for illustration and are not intended to be limiting.

Examples

Some examples of the provided techniques (compounds (oligonucleotides, reagents, etc.), compositions, methods (methods of preparation, methods of use, methods of evaluation, etc.) are described below.

EXAMPLE 1 oligonucleotide Synthesis

Various techniques for preparing oligonucleotides and oligonucleotide compositions (stereorandom and chirally controlled) are known and may be used in accordance with the present disclosure, including, for example, those in US 9394333、US 9744183、US 9605019、US 9598458、US 9982257、US 10160969、US 10479995、US 2020/0056173、US 2018/0216107、US 2019/0127733、US 10450568、US 2019/0077817、US 2019/0249173、US 2019/0375774、WO 2018/223056、WO 2018/223073、WO 2018/223081、WO 2018/237194、WO 2019/032607、WO 2019/055951、WO 2019/075357、WO 2019/200185、WO 2019/217784、WO 2019/032612、 and/or WO 2020/191252, the methods and reagents in each of which are incorporated herein by reference.

Abbreviations:

1X reagent: TEA-3HF: TEA: H2O: DMSO = 5.0:1.8:15.5:77.7 (v/v/v/v)

ADIH: 2-azido-1, 3-dimethylimidazolium hexafluorophosphate

CMIMT: n-cyanomethylimidazolium triflate salt

CPG: hole-controllable glass

DCM: dichloromethane, CH2Cl2

DIPEA: diisopropylethylamine

DMSO: dimethyl sulfoxide

DMTr:4,4' -Dimethoxytrityl radical

GalNAc: n-acetylgalactosamine

HF: hydrogen fluoride

HATU:1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

IBN: isobutyronitrile (i-butyronitrile)

MeCN: acetonitrile

MeIm: n-methylimidazole

TCA: trichloroacetic acid

TEA: triethylamine

XH: hydrogenation Huang Yuansu

The HSD17B13 sequences (guide and passenger) are both sterically defined oligonucleotides with mixed PS/PO/PN backbones. The respective stereorandom sequences were synthesized as positive controls. The number of PS/PO/PN linkages varies from sequence to sequence. Different imides and synthesis cycles were used for the synthesis of PO, PS and PN linkages. For example, to synthesize an oligonucleotide, a defined number of Phosphodiester (PO) linkages are formed by cyanoethyl protected imide using an oxidation step, and a stereo-random Phosphorothioate (PS) linkage is formed by cyanoethyl protected imide using a sulfidation step. A defined number of chiral Phosphorothioates (PS) (Sp and Rp linkages) are formed by DPSE protected chiral imides using a vulcanization step, and a defined number of chiral Phosphoramidates (PN) (Sp and Rp) linkages are formed by PSM chiral auxiliary imides using an imidization step. All sequences carry various modifications, in particular 2' -modifications (including 2' -OMe and 2' -F modified nucleotides). The passenger strand oligonucleotide has a 5'-GalNAc modification (a three-branched GalNAc moiety) at the 5' end. To introduce GalNAc moieties at the 5' end, sequences were synthesized by coupling with C-6 amino modifiers as the final coupling cycle and conjugated with tri-branched galnacs after purification and desalting to prepare the respective conjugates. For example, WV-42597 is first synthesized and then conjugated thereto to prepare WV-42589. The synthesis process is described in detail herein.

Procedure for the synthesis of WV-42597:

the synthesis of WV-42597 was performed on an AKTA OP100 synthesizer (general medical group (GE HEALTHCARE)) using CPG support (loaded with 72. Mu. Mol/g) using a 3.5cm diameter SS column at 400. Mu. Mol scale. The process comprises five steps: detritylation, coupling, capping 1, oxidation/vulcanization/imidization and capping 2.

Detritylation step: detritylation was performed using 3% DCA in toluene and UV viewing commands at 436nm were set. After the detritylation step, the CPG support was subjected to a 2CV wash cycle using acetonitrile.

And (3) a coupling step: DPSE and PSM chiral imides (ACN or 20% IBN in ACN) were prepared at 0.2M concentration. The imide was mixed in-line with CMIMT M activator (0.5M acetonitrile solution) in a ratio of 5.83 before addition to the column. The coupling mixture was cycled for 10 minutes to maximize coupling efficiency, followed by column washing with 2CV ACN.

Cyanoethylimides (ACN or 20% ibn in ACN) were prepared at a concentration of 0.2M. The imide was mixed in-line with ETT activator (0.5M acetonitrile solution) in a ratio of 4.07 prior to addition to the column. The coupling mixture was cycled for 10 minutes to maximize coupling efficiency, followed by column washing with 2CV ACN.

And (3) end capping 1: for the stereodefining coupling, column 1CV was then treated with a cap 1 solution (acetic anhydride, lutidine, ACN) to acetylate the chiral auxiliary amine in 2 minutes. After this step, the column was washed with 1.5CV of acetonitrile. For the stereorandom coupling, the end-capping 1 step was not performed.

Vulcanization/imidization/oxidation step:

Sulfiding was carried out with 0.1M hydrogenation Huang Yuansu in pyridine/acetonitrile (1.2 eq.) for a contact time of 6 minutes followed by a 2CV washing step.

Imidization step was performed with 0.3M ADIH reagent in acetonitrile (18 eq.) for 15min followed by a 2CV wash step.

The oxidation step was carried out using 3.5eq. Oxidant (50 mM I2/pyridine-H2O (9:1, v/v)) for 2.5 minutes followed by a 2CV acetonitrile wash.

And end capping 2: the capping 2 step was performed using an inline mix (1:1) of capping A and capping B reagents followed by a 2CV ACN wash.

General procedure for C & D conditions:

after the synthesis was completed, the CPG support was finally treated with a 20% diethylamine/acetonitrile wash step, 5 column volumes/15 minutes, to remove only the cyanoethyl protecting group from the phosphate backbone, followed by an ACN wash cycle. The CPG solid support was dried and transferred to a pressure vessel.

The following cleavage and deprotection protocol is described for (WV-42597): the DPSE protecting groups on SERPINA1-1159 were removed by treating the oligo-bound support with desilylation reagent at a rate of 100 μl of desilylation reagent per μl of support. The desilylation reagent was prepared by mixing DMSO, water, TEA, TEA.3HF in a ratio of 7.33:1.47:0.7:0.5. CPG support was incubated with desilylation reagents in an incubator shaker at 27℃for 3 hours. Thereafter, concentrated aqueous ammonia was added at a rate of 200. Mu.L of concentrated aqueous ammonia per mu.mol of support to remove PSM aid, protecting group on nucleobase and oligonucleotide from CPG support. The mixture was incubated at 37℃and shaken for 24 hours. The mixture was cooled and filtered using a 0.2-0.45 micron filter and the CPG support was rinsed three times to collect all the desired material as filtrate. The filtrate containing the crude oligonucleotide was analyzed by RP-UPLC and quantified using a Nanodrop One spectrophotometer (Sesamer technologies Co. (Thermo Scientific)), yielding a yield of 110,000OD/. Mu.mol.

Purification and desalting of WV-42597: the crude WV-42597 was loaded onto a Waters AP-2 glass column (2.0 cm. Times.20 cm) equipped with Source 15Q (Si Tuo Va. Co., ltd. (Cytiva)). Purification was performed on AKTA150 Pure (general medical group) using the following buffers: (buffer A:20mM NaOH, 20% acetonitrile v/v) (buffer B:20mM NaOH, 2.5M NaCl, 20% acetonitrile v/v). Desired fractions with full length product in the range of 70% -80% were pooled together. The combined material was then desalted on a 2KD regenerated cellulose membrane, followed by lyophilization to obtain SERPINA1-1159 as a fluffy white cake for conjugation.

Synthesis of WV-42589:

GalNAc conjugation protocol:

precursor material: WV-42597.01 (.01 indicates a lot number)

Final conjugated material: WV-42589.01

Oligonucleotides/reagents	Molecular weight	Corresponding to oligonucleotides	mL
				WV-42597	10050.80	1	-
Tri-branched GalNAc acid	2006	1.8	-
				HATU	382	1.4	-
[00723]DIEA	129	10	-
				Acetonitrile		-	4

Three-branched GalNAc acid and HATU were weighed out in 50mL plastic tubes and dissolved in anhydrous acetonitrile before DIEA was added to the tubes. The resulting mixture was stirred at 37℃for 10min. Lyophilized WV-42597 was reconstituted in water in a separate tube and GalNAc mixture was added to the oligonucleotide solution and stirred at 37 ℃ for 60min. The reaction was monitored by RP-UPLC. The reaction was found to complete within 1 h. The reaction mixture was concentrated under vacuum to remove acetonitrile, and the resulting GalNAc-conjugated oligonucleotide was treated with concentrated aqueous ammonia at 37 ℃ for 2h. The formation of the final product was confirmed by mass spectrometry and RP-UPLC. The conjugated material was purified by anion exchange chromatography and desalted using Tangential Flow Filtration (TFF) to obtain the final product (target mass: 8708.54; observed mass: 8709.9).

Example 2 oligonucleotides and compositions provided are effective in knocking down HSD17B13 in vitro

Various double-stranded oligonucleotides and compositions targeting HSD17B13 were designed, constructed, characterized, and evaluated. As will be appreciated by those of skill in the art, various techniques may be utilized to evaluate the identity and/or activity of the provided oligonucleotides and compositions thereof. Some such techniques are described in this example. Those skilled in the art will appreciate that many other techniques may be readily utilized in accordance with the present disclosure. As demonstrated herein, double-stranded oligonucleotides and compositions targeting HSD17B13 may have high activity, e.g., in reducing the level of their target nucleic acids and proteins encoded thereby, among other things.

Many siRNAs were tested in vitro in human or NHP primary hepatocytes at one or a range of concentrations. Exemplary protocol for in vitro siRNA activity assay: to determine siRNA activity, specific concentrations of siRNA were delivered in either naked form or by transfection with lipofectamine (lipofectamine) to human or NHP primary hepatocytes plated in 96-well plates (10,000 cells (human) or 40,000 cells (NHP) per well). After 48 hours of treatment, total RNA was extracted using SV96 total RNA isolation kit (plagmatogen). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Siemens Feier Co., thermo Fisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle Co., bio-Rad)) according to the manufacturer's instructions. For human HSD17B13mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; probe 5'ATTTGCCGCTGTTGGC TTTCACAG-3'. For NHP HSD17B13, the following qPCR assay was used: race feeichhorns Mf02888851_m1. Human SFRS9 was used as normalization agent (forward, 5'-TGGAATATGCCCTGCGTAAA-3'; reverse, 5'-TGGTGCTTCTC TCAGGATAAAC-3', probe, 5'-TGGATGACACCAAATTCCGC TCTCA-3'. MRNA knockdown level was calculated as% remaining relative to mock-treated mRNA.

Table 2 shows the remaining% of human HSD17B13 mRNA relative to human SFRS9 control (1 nM siRNA treatment by transfection). N=2. N.d.: not determined.

TABLE 2

Table 14 shows the IC50 for knockdown of human HSD17B13 mRNA in human primary hepatocytes.

TABLE 14

Guidance	Passenger carrier	IC50(pM)	95％CI
				WV-43135	WV-44173	44.94	17.77 To 105.8
WV-42383	WV-42427	92.7	54.70 To 159.0
				WV-45041	WV-42589	22.61	10.14 To 49.82
WV-46490	WV-42589	20.85	12.62 To 34.38
				WV-47138	WV-42589	17.74	7.51 To 40.83
WV-46487	WV-42589	42.27	20.98 To 83.38
				WV-47158	WV-42589	39.69	23.27 To 67.98
WV-45042	WV-42589	26.68	14.09 To 50.38
				WV-46496	WV-42589	24.9	12.74 To 48.46
WV-47139	WV-42589	19.43	11.97 To 31.42
				WV-46493	WV-42589	18.95	10.53 To 34.62
WV-47159	WV-42589	20.27	8.63 To 48.06

Table 15 shows the IC50 for knockdown of human HSD17B13 mRNA in human primary hepatocytes.

TABLE 15

Example 3 oligonucleotides and compositions provided are active in vivo

Among other things, the present disclosure demonstrates that the provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess efficacy, human HSD17B13 transgenic mice were dosed at 3mg/kg by subcutaneous administration on day 1. On day 8 animals were euthanized by CO ₂ asphyxiation, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. The treated serum samples were kept at-70 ℃. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13 mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; probe 5'ATTTGCCGCTGTTGGCTTT CACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAA CACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3').

Table 16 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=5. N.d.: not determined.

Table 16

Example 4 oligonucleotides and compositions provided are active in vivo

Among other things, the present disclosure demonstrates that the provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess efficacy, human HSD17B13 transgenic mice were dosed at 5mg/kg by subcutaneous administration on day 1. On day 8 animals were euthanized by CO ₂ asphyxiation, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. The treated serum samples were kept at-70 ℃. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13 mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; probe 5'ATTTGCCGCTGTTGGC TTTCACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAACACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3').

Table 17 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=7. N.d.: not determined.

TABLE 17

Example 5 oligonucleotides and compositions provided are active in vivo

Among other things, the present disclosure demonstrates that the provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess efficacy, human HSD17B13 transgenic mice were dosed at the desired oligonucleotide concentration by subcutaneous administration on day 1. On day 8 animals were euthanized by CO ₂ asphyxiation, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. The treated serum samples were kept at-70 ℃. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13 mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; the probe, 5'ATTTGCCGCTGTTGGCTTTCACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAAC ACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3').

Table 18 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=5. N.d.: not determined.

TABLE 18

Example 6 oligonucleotides and compositions provided are active in vivo

Among other things, the present disclosure demonstrates that the provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess the efficacy and liver exposure of the provided oligonucleotides and compositions, HSD17B13 transgenic mice were dosed at day 1 by subcutaneous administration at 3 mg/kg. On day 8 animals were euthanized by CO ₂ asphyxiation, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. The treated serum samples were kept at-70 ℃. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; the probe, 5'ATTTGCCGCTGTTGGCTTTCACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAACACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3'). Oligonucleotide accumulation in the liver was determined by hybridization ELISA.

Table 19 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=5. N.d.: not determined.

TABLE 19

Table 20 shows the% accumulation of antisense strand in liver tissue. N=5. N.d.: not determined.

Table 20

Example 7. In vivo activity of provided oligonucleotides and compositions the present disclosure demonstrates, among other things, that provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess the efficacy and liver exposure of the provided oligonucleotides and compositions, human HSD17B13 transgenic mice were dosed at day 1 by subcutaneous administration at 3 mg/kg. Animals were euthanized by CO ₂ asphyxiation on days 8, 22, and 48, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. The treated serum samples were kept at-70 ℃. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13 mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; the probe, 5'ATTTGCCGCTGTTGGCTTTCACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAA CACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3'). Oligonucleotide accumulation in the liver was determined by hybridization ELISA.

Ago2 immunoprecipitation assay: the tissues (administered at 1 mpk) were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 200mM NaCl, 0.5% Triton X-100, 2mM EDTA, 1mg/mL heparin) containing protease inhibitors (Sigma Aldrich). Lysate concentration was measured using protein BCA kit (Pierce BCA protein assay kit or Bradford protein assay kit). anti-Ago 2 antibodies were purchased from Wako chemical company. Control mouse IgG was from eBioscience. Dai Nuozhu particles (England Inc.) were used to precipitate the antibodies. Ago 2-related siRNA and endogenous miR122 were measured by Stem-Loop RT and then analyzed TAQMAN PCR using TAQMAN MIRNA and an siRNA assay kit (Sesameiser) according to the manufacturer's methods.

Table 21 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=4 or 5.N.d.: not determined.

Table 21

Table 22 shows the% accumulation of antisense strand in liver tissue. N=4 or 5.N.d.: not determined.

Table 22

Table 23 shows the Ago2 load% relative to the antisense strand of miR-122. N=3.

Table 23

Example 8 oligonucleotides and compositions provided are active in vivo

Among other things, the present disclosure demonstrates that the provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess the efficacy and liver exposure of the provided oligonucleotides and compositions, human HSD17B13 transgenic mice were dosed at day 1 by subcutaneous administration at 3 mg/kg. Animals were euthanized by CO ₂ asphyxiation on days 15, 50, and 99, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. The treated serum samples were kept at-70 ℃. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13 mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCT GAAGTTCTGAT-3'; the probe, 5'ATTTGCCGCTGTTGGCTTTCACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAACACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3'). Oligonucleotide accumulation in the liver was determined by hybridization ELISA.

Ago2 immunoprecipitation assay: the tissues (administered at 1 mpk) were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 200mM NaCl, 0.5% Triton X-100, 2mM EDTA, 1mg/mL heparin) containing protease inhibitors (Sigma Aldrich). Lysate concentration was measured using protein BCA kit (Pierce BCA protein assay kit or Bradford protein assay kit). anti-Ago 2 antibodies were purchased from Wako chemical company. Control mouse IgG was from eBioscience. Dai Nuozhu particles (England Inc.) were used to precipitate the antibodies. Ag 02-related siRNA and endogenous miR122 were measured by Stem-Loop RT and then analyzed TAQMAN PCR using TAQMAN MIRNA and siRNA assay kit (sameiser) according to the manufacturer's method.

Table 24 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=4 or 5.N.d.: not determined.

Table 24

Table 25 shows the% accumulation of antisense strand in liver tissue. N=4 or 5.N.d.: not determined.

Table 25

Table 26 shows the Ago2 load% relative to the antisense strand of miR-122. N=2.

Table 26

Example 9 oligonucleotides and compositions provided are effective in knocking down HSD17B13 in vitro

Many siRNAs were tested in vitro in NHP primary hepatocytes at a range of concentrations. Exemplary protocol for in vitro siRNA activity assay: to determine siRNA activity, specific concentrations of siRNA were delivered in bare form to NHP primary hepatocytes plated in 96-well plates (40,000 cells per well). After 48 hours of treatment, total RNA was extracted using SV96 total RNA isolation kit (plagmatogen). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For NHP HSD17B13, the following qPCR assay was used: race feeichhorns Mf02888851_m1.SFRS9 was used as normalization agent (forward, 5'-TGGAATATGCCCTGCGTAAA-3'; reverse, 5'-TGGTGCTTCTCTCAGGATAAAC-3', probe, 5'-TGGATGACACCAAATTCCGCTCTCA-3'. MRNA knockdown level was calculated as% remaining relative to mock-treated mRNA.

Table 27 shows the IC50 for knockdown of NHP HSD17B13 mRNA in NHP primary hepatocytes.

Table 27

Example 10 oligonucleotides and compositions are provided that are effective in knocking down HSD17B13 in vitro

Many siRNAs were tested in vitro in human primary hepatocytes at a range of concentrations. Exemplary protocol for in vitro siRNA activity assay: to determine siRNA activity, specific concentrations of siRNA were delivered in bare form to human primary hepatocytes plated in 96-well plates (10,000 cells per well). After 48 hours of treatment, total RNA was extracted using SV96 total RNA isolation kit (plagmatogen). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGT TCTGAT-3'; the probe, 5'ATTTGCCGCTGTTGGCTTTCACAG-3'. SFRS9 was used as normalization agent (forward, 5'-TGGAATATGCCCTGCGTAAA-3'; reverse, 5'-TGGTGCTTCTCTCAGGATAAAC-3', probe, 5'-TGGATGACACCAAATTCCGCTCTCA-3'. MRNA knockdown level was calculated as% remaining relative to mock-treated mRNA.

Table 28 shows the IC50 for knockdown of human HSD17B13 mRNA in human primary hepatocytes.

Table 28

Example 11 oligonucleotides and compositions provided are active in vivo

Among other things, the present disclosure demonstrates that the provided oligonucleotides and compositions are active in vivo. In this example, animal procedures were all performed according to IACUC guidelines. To assess the efficacy and liver exposure of the provided oligonucleotides and compositions, human HSD17B13 transgenic mice were dosed at day 1 by subcutaneous administration at 3 or 1.5 mg/kg. Animals were euthanized by CO ₂ asphyxiation on day 90, followed by open chest surgery. After cardiac perfusion with PBS, liver samples were collected and quick frozen in dry ice. After tissue lysis with TRIzol and bromochloropropane, liver total RNA was extracted using SV96 total RNA isolation kit (prolog). cDNA was generated from RNA samples using a high-capacity cDNA reverse transcription kit (Sieimer's Feisher) and qPCR analysis was performed in a CFX system using iQ Multiplex Powermix (Berle's) according to the manufacturer's instructions. For human HSD17B13 mRNA, the following qPCR assay was used: forward direction 5'-CGAAGGGATTCCTTACCTCATC-3'; reverse 5'-CCAAGGCCTGAAGTTCTGAT-3'; the probe, 5'ATTTGCCGCTGTTGGCTTTCACAG-3'. Mouse HPRT was used as normalization agent (forward 5'-CAAACTTTGCTTTCCCTGGTT-3', reverse 5'-TGGCCTGTATCCAA CACTTC-3', probe 5'-ACCAGCAAGCTTGCAACCTTAACC-3'). Oligonucleotide accumulation in the liver was determined by hybridization ELISA.

Table 29 shows the% remaining human HSD17B13 mRNA relative to PBS control. N=4 or 5.N.d.: not determined.

Table 29

Table 30 shows the% accumulation of antisense strand in liver tissue. N=4 or 5.N.d.: not determined. Table 30

Table 31 shows the Ago2 load% relative to the antisense strand of miR-122. N=4 or 5.

Table 31

Although various embodiments have been described and illustrated herein, those of ordinary skill in the art will readily recognize a variety of other methods and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described in the present disclosure, and each of such variations and/or modifications are contemplated as being included. More generally, one of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be examples and that the actual parameters, dimensions, materials, and/or configurations may depend upon the specific application or applications for which the teachings of the present disclosure are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments of the disclosure. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the claimed technology may be practiced otherwise than as specifically described and claimed. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods is included within the scope of the present disclosure if such features, systems, articles, materials, kits, and/or methods are not mutually incompatible.

Claims

1. A double stranded RNAi (dsRNAi) agent comprising a guide strand and a passenger strand, wherein:

a) The guide strand is complementary or substantially complementary to the HSD17B13 target RNA sequence and comprises:

i. A backbone phosphorothioate chiral centre in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide,

A backbone phosphorothioate chiral center in Rp, sp or alternating configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide;

One or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide; and/or

Backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide and in one or more of the following: (a) between the (+ 3) and (+ 4) nucleotides; and (b) between the +5 and +6 nucleotides;

b) The guide strand comprises one or more Rp, sp or stereotactically non-negatively charged internucleotide linkages between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and any two adjacent nucleotides between the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide;

c) The guide strand comprises a2 'modification of the 3' nucleotide of the nucleotide pair linked by Rp, sp or a stereotactically nonnegatively charged internucleotide linkage;

d) The passenger chain comprises one or two of the following:

i.0-n Rp, sp or stereorandom nonnegatively charged internucleotide linkages, wherein n is about 1 to 49, and

One or more chiral centers of the backbone in Rp or Sp configuration,

E) Each strand of the dsRNAi agent independently has a length of about 15 to about 49 nucleotides,

F) The dsRNAi is capable of directing HSD17B13 specific RNA interference.

2. A chirally controlled oligonucleotide composition comprising double stranded oligonucleotides, wherein the guide strand and the passenger strand of the double stranded oligonucleotides are independently characterized by:

a) Common base sequence and length;

b) A common backbone linkage pattern; and

C) A common backbone chiral center pattern;

The composition is chirally controlled in that the composition is enriched for oligonucleotides having a common chiral center pattern relative to a substantially racemic preparation of guide strands having the same common base sequence and length; and

A) Wherein the guide strands are complementary or substantially complementary to the HSD17B13 target RNA sequence and comprise:

Backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide and in one or more of the following: (a) between the (+ 3) and (+ 4) nucleotides; and (b) between the +5 and +6 nucleotides; or (b)

d) These passenger chains contain one or both of the following:

One or more chiral centers of the backbone in Rp or Sp configuration,

E) The guide strand and the passenger strand have a length of about 15 to about 49 nucleotides; and

F) The guide and passenger strands are capable of directing HSD17B 13-specific RNA interference.

3. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-N Rp, sp or stereotactic non-negatively charged internucleotide linkages, wherein N is about 1 to 49.

4. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in Rp, sp or alternating configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises 0-n Rp, sp or stereotactically non-negatively charged internucleotide linkages, wherein n is about 1 to 49.

5. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-N Rp, sp or stereoscopically nonnegatively charged internucleotide linkages, where N is about 1 to 49.

6. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more Rp, sp or stereotactically non-negatively charged internucleotide linkages between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide, and the passenger strand comprises 0-N Rp, sp or stereotactically non-negatively charged internucleotide linkages, wherein N is about 1 to 49.

7. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises one or more backbone chiral centers in the Rp or Sp configuration.

8. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in Rp, sp or alternating configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration.

9. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration.

10. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration between the 5' terminus (+1) nucleotide and the immediately downstream (+2) nucleotide and between the (+2) nucleotide and the immediately downstream (+3) nucleotide and in one or both of: (a) between the (+ 3) and (+ 4) nucleotides; and (b) between the +5 and +6 nucleotides; or (b)

11. The double stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a 5' terminal modification selected from the group consisting of:

base: A. c, G, T, U, abasic and modified nucleobases;

R: H. OH, O-alkyl, F, MOE, LNA bridge to the 4 'position, BNA bridge to the 4' position.

12. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more Rp, sp or stereotactically non-negatively charged internucleotide linkages between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide, and the passenger strand comprises one or more backbone chiral centers in Rp or Sp configuration.

13. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in the Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-N Rp, sp or stereotactically non-negatively charged internucleotide linkages, wherein N is about 1 to 49, and one or more backbone chiral centers in the Rp or Sp configuration.

14. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises a backbone phosphorothioate chiral center in Rp, sp or alternating configuration between the 5' terminal (+1) nucleotide and the immediately downstream (+2) nucleotide and between the +2 nucleotide and the immediately downstream (+3) nucleotide, and the passenger strand comprises 0-n Rp, sp or stereotactically non-negatively charged internucleotide linkages, wherein n is about 1 to 49, and one or more backbone chiral centers in Rp or Sp configuration.

15. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more backbone phosphorothioate chiral centers in Rp or Sp configuration upstream of the backbone phosphorothioate chiral center in Sp configuration between the 3' terminal nucleotide and the penultimate (N-1) nucleotide and between the penultimate (N-1) nucleotide and the immediately upstream (N-2) nucleotide, and the passenger strand comprises 0-N Rp, sp or stereotactic nonnegatively charged internucleotide linkages, wherein N is about 1 to 49, and one or more backbone chiral centers in Rp or Sp configuration.

16. The double-stranded oligonucleotide of claim 1 or the composition of claim 2, wherein the guide strand comprises one or more Rp, sp or stereotactic non-negatively charged internucleotide linkages between any two adjacent nucleotides between the second (+2) nucleotide of the guide strand relative to the 5' terminal nucleotide and the penultimate 3' (N-1) nucleotide of the guide strand, wherein N is the 3' terminal nucleotide, and the passenger strand comprises 0-N non-negatively charged internucleotide linkages, wherein N is about 1 to 49, and one or more backbone chiral centers in Rp or Sp configuration.

17. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the Rp, sp or stereotactically non-negatively charged backbone internucleotide linkages are neutral-charged.

18. The double-stranded oligonucleotide or composition of claim 17, wherein the neutral backbone internucleotide linkage is

19. The double-stranded oligonucleotide or composition of claim 18, wherein the guide strand is between the third (+3) and fourth (+4) nucleotides of the guide strand, between the tenth (+10) and eleventh (+11) nucleotides of the guide strand, or both comprise a structure havingIs a bond of (a).

20. The double-stranded oligonucleotide or composition of claim 19, wherein the passenger strand is 5 'of the central nucleotide of the passenger strand, 3' of the central nucleotide of the passenger strand, or both comprise a structure havingIs a bond of (a).

21. The composition of claim 2, wherein the guide and passenger strands in the composition that independently share a common base sequence, a common base modification pattern, a common sugar modification pattern, and/or a common internucleotide linkage pattern are at least 90% of all guide and passenger strands in the composition.

22. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the double-stranded oligonucleotide comprises a carbohydrate moiety at a nucleoside, internucleotide linkage optionally via a linker.

23. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the double-stranded oligonucleotide comprises a lipid moiety attached to the double-stranded oligonucleotide at a nucleoside, internucleotide linkage, optionally via a linker.

24. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein one or both strands of the double-stranded oligonucleotide comprise a target moiety at a nucleoside, optionally linked via a linker, at an internucleotide linkage.

25. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the internucleotide linkages of the double-stranded oligonucleotide are independently chiral internucleotide linkages.

26. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% of the nucleotide units of the double-stranded oligonucleotide independently comprise a 2' -substitution.

27. The double stranded oligonucleotide or composition of any one of the preceding claims, wherein the 2 '-substitution of the oligonucleotide is 2' -F.

28. The double stranded oligonucleotide OR composition of any one of the preceding claims, wherein the 2 '-substitution of the oligonucleotide is 2' -OR1.

29. The double stranded oligonucleotide or composition of any one of the preceding claims, wherein the 2' -substitution of the oligonucleotide is-L-, wherein L connects C2 and C4 of the sugar unit.

30. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein at least 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 97% of the nucleotide units of the double-stranded oligonucleotide do not comprise a 2' -substitution.

31. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the guide strand comprises an HSD17B13 target binding sequence that is fully complementary to an HSD17B13 target sequence, wherein the HSD17B13 target binding sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases in length, wherein each base is an optionally substituted adenine, cytosine, guanosine, thymine or uracil, and wherein the HSD17B13 target sequence comprises one or more allelic loci, wherein an allelic locus is a SNP or mutation.

32. The double-stranded oligonucleotide or composition of any one of the preceding claims, wherein the HSD17B13 target sequence comprises two SNPs.

33. The double stranded oligonucleotide or composition of any one of the preceding claims, wherein the HSD17B13 target sequence comprises an allelic site and the HSD17B13 target binding sequence is fully complementary to the HSD17B13 target sequence of a disease-associated allele but not fully complementary to the HSD17B13 target sequence of a less disease-associated allele.

34. The double stranded oligonucleotide or composition of any one of the preceding claims, wherein

The double stranded oligonucleotide comprises a guide strand that binds to a transcript of an HSD17B13 target nucleic acid sequence for which there are multiple alleles in a population, each of the multiple alleles containing a specific nucleotide signature sequence element defining an allele relative to other alleles of the same HSD17B13 target nucleic acid sequence,

Wherein the base sequence of the guide strand is or comprises a sequence complementary to a characteristic sequence element defining the target allele, and

The guide strand is characterized in that it exhibits a level of inhibition of the transcript of the target allele or the protein encoded thereby that is higher than the level of inhibition observed for another allele of the same nucleic acid sequence when it is contacted with a cell comprising the transcript of the HSD17B13 target nucleic acid sequence.

35. The double stranded oligonucleotide of claim 1 or the composition of claim 2 comprising:

A guide chain comprising WV-47139 and a passenger chain comprising WV-42589;

a guide chain comprising WV-47159 and a passenger chain comprising WV-42589;

a guide chain comprising WV-49590 and a passenger chain comprising WV-42589; or (b)

A guide chain containing WV-49591 and a passenger chain containing WV-42589.

36. A method for reducing the level and/or activity of an HSD17B13 transcript or a protein encoded thereby, the method comprising administering to a cell expressing the HSD17B13 transcript a double stranded oligonucleotide or composition according to any one of the preceding claims, wherein the guide strand of the double stranded oligonucleotide or composition comprises an HSD17B13 binding sequence that is fully complementary to an HSD17B13 target sequence in the transcript.

37. The method of claim 36, wherein the cell is an immune cell, a blood cell, a cardiomyocyte, a lung cell, an optic cell, a muscle cell, a liver cell, a kidney cell, a brain cell, a central nervous system cell, or a peripheral nervous system cell.

38. A method for allele-specific suppression of HSD17B13 transcripts from a nucleic acid sequence for which a plurality of alleles are present in a population, each of the plurality of alleles containing a specific nucleotide signature sequence element defining an allele relative to other alleles of the same HSD17B13 target nucleic acid sequence, the method comprising the steps of:

contacting a sample comprising transcripts of the HSD17B13 target nucleic acid sequence with a double stranded oligonucleotide or composition as claimed in any one of the preceding claims,

Wherein the guide strand of the double-stranded oligonucleotide or composition comprises an HSD17B13 binding sequence that is identical or fully complementary to an HSD17B13 target sequence in the nucleic acid sequence, the HSD17B13 target sequence comprising a characteristic sequence element defining a target allele, and

Wherein when the guide strand of the double stranded oligonucleotide or composition is contacted with a cell comprising transcripts of both the target allele and another allele of the same nucleic acid sequence, the level of inhibition of transcripts of the target allele is greater than that observed for the other allele of the same nucleic acid sequence.

39. A method for allele-specific suppression of HSD17B13 transcripts from a nucleic acid sequence for which a plurality of alleles are present in a population, each of the plurality of alleles containing a specific nucleotide signature sequence element defining an allele relative to other alleles of the same HSD17B13 target nucleic acid sequence, the method comprising the steps of:

the method of claim, wherein the double stranded oligonucleotide or composition of any one of the preceding claims is administered to a subject comprising transcripts of the HSD17B13 target nucleic acid sequence,

Wherein when the guide strand of the double stranded oligonucleotide or composition is contacted with a cell comprising transcripts of both the HSD17B13 target allele and the other allele of the same nucleic acid sequence, the level of inhibition of transcripts of the target allele is greater than that observed for the other allele of the same nucleic acid sequence.

40. The method of any one of claims 36-39, wherein the oligonucleotide or the oligonucleotide of the composition exhibits a level of inhibition of a transcript of the target allele of HSD17B13 when contacted with a cell comprising transcripts of both the target allele and another allele of the same nucleic acid sequence:

a) Greater than the level of inhibition in the absence of the composition;

b) Greater than the level of inhibition observed for another allele of the same nucleic acid sequence; or (b)

C) Greater than both the level of inhibition in the absence of the composition and the level of inhibition observed for another allele of the same nucleic acid sequence.

41. The method of claim 40, wherein the cell is an immune cell, a blood cell, a myocardial cell, a lung cell, an optic cell, a muscle cell, a liver cell, a kidney cell, a brain cell, a central nervous system cell, or a peripheral nervous system cell.

42. The method of any one of claims 36-39, wherein the level of inhibition of HSD17B13 transcript of the target allele is greater than both the level of inhibition in the absence of the composition and the level of inhibition observed for another allele of the same nucleic acid sequence.

43. A non-human animal engineered to comprise an HSD17B13 polypeptide or a characteristic portion thereof.

44. A non-human animal engineered to comprise and/or express a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof.

45. The animal of claim 43 or 44, wherein the genome of the animal comprises a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof.

46. The animal of any one of claims 43-45, wherein the germline genome of the animal comprises a polynucleotide whose sequence encodes an HSD17B13 polypeptide or a characteristic portion thereof.

47. The animal of any one of claims 43-46, wherein the animal is a rodent.

48. The rodent of claim 47, wherein the rodent is a mouse.

49. The rodent of claim 47, wherein the rodent is a rat.