Nucleotide-based enhancers for RNA delivery and therapy

Abstract

The present invention provides a nucleotide-based enhancer that is a compound or a pharmaceutically acceptable salt thereof, comprising 2 to 30 nucleotides, each nucleotide independently represented by formula (I) or (II): JPEG2025541956000094.jpg92170, pharmaceutically acceptable salts thereof, and related conjugates. The present disclosure also relates to the use of the nucleotide-based enhancers and conjugates, for example, in delivering nucleic acids and / or treating or preventing disease.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Patent Application No. 63 / 421,799, filed November 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference The contents of the electronic sequence listing (SANB_011_001WO_SeqList_ST26.xml, size: 7,063 bytes, created date: October 31, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Efficient delivery of genetic material, such as RNA, into cells in vivo requires specific targeting and protection from the extracellular environment, particularly serum proteins. One way to achieve specific targeting is to conjugate a targeting moiety to a nucleic acid (e.g., an oligonucleotide). The targeting moiety serves to guide the nucleic acid to the desired site. Targeting moieties can improve delivery by receptor-mediated endocytosis. This process is initiated by activation of a cell surface or membrane receptor after binding of a specific ligand to the receptor. Many receptor-mediated endocytosis systems are known, including those that recognize sugars such as galactose, mannose, and mannose-6-phosphate, peptides, and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin, and epidermal growth factor (EGF). The asialoglycoprotein receptor (ASGP-R) is a high-capacity receptor highly abundant on hepatocytes. ASGP-R exhibits a higher affinity for N-acetyl-D-galactosylamine (GalNAc) than for D-Gal. Similarly, the glucagon-like peptide-1 receptor (GLP-1 receptor) is present in pancreatic beta cells and can be targeted by appropriate ligands to direct delivery. Recently, specific carbohydrate conjugates have been shown to be valuable alternatives to liposomes for nucleic acid delivery. Furthermore, after successful delivery into cells, the stability of nucleic acids in the cellular environment is crucial to achieving the desired therapeutic effect.

[0004] Thus, there remains a need for novel modifications to enhance delivery of nucleic acids. The present disclosure addresses this need. Summary of the Invention

[0005] In some aspects, the disclosure provides a nucleotide-based enhancer that is a compound or a pharmaceutically acceptable salt thereof, comprising 2 to 30 nucleotides, each nucleotide independently represented by Formula (I) or (II): [ka] And, wherein each * independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer, or represents H when the nucleotide is at the 5' terminus of the nucleotide-based enhancer; each ** independently represents an attachment of the nucleotide-based enhancer to the 5' position of another nucleotide, or represents H if the nucleotide is at the 2' or 3' terminus of the nucleotide-based enhancer; B is H, C1-C6 alkyl, or a nucleobase moiety; V is -O-, -NR V - or -C(R V )2-, Each R V are independently H or C1-C6 alkyl; X is H, halogen, or -OR X That is, R X is H, C1-C 12 Alkyl, or -(C1-C6 alkyl)-(C6-C 10 aryl), and the C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) optionally includes one or more R Xa or replaced by R X and R 4 together form a C1-C6 alkylene, Each R Xa are independently halogen, C1-C6 alkyl, or —O—(C1-C6 alkyl), wherein said C1-C6 alkyl or —O—(C1-C6 alkyl) is optionally substituted with one or more halogens; Y is -P(R Y )-, -P(OR Y )-, -P(N(R Y )2)-, -P(=O)(OR Y )-, -P(=O)(R Y )-, -P(=S)(OR Y )-, -P(=S)(R Y )-, -P(=O)(SR Y )-, or -P(=S)(SR Y )-is, Each RY are independently H or C1-C6 alkyl optionally substituted with one or more halogens or cyano; R 1 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens, or R 4 and R X together form a C1-C6 alkylene, and Each R 5 are independently H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens.

[0006] In some aspects, the present disclosure provides: (i) one or more nucleic acid agents; (ii) one or more ligands, and (iii) one or more nucleotide-based enhancer units, each nucleotide-based enhancer unit independently comprising 2 to 30 nucleotides, each nucleotide independently comprising [ka] A complex or a pharmaceutically acceptable salt thereof, comprising a nucleotide-based enhancer unit represented by Variables B, V, X, Y, R 1 , R 2 , R 3 , R 4 , and R 5 is described herein, wherein each # independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 5' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the complex; and provides a conjugate or a pharmaceutically acceptable salt thereof, wherein each ## independently represents a bond to the 5' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the conjugate.

[0007] In some aspects, the present disclosure provides isotopic derivatives of the nucleotide-based enhancers described herein.

[0008] In some aspects, the present disclosure provides pharmaceutical compositions comprising the nucleotide-based enhancers or conjugates described herein.

[0009] In some aspects, the present disclosure provides a method of modulating expression of a target gene in a subject, comprising administering to the subject a conjugate described herein.

[0010] In some aspects, the present disclosure provides a method of delivering a nucleic acid agent to a subject, comprising administering to the subject a complex described herein.

[0011] In some aspects, the present disclosure provides a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein.

[0012] In some aspects, the present disclosure provides a conjugate as described herein for modulating expression of a target gene in a subject.

[0013] In some aspects, the present disclosure provides a conjugate as described herein for delivering a nucleic acid agent to a subject.

[0014] In some aspects, the present disclosure provides a conjugate as described herein for treating or preventing a disease in a subject in need thereof.

[0015] In some aspects, the present disclosure provides for the use of a conjugate described herein in the manufacture of a medicament for modulating expression of a target gene in a subject.

[0016] In some aspects, the disclosure provides for the use of a conjugate described herein in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0017] In some aspects, the disclosure provides for the use of a conjugate described herein in the manufacture of a medicament for treating or preventing a disease in a subject in need thereof.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular also encompasses the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. References cited herein are not admitted to be prior art to the claimed invention(s). In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not limiting. In the event of a conflict between the chemical structure and the name of a compound disclosed herein, the chemical structure will control.

[0019] Other features and advantages of the present disclosure will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0020] [Figure 1]FIG. 1 is a graph illustrating various exemplary configurations of the conjugates described herein.

[0021] [Figure 2] 2 is a graph showing the chemical structures of exemplary complexes of duplexes 1 to 5.

[0022] [Figure 3] FIG. 3 is a graph showing the in vivo KD activity of duplexes 1 to 5 in tissues of interest.

[0023] [Figure 4] Figure 4 is an image of RNASCOPE-based mRNA silencing assessment of duplex 15 in tissues of interest.

[0024] [Figure 5] Figure 5 shows miRNASCOPE-based imaging of siRNA molecules accumulated in tissues of interest. DETAILED DESCRIPTION OF THE INVENTION

[0025] In order to efficiently deliver genetic material such as RNA to cells in vivo, it requires specific targeting and protection from extracellular environment, especially serum protein.One way to achieve specific targeting is to conjugate targeting moiety with nucleic acid (for example, oligonucleotide).Targeting moiety can be useful for guiding nucleic acid to desired site.

[0026] It is known that to achieve potent gene knockdown in specific tissues or cell types, oligonucleotides must circumvent several extracellular and intracellular barriers. Chemical modification and conjugation with targeting ligands can improve the pharmacological properties of oligonucleotides and potentially enhance tissue-specific delivery. However, despite the testing of various conjugates with peptides, antibodies, small molecules, carbohydrates, and adapters, oligonucleotides conjugated to a single targeting ligand have not been able to achieve clinically relevant knockdown activity and specificity. These suboptimal delivery approaches may be due to poor pharmacokinetic and ADME profiles of the conjugates, lack of efficient tissue accumulation and cellular uptake, and inefficient intracellular sorting and endosomal escape.

[0027] The present disclosure provides nucleotide-based enhancers that can improve targeted delivery efficiency and enhance cellular activity of nucleic acids, for example, by improving PK, stability, cellular uptake and transport, or other mechanisms. The present disclosure also provides complexes containing the nucleotide-based enhancers. The present disclosure also relates to the use of the nucleotide-based enhancers and complexes, for example, in delivering nucleic acids and / or treating or preventing diseases.

[0028] In some embodiments, the complexes comprising the nucleotide-based enhancer have a higher targeted delivery efficiency compared to complexes not comprising the nucleotide-based enhancer.

[0029] In some aspects, the present disclosure provides a general-purpose delivery-enhancing agent that can further improve the delivery efficiency, specificity, and knockdown activity of oligonucleotides in the presence or absence of a targeting ligand. The delivery-enhancing agent can increase the binding affinity of the complex to serum proteins, enhance nuclease resistance, and improve pharmacokinetic properties. The delivery-enhancing agent can also promote the passage of oligonucleotides through the capillary endothelium into the tissue interstitium. The delivery-enhancing agent can improve the binding of the oligonucleotide complex to the target receptor, thereby improving receptor-mediated endocytosis. The delivery-enhancing agent can also promote intracellular transport and endosomal escape of the oligonucleotide, improving intracellular activity. The delivery-enhancing agent can also improve oligonucleotide delivery and gene knockdown activity via other mechanisms or pathways.

[0030] Nucleotide enhancers In some aspects, the disclosure provides a nucleotide-based enhancer that is a compound, or a pharmaceutically acceptable salt thereof, comprising 2 to 30 nucleotides, each nucleotide independently having the formula (I) or (II): [ka] is expressed as wherein each * independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer, or represents H when the nucleotide is at the 5' terminus of the nucleotide-based enhancer; each ** independently represents an attachment of the nucleotide-based enhancer to the 5' position of another nucleotide, or represents H if the nucleotide is at the 2' or 3' terminus of the nucleotide-based enhancer; B is H, C1-C6 alkyl, or a nucleobase moiety; V is -O-, -NR V - or -C(R V )2-, Each R V are independently H or C1-C6 alkyl; X is H, halogen, or -OR X That is, R X is H, C1-C 12 Alkyl, or -(C1-C6 alkyl)-(C6-C 10 aryl), and the C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) optionally includes one or more R Xa or replaced by R X and R 4 together form a C1-C6 alkylene, Each R Xa are independently halogen, C1-C6 alkyl, or —O—(C1-C6 alkyl), wherein said C1-C6 alkyl or —O—(C1-C6 alkyl) is optionally substituted with one or more halogens; Y is -P(R Y )-, -P(OR Y )-, -P(N(R Y )2)-, -P(=O)(OR Y )-, -P(=O)(R Y )-, -P(=S)(OR Y )-, -P(=S)(R Y )-, -P(=O)(SR Y )-, or -P(=S)(SR Y )-is, Each R Y are independently H or C1-C6 alkyl optionally substituted with one or more halogens or cyano; R 1 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens, or R 4 and R Xtogether form a C1-C6 alkylene, and Each R 5 are independently H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens.

[0031] In some embodiments, the nucleotide-based enhancer comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides.

[0032] In some embodiments, the nucleotide-based enhancer comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides.

[0033] In some embodiments, the nucleotide-based enhancer comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.

[0034] In some embodiments, the nucleotide-based enhancer comprises 5 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 6 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 7 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 8 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 9 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 10 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 11 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 12 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 13 nucleotides.

[0035] In some embodiments, the nucleotide-based enhancer comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides.

[0036] In some embodiments, the nucleotide-based enhancer comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides.

[0037] In some embodiments, the nucleotide-based enhancer comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, at least two nucleotides of the nucleotide-based enhancer are linked via a phosphodiester linker or a phosphorothioate linker. In some embodiments, at least two nucleotides of the nucleotide-based enhancer are linked via a phosphorothioate linker. In some embodiments, two adjacent nucleotides of the nucleotide-based enhancer are each linked via a phosphodiester linker or a phosphorothioate linker.

[0038] In some embodiments, the nucleotide-based enhancer comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0039] In some embodiments, the nucleotide-based enhancer comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0040] In some embodiments, the nucleotide-based enhancer comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0041] In some embodiments, the nucleotide-based enhancer comprises five nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises six nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises seven nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises eight nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises nine nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 10 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 11 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 12 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 13 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0042] In some embodiments, the nucleotide-based enhancer comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0043] In some embodiments, the nucleotide-based enhancer comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0044] In some embodiments, the nucleotide-based enhancer comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0045] In some embodiments, the nucleotide-based enhancer comprises 4-30 nucleotides, 6-30 nucleotides, 8-30 nucleotides, 10-30 nucleotides, 12-30 nucleotides, 14-30 nucleotides, 16-30 nucleotides, 18-30 nucleotides, 20-30 nucleotides, 21-30 nucleotides, 22-30 nucleotides, 23-30 nucleotides, 24-30 nucleotides, 25-30 nucleotides, 26-30 nucleotides, 27-30 nucleotides, 28-30 nucleotides, or 29-30 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0046] In some embodiments, the nucleotide-based enhancer comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0047] In some embodiments, the nucleotide-based enhancer comprises a 5 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises a 6 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises a 7 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises an 8 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises a 9 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises a 10 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises an 11 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises a 12 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 13 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0048] In some embodiments, the nucleotide-based enhancer comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0049] In some embodiments, the nucleotide-based enhancer comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0050] In some embodiments, the nucleotide-based enhancer comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0051] In some embodiments, the nucleotide-based enhancer comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0052] In some embodiments, the nucleotide-based enhancer comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0053] In some embodiments, the nucleotide-based enhancer comprises a 5 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises a 6 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises a 7 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises an 8 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises a 9 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises a 10 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises an 11 nucleotide, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 12 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 13 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0054] In some embodiments, the nucleotide-based enhancer comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0055] In some embodiments, the nucleotide-based enhancer comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides, and the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0056] In the compounds of the present disclosure (e.g., nucleotide-based enhancers or conjugates), the variables B, V, R V , X, R X , R Xa , Y, R Y , R 1 , R 2 , R 3 , R 4 , and R 5 may each be selected from the groups described herein, where applicable, and the variables B, V, R V , X, R X , R Xa , Y, R Y , R 1 , R 2 , R 3 , R 4 , and R 5 Any group described herein for any of the variables B, V, R, V , X, R X , R Xa , Y, R Y , R 1 , R 2 , R 3 , R 4 , and R 5 It is understood that one or more of the remainders of may be combined with any group described herein.

[0057] Variable B In some embodiments, B is H.

[0058] In some embodiments, B is C1-C6 alkyl (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0059] In some embodiments, B is methyl, ethyl, or propyl.

[0060] In some embodiments, B is a nucleobase moiety.

[0061] The term "nucleobase moiety" as used herein refers to a nucleobase that is attached to the remainder of a compound, for example, via an atom of the nucleobase or a functional group thereof.

[0062] In some embodiments, the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0063] In some embodiments, the nucleobase moiety is [ka] where: [ka] indicates the point of attachment to the remainder of the nucleotide-based enhancer.

[0064] In some embodiments, the nucleobase moiety is a modified nucleobase.

[0065] In some embodiments, the modified nucleobase is 5-methylcytosine.

[0066] In some embodiments, the modified nucleobase is hypoxanthine, xanthine, or 7-methylguanine.

[0067] In some embodiments, the modified nucleobase is 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethylcytosine.

[0068] In some embodiments, the nucleobase moiety is an artificial nucleobase.

[0069] In some embodiments, the artificial nucleobase is isoguanine, isocytosine, 2-amino-6-(2-thienyl)purine, or pyrrole-2-carbaldehyde.

[0070] Variables V and R V In some embodiments, V is —O—.

[0071] In some embodiments, V is —NR V -It is.

[0072] In some embodiments, V is —NH—.

[0073] In some embodiments, V is —C(R V )2-.

[0074] In some embodiments, V is —CH 2 —.

[0075] In some embodiments, at least one R V is H.

[0076] In some embodiments, each R V is H.

[0077] In some embodiments, at least one R V is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0078] In some embodiments, each R Vis C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0079] Variables X and R X , R Xa , Y, and R Y In some embodiments, X is H.

[0080] In some embodiments, X is a halogen (eg, F, Cl, Br, or I).

[0081] In some embodiments, X is F or Cl.

[0082] In some embodiments, X is F.

[0083] In some embodiments, X is -OR X is.

[0084] In some embodiments, X is —OH.

[0085] In some embodiments, X is —O—(C 12 alkyl).

[0086] In some embodiments, X is -O-(C1-C6 alkyl) (e.g., wherein said C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0087] In some embodiments, X is —OCH 3 .

[0088] In some embodiments, X is —O—(C1-C6 alkyl)-O—(C1-C6 alkyl) (e.g., wherein the C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0089] In some embodiments, X is —OCH 2 CH 2 OCH 3 .

[0090] In some embodiments, X optionally represents one or more R Xa -O-(C1-C6 alkyl)-(C6-C 10 aryl).

[0091] In some embodiments, X is —O—(C1-C6 alkyl)-(C6-C 10 aryl).

[0092] In some embodiments, X is [ka] where: [ka] indicates the point of attachment to the remainder of the nucleotide-based enhancer.

[0093] In some embodiments, X optionally represents one or more R Xa is replaced by [ka] where: [ka] indicates the point of attachment to the remainder of the nucleotide-based enhancer.

[0094] In some embodiments, X is optionally substituted with one or more halogens. [ka] where: [ka] indicates the point of attachment to the remainder of the nucleotide-based enhancer.

[0095] In some embodiments, X is optionally substituted with one or more C1-C6 alkyl or -O-(C1-C6 alkyl). [ka] wherein said C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens; [ka] indicates the point of attachment to the remainder of the nucleotide-based enhancer.

[0096] In some embodiments, R X is H.

[0097] In some embodiments, R X optionally one or more R Xa C1-C substituted with 12 It is alkyl.

[0098] In some embodiments, R X is C1-C 12 It is alkyl.

[0099] In some embodiments, R X optionally one or more R Xa and C1-C6 alkyl substituted with (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0100] In some embodiments, R Xis C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I), or —O—(C1-C6 alkyl) optionally substituted with one or more halogens (e.g., said C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0101] In some embodiments, R X is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0102] In some embodiments, R X is methyl, ethyl, or propyl.

[0103] In some embodiments, R X is methyl.

[0104] In some embodiments, R X is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0105] In some embodiments, R X is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more -O-(C1-C6 alkyl) (e.g., said C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), wherein said -O-(C1-C6 alkyl) is optionally substituted with one or more halogens.

[0106] In some embodiments, R X optionally one or more R Xa -(C1-C6 alkyl)-(C6-C 10 aryl).

[0107] In some embodiments, R X is optionally substituted with one or more halogens (e.g., F, Cl, Br, or I), C-C alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), -(C-C alkyl)-(C-C 10 aryl) or -O-(C1-C6 alkyl) (e.g., said C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), wherein said C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens.

[0108] In some embodiments, R X -(C1-C6 alkyl)-(C6-C 10 aryl).

[0109] In some embodiments, R X and R 4 together form a C1-C6 alkylene (e.g., methylene, ethylene, propylene, butylene, pentylene, or hexylene).

[0110] In some embodiments, R X and R 4 together form methylene, ethylene, or propylene.

[0111] In some embodiments, R X and R 4 together form methylene.

[0112] In some embodiments, R X and R 4 together to form ethylene.

[0113] In some embodiments, R X and R 4 together to form propylene.

[0114] In some embodiments, Y is -P(R Y )-.

[0115] In some embodiments, Y is -PH-.

[0116] In some embodiments, Y is -P(OR Y )-.

[0117] In some embodiments, Y is —P(OH)—.

[0118] In some embodiments, Y is -P(N(R Y )2)-.

[0119] In some embodiments, Y is —P(NH 2 )—.

[0120] In some embodiments, Y is -P(=O)(OR Y )-.

[0121] In some embodiments, Y is —P(═O)(OH)—.

[0122] In some embodiments, Y is —P(═O)(R Y )-.

[0123] In some embodiments, Y is -P(=O)H-.

[0124] In some embodiments, Y is -P(=S)(OR Y )-.

[0125] In some embodiments, Y is —P(═S)(OH)—.

[0126] In some embodiments, Y is -P(=S)(R Y )-.

[0127] In some embodiments, Y is -P(=S)H-.

[0128] In some embodiments, Y is -P(=O)(SR Y )-.

[0129] In some embodiments, Y is -P(=O)(SH)-.

[0130] In some embodiments, Y is -P(=S)(SR Y )-.

[0131] In some embodiments, Y is -P(=S)(SH)-.

[0132] In some embodiments, at least one R Y is H.

[0133] In some embodiments, each R Y is H.

[0134] In some embodiments, at least one R Y is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0135] In some embodiments, each R Yis C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0136] In some embodiments, at least one R Y is H and at least one R Y is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen or cyano.

[0137] Variable R 1 , R 2 , R 3 , R 4 , and R 5 In some embodiments, R 1 is H.

[0138] In some embodiments, R 1 is a halogen (e.g., F, Cl, Br, or I).

[0139] In some embodiments, R 1 is F or Cl.

[0140] In some embodiments, R 1 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0141] In some embodiments, R 1 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0142] In some embodiments, R 1 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0143] In some embodiments, R 2 is H.

[0144] In some embodiments, R 2 is a halogen (e.g., F, Cl, Br, or I).

[0145] In some embodiments, R 2 is F or Cl.

[0146] In some embodiments, R 2 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0147] In some embodiments, R 2 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0148] In some embodiments, R 2 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0149] In some embodiments, R 3 is H.

[0150] In some embodiments, R 3 is a halogen (e.g., F, Cl, Br, or I).

[0151] In some embodiments, R 3 is F or Cl.

[0152] In some embodiments, R 3 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0153] In some embodiments, R 3 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0154] In some embodiments, R 3 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0155] In some embodiments, R 4 is H.

[0156] In some embodiments, R 4 is a halogen (e.g., F, Cl, Br, or I).

[0157] In some embodiments, R 4 is F or Cl.

[0158] In some embodiments, R 4is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0159] In some embodiments, R 4 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0160] In some embodiments, R 4 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0161] In some embodiments, R 4 and R X together form a C1-C6 alkylene (e.g., methylene, ethylene, propylene, butylene, pentylene, or hexylene).

[0162] In some embodiments, R 4 and R X together form methylene, ethylene, or propylene.

[0163] In some embodiments, R 4 and R X together form methylene.

[0164] In some embodiments, R 4 and R X together to form ethylene.

[0165] In some embodiments, R 4 and R X together to form propylene.

[0166] In some embodiments, each R 5 is H.

[0167] In some embodiments, at least one R 5 is halogen (e.g., F, Cl, Br, or I) or C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0168] In some embodiments, at least one R 5 is a halogen (e.g., F, Cl, Br, or I).

[0169] In some embodiments, at least one R 5 is F or Cl.

[0170] In some embodiments, at least one R 5 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0171] In some embodiments, at least one R 5 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0172] In some embodiments, at least one R 5 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0173] In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 are H respectively.

[0174] Exemplary Embodiments of Nucleotides of Nucleotide-Based Enhancers In some embodiments, at least one nucleotide of the nucleotide-based enhancer has Formula (I') or (II'): [ka] It is expressed as:

[0175] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (IA), (II-A), (III-A), or (IV-A): [ka] It is expressed as:

[0176] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I'-A), (II'-A), (III'-A), or (IV'-A): [ka] It is expressed as:

[0177] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (IB) or (II-B): [ka] It is expressed as:

[0178] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I'-B) or (II'-B): [ka] It is expressed as:

[0179] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (IC), (II-C), (III-C), or (IV-C): [ka] It is expressed as:

[0180] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I'-C), (II'-C), (III'-C), or (IV'-C): [ka] It is expressed as:

[0181] In some embodiments, at least one nucleotide of the nucleotide-based enhancer is selected from the nucleotides listed in Table L. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0182] In some aspects, the present disclosure provides isotopic derivatives (eg, isotopically labeled compounds) of the nucleotide-based enhancers disclosed herein.

[0183] It will be appreciated that such isotopic derivatives can be prepared using any of a variety of art-recognized techniques, for example, such isotopic derivatives can be prepared generally by following the procedures disclosed in the schemes and / or examples herein, substituting isotopically labeled reagents for non-isotopically labeled reagents.

[0184] In some embodiments, the isotope derivative is a deuterium-labeled derivative.

[0185] As used herein, the term "isotopic derivative" refers to a derivative of a chemical structure in which one or more atoms are isotopically enriched or labeled. For example, an isotopic derivative of a drug is isotopically enriched with or labeled with one or more isotopes compared to the corresponding drug. In some embodiments, an isotopic derivative is 2 H, 13 C. 14 C. 15 N, 18 O.29 Si, 32 P, and 34 In some embodiments, the isotopic derivatives are deuterium-labeled derivatives (i.e., enriched with or labeled at one or more atoms selected from S and S). 2 In some embodiments, the derivative is 2 In some embodiments, the derivative is 13 C-labeled derivatives or 14 In some embodiments, the derivative is a C-labeled derivative. 18 In some embodiments, the derivative is 123 I-labeled derivative, 124 I-labeled derivative, 125 I-labeled derivative, 129 I-labeled derivative, 131 I-labeled derivative, 135 I-labeled derivatives, or any combination thereof. In some embodiments, the derivatives are 32 P-labeled derivatives or 32 In some embodiments, the derivative is 33 S-labeled derivative, 34 S-labeled derivative, 35 S-labeled derivative, 36 S-labeled derivatives, or any combination thereof.

[0186] It has also been found that increased metabolic stability through isotopic substitution may confer certain therapeutic advantages, such as increased in vivo half-life or reduced dosage requirements.

[0187] For the avoidance of doubt, when a group is qualified herein by the modifier "as described herein," the group is to be understood to include the broadest definition appearing first and all of the individual definitions of that group.

[0188] It will be understood that the compounds disclosed herein may be presented in one particular configuration. Such a particular configuration should not be construed as limiting the disclosure to one or another isomer, tautomer, regioisomer, or stereoisomer, nor does it exclude mixtures of isomers, tautomers, regioisomers, or stereoisomers. In some embodiments, the presentation of a compound herein in a particular configuration is intended to encompass and refer to each of the available isomers, tautomers, regioisomers, and stereoisomers of that compound, or any mixtures thereof, and the presentation is intended to refer to a particular configuration of the compound.

[0189] It will be understood that the compounds disclosed herein may be presented without a specific configuration (e.g., without a specific stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, regioisomers, and stereoisomers of the compound. In some embodiments, presentation of a compound herein without a specific configuration is intended to refer to each of the available isomers, tautomers, regioisomers, and stereoisomers of the compound, or any mixture thereof.

[0190] As used herein, the term "isomers" refers to compounds that have the same molecular formula but differ in the bonding sequence of their atoms or the spatial arrangement of their atoms. Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space are called "isomers." Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers." When a compound has an asymmetric center, for example, when it is bonded to four different groups, a pair of enantiomers can exist. Enantiomers are characterized by the absolute configuration of their asymmetric center and are described by the R- and S-sequencing rules of Cahn and Prelog, or by the way the molecule rotates the plane of polarized light and are designated as dextrorotatory or levorotatory ((+) or (-) isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal parts of enantiomers is called a "racemic mixture".

[0191] The compounds of the present disclosure may have one or more asymmetric centers; therefore, such compounds can be prepared as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless otherwise specified, the description or naming of a particular compound in the specification and claims is intended to encompass both individual enantiomers and their racemic or other mixtures. Methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (see the "Discussion" in Chapter 4 of "Advanced Organic Chemistry," 4th edition J. March, John Wiley and Sons, New York, 2001), for example, by synthesis from optically active starting materials or resolution of racemates. Some compounds of the present disclosure have geometric isomeric centers (E-isomers and Z-isomers). It is understood that the present disclosure encompasses all optical isomers, diastereoisomers, geometric isomers, and mixtures thereof that possess inflammasome inhibitory activity.

[0192] As used herein, the term "chiral center" refers to a carbon atom bonded to four nonidentical substituents.

[0193] As used herein, the term "chiral isomer" refers to a compound having at least one chiral center. Compounds with multiple chiral centers may exist as individual diastereomers or as a mixture of diastereomers, termed a "diastereomeric mixture." When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked according to the Cahn-Ingold-Prelog ranking rules. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).

[0194] As used herein, the term "geometric isomer" refers to diastereomers that exist due to hindered rotation about a double bond or a cycloalkyl linker (e.g., 1,3-cyclobutyl). These configurations are distinguished in names by the prefixes cis and trans, or Z and E, which indicate that groups are on the same or opposite sides of a double bond in a molecule according to the Cahn-Ingold-Prelog precedence rules.

[0195] It is understood that the compounds of the present disclosure may be represented as different chiral or geometric isomers, and when a compound has chiral or geometric isomers, all isomers are intended to be included within the scope of the disclosure, and the naming of the compound does not exclude any isomer, and it is also understood that not all isomers have the same level of activity.

[0196] It is understood that the structures and other compounds discussed in this disclosure include all atropisomers thereof. It is also understood that not all atropisomers have the same level of activity.

[0197] As used herein, the term "atropisomer" refers to a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropisomers exist due to restricted rotation, which occurs when large groups are prevented from rotating around a central bond. Such atropisomers usually exist as mixtures, but recent advances in chromatography techniques have made it possible to separate mixtures of two atropisomers in certain cases.

[0198] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This conversion involves the formal migration of a hydrogen atom accompanied by the switching of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomeric sets in solution. In solutions where tautomerization can occur, a chemical equilibrium of tautomers is achieved. The exact ratio of tautomers varies depending on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerization is called tautomerism. Of the various types of tautomerism that can occur, two are commonly observed: keto-enol tautomerism, in which the simultaneous migration of an electron and a hydrogen atom occurs. Ring-chain tautomerism occurs when an aldehyde group (-CHO) in a sugar molecule reacts with one of the hydroxyl groups (-OH) in the same molecule to form a cyclic (ring-like) form, similar to glucose.

[0199] It should be understood that the compounds of the present disclosure may be represented as different tautomers. It should also be understood that, where a compound has tautomers, all tautomers are intended to be included within the scope of the present disclosure, and the naming of a compound does not exclude any tautomer. It will be understood that certain tautomers may have a higher level of activity than other tautomers.

[0200] It should be understood that the compounds of any formula described herein include not only the compounds themselves, but also their salts and solvates, if applicable. For example, a salt can be formed between an anion on a substituted compound disclosed herein and a positively charged group (e.g., amino). Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).

[0201] As used herein, the term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a substituted compound disclosed herein. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations such as tetramethylammonium or diethylamine. The substituted compounds disclosed herein also include salts containing a quaternary nitrogen atom.

[0202] It is understood that compounds of the present disclosure, such as salts of the compounds, can exist in hydrated or non-hydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0203] The term "solvate" as used herein refers to a solvent addition form containing a stoichiometric or non-stoichiometric amount of solvent. Some compounds tend to trap a fixed molar ratio of solvent molecules in the crystalline solid state to form a solvate. When the solvent is water, the solvate formed is a hydrate, and when the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is formed by the combination of one or more water molecules with one molecule of a substance, where the water maintains its molecular state as HO.

[0204] As used herein, the term "analog" refers to a compound that is structurally similar but differs slightly in composition (such as the replacement of one atom with an atom of a different element, the presence of a particular functional group, or the replacement of one functional group with another). Thus, an analog is a compound that is similar or comparable in function and appearance, but whose structural origin is not that of the reference compound.

[0205] The term "derivative," as used herein, refers to compounds that have a common core structure and are substituted with various groups as described herein.

[0206] As used herein, the term "bioisostere" refers to a compound produced by exchanging an atom or group of atoms for another broadly similar atom or group of atoms. The purpose of bioisostere substitution is to create a new compound with similar biological properties as the parent compound. Bioisostere substitution can be physicochemically or topologically based. Examples of carboxylic acid bioisosteres include, but are not limited to, acylsulfonamides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.

[0207] It is also understood that any particular compound of the formula disclosed herein can exist in solvated as well as unsolvated forms, such as, for example, hydrated forms. Suitable pharmaceutically acceptable solvates include, for example, hydrates, such as hemihydrates, monohydrates, dihydrates, or trihydrates. It is understood that the present invention encompasses all such solvated forms that possess inflammasome inhibitory activity.

[0208] It is also understood that certain compounds of any of the formulas disclosed herein may exhibit polymorphism, and the present disclosure encompasses all such forms, or mixtures thereof, that have inflammasome inhibitory activity. It is generally known that crystalline materials can be analyzed using conventional techniques, such as X-ray powder diffraction analysis, differential scanning calorimetry, thermogravimetry, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, near-infrared (NIR) spectroscopy, and solution and / or solid-state nuclear magnetic resonance spectroscopy. The water content of such crystalline materials can be measured by Karl Fischer analysis.

[0209] Compounds of any of the formulas disclosed herein may exist in a number of different tautomeric forms, and a reference to a compound of any of the formulas includes all such forms. For the avoidance of doubt, if a compound exists in one of several tautomeric forms and only one is specifically described or shown, all other tautomeric forms are also encompassed by the formulas disclosed herein. Examples of tautomers include keto, enol, and enolate forms, including tautomeric pairs such as keto / enol (illustrated below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, and nitro / acinitro. [ka]

[0210] Any compound of any formula disclosed herein containing an amine functional group can also form an N-oxide. Reference herein to any compound of any formula disclosed herein containing an amine functional group also includes the N-oxide. When a compound contains multiple amine functional groups, one or more nitrogen atoms may be oxidized to form an N-oxide. Specific examples of N-oxides include the N-oxide of a tertiary amine or the nitrogen atom of a nitrogen-containing heterocycle. N-oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., a peroxycarboxylic acid). See, for example, Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages 1977-1979. More specifically, N-oxides can be prepared by the procedure of LW Deady (Syn. Comm. 1977, 7, 509-514), in which an amine compound is reacted with metachloroperoxybenzoic acid (mCPBA) in an inert solvent such as dichloromethane.

[0211] The compounds of any of the formulas disclosed herein can be administered in the form of prodrugs that are broken down in the human or animal body to release the compounds of the present invention. Prodrugs can be used to modify the physical and / or pharmacokinetic properties of the compounds of the present disclosure. A prodrug can be formed when the compounds of the present disclosure contain a suitable group or substituent to which a property-modifying group can be attached.

[0212] Thus, the present disclosure includes compounds of any of the above-defined formulas disclosed herein when made available by organic synthesis and when made available in the human or animal body by cleavage of a prodrug thereof. Thus, the present disclosure includes compounds of any of the formulas disclosed herein produced by organic synthetic means, as well as compounds produced in the human or animal body by metabolism of a precursor compound, and any of the formulas disclosed herein may be synthetically produced or metabolically produced.

[0213] Suitable pharmaceutically acceptable prodrugs of compounds of any of the formulae disclosed herein are those that are based on sound medical judgment and are suitable for administration to the human or animal body without undesirable pharmacological effects and undue toxicity. Various forms of prodrugs are described, for example, in the following documents: a) Methods in Enzymology, Vol. 42, pp. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and Application of Prodrugs", by H. Bundgaard, pp. 113-191 (1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya, et al., Chem. Pharm. Bull. 32, 692 (1984); and g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, ACS Symposium Series, Volume 14; and h) E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.

[0214] The in vivo effects of a compound of any of the formulas disclosed herein may be exerted in part by one or more metabolites formed in the human or animal body after administration of a compound of any of the formulas disclosed herein. As noted above, the in vivo effects of a compound of any of the formulas disclosed herein may also be exerted by metabolism of a precursor compound (prodrug).

[0215] Preferably, the present disclosure excludes individual compounds that do not have biological activity as defined herein.

[0216] Nucleotide-based enhancer unit-containing complexes As used herein, the term "conjugate" refers to a compound or complex that includes a nucleic acid agent covalently attached to a ligand. In some embodiments, the conjugate further includes a nucleotide-based enhancer unit as described herein.

[0217] In some aspects, the present disclosure provides: (i) one or more nucleic acid agents; (ii) one or more ligands, and (iii) one or more nucleotide-based enhancer units, each nucleotide-based enhancer unit independently comprising 2 to 30 nucleotides, each nucleotide independently comprising [ka] A complex or a pharmaceutically acceptable salt thereof, comprising a nucleotide-based enhancer unit represented by Variables B, V, X, Y, R 1 , R 2 , R 3 , R 4 , and R 5 is described herein, wherein each # independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 5' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the complex; and provides a conjugate or a pharmaceutically acceptable salt thereof, wherein each ## independently represents a bond to the 5' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the conjugate.

[0218] It is understood that the one or more ligands and the one or more nucleotide-based enhancer units are each independently attached to a terminal or internal position of one or more nucleic acid agents. Furthermore, when a nucleic acid agent includes two or more strands (e.g., a sense strand and an antisense strand), it is understood that the one or more ligands and the one or more nucleotide-based enhancer units can be attached to the same strand or different strands of the nucleic acid agent.

[0219] In some embodiments, the conjugate further comprises one or more linker units.

[0220] In some embodiments, the complex comprises a double-stranded RNA (eg, a double-stranded siRNA), one or more ligands, and one or more nucleotide-based enhancer units.

[0221] In some embodiments, at least one nucleotide-based enhancer unit is directly attached to the nucleic acid agent (eg, siRNA).

[0222] In some embodiments, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (eg, siRNA) via a linker unit.

[0223] In some embodiments, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0224] In some embodiments, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) at a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal) directly or via a linker unit.

[0225] In some embodiments, at least one nucleotide-based enhancer unit is attached to a terminal position (eg, the 3'- or 5'-terminal nucleotide) of the nucleic acid agent (eg, siRNA) either directly or via a linker unit.

[0226] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' terminal nucleotide of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0227] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' terminal nucleotide of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0228] In some embodiments, at least one nucleotide-based enhancer unit is attached to an internal position (e.g., a nucleotide other than the 3' or 5' terminus) of the nucleic acid agent (e.g., siRNA) either directly or via a linker unit.

[0229] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense or antisense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0230] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0231] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0232] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0233] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0234] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the sense strand of the nucleic acid agent (e.g., siRNA).

[0235] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the sense strand of the nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0236] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 3'-terminal nucleotide of the sense strand of the nucleic acid agent (eg, siRNA).

[0237] In some embodiments, at least one nucleotide-based enhancer unit is bound to the 3'-terminal nucleotide of the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0238] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 5'-terminal nucleotide of the sense strand of the nucleic acid agent (eg, siRNA).

[0239] In some embodiments, at least one nucleotide-based enhancer unit is bound to the 5'-terminal nucleotide of the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0240] In some embodiments, at least one nucleotide-based enhancer unit is attached to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the sense strand of the nucleic acid agent (e.g., siRNA) either directly or via a linker unit.

[0241] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the sense strand of the nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0242] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0243] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the antisense strand of the nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0244] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 3'-terminal nucleotide of the antisense strand of the nucleic acid agent (eg, siRNA).

[0245] In some embodiments, at least one nucleotide-based enhancer unit is bound to the 3'-terminal nucleotide of the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0246] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 5'-terminal nucleotide of the antisense strand of the nucleic acid agent (eg, siRNA).

[0247] In some embodiments, at least one nucleotide-based enhancer unit is bound to the 5'-terminal nucleotide of the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0248] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0249] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the antisense strand of the nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0250] In some embodiments, at least one nucleotide-based enhancer unit is attached to an overhang (eg, a 3' or 5' overhang) of the nucleic acid agent (eg, an siRNA) directly or via a linker unit.

[0251] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' overhang of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0252] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' overhang of the nucleic acid agent (eg, siRNA) either directly or via a linker unit.

[0253] In some embodiments, at least one nucleotide-based enhancer unit is attached to an overhang (e.g., a 3' or 5' overhang) of the antisense strand of the nucleic acid agent (e.g., an siRNA) directly or via a linker unit.

[0254] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' overhang of the antisense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0255] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' overhang of the antisense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0256] In some embodiments, at least one nucleotide-based enhancer unit is attached to an overhang (eg, a 3' or 5' overhang) of the sense strand of the nucleic acid agent (eg, an siRNA) directly or via a linker unit.

[0257] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' overhang of the sense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0258] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' overhang of the sense strand of the nucleic acid agent (eg, siRNA) directly or via a linker unit.

[0259] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (e.g., siRNA) and is not attached to any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0260] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (e.g., siRNA) and is not attached to any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0261] In some embodiments, at least one nucleotide-based enhancer unit is not associated with any portion of the antisense strand of the nucleic acid agent (eg, siRNA).

[0262] In some embodiments, at least one nucleotide-based enhancer unit is not associated with any portion of the sense strand of the nucleic acid agent (eg, siRNA).

[0263] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (eg, siRNA).

[0264] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (eg, siRNA).

[0265] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal) of the sense or antisense strand of the nucleic acid agent (e.g., siRNA).

[0266] In some embodiments, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0267] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal) of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0268] In some embodiments, at least one nucleotide-based enhancer unit is attached to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the nucleic acid agent (e.g., siRNA) either directly or via a linker unit, and is attached to the ligand either directly or via a linker unit.

[0269] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3'-terminal nucleotide of the nucleic acid agent (e.g., siRNA) either directly or via a linker unit, and the ligand is attached directly or via a linker unit.

[0270] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5'-terminal nucleotide of the nucleic acid agent (e.g., siRNA) either directly or via a linker unit, and the ligand is attached directly or via a linker unit.

[0271] In some embodiments, at least one nucleotide-based enhancer unit is attached to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to the ligand directly or via a linker unit.

[0272] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense or antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0273] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0274] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (e.g., siRNA) directly or through a linker unit, and attached to the ligand directly or through a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0275] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0276] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (e.g., siRNA) directly or through a linker unit, and attached to the ligand directly or through a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0277] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the sense strand of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0278] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the sense strand of the nucleic acid agent (e.g., siRNA) and is attached, either directly or via a linker unit, to the ligand, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0279] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 3'-terminal nucleotide of the sense strand of the nucleic acid agent (e.g., siRNA), and the ligand is attached directly or via a linker unit.

[0280] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3'-terminal nucleotide of the sense strand of the nucleic acid agent (e.g., siRNA) directly or through a linker unit, and attached to the ligand directly or through a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0281] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 5'-terminal nucleotide of the sense strand of the nucleic acid agent (e.g., siRNA), and the ligand is attached directly or via a linker unit.

[0282] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5'-terminal nucleotide of the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0283] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the sense strand of the nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to the ligand.

[0284] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the sense strand of the nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to the ligand, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0285] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the antisense strand of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0286] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the antisense strand of the nucleic acid agent (e.g., siRNA) and is attached, either directly or via a linker unit, to the ligand, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0287] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 3'-terminal nucleotide of the antisense strand of the nucleic acid agent (e.g., siRNA), and the ligand is attached directly or via a linker unit.

[0288] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3'-terminal nucleotide of the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0289] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to the 5'-terminal nucleotide of the antisense strand of the nucleic acid agent (e.g., siRNA), and the ligand is attached directly or via a linker unit.

[0290] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5'-terminal nucleotide of the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0291] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the antisense strand of the nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to the ligand.

[0292] In some embodiments, at least one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the antisense strand of the nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to the ligand, and the nucleotide-based enhancer unit does not interact with (e.g., bind to) any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0293] In some embodiments, at least one nucleotide-based enhancer unit is attached to an overhang (e.g., a 3' or 5' overhang) of the nucleic acid agent (e.g., an siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0294] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' overhang of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0295] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' overhang of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0296] In some embodiments, at least one nucleotide-based enhancer unit is attached to an overhang (e.g., a 3' or 5' overhang) of the antisense strand of the nucleic acid agent (e.g., an siRNA), either directly or via a linker unit, and is attached to the ligand, either directly or via a linker unit.

[0297] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' overhang of the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0298] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' overhang of the antisense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0299] In some embodiments, at least one nucleotide-based enhancer unit is attached to an overhang (e.g., a 3' or 5' overhang) of the sense strand of the nucleic acid agent (e.g., an siRNA) directly or via a linker unit, and is attached to the ligand directly or via a linker unit.

[0300] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 3' overhang of the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0301] In some embodiments, at least one nucleotide-based enhancer unit is attached to the 5' overhang of the sense strand of the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0302] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (e.g., siRNA) and is not attached to any portion of the antisense strand of the nucleic acid agent (e.g., siRNA), and is attached to the ligand directly or via a linker unit.

[0303] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (e.g., siRNA) and is not attached to any portion of the sense strand of the nucleic acid agent (e.g., siRNA), and is attached to the ligand directly or via a linker unit.

[0304] In some embodiments, at least one nucleotide-based enhancer unit is not attached to any portion of the antisense strand of the nucleic acid agent (e.g., siRNA) and is attached to the ligand directly or via a linker unit.

[0305] In some embodiments, at least one nucleotide-based enhancer unit is not attached to any portion of the sense strand of the nucleic acid agent (e.g., siRNA) and is attached to the ligand directly or via a linker unit.

[0306] In some embodiments, at least one nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent (e.g., siRNA) and is attached to the ligand directly or via a linker unit.

[0307] In some embodiments, at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent (e.g., siRNA) and is attached to the ligand either directly or via a linker unit.

[0308] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal) of the sense or antisense strand of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0309] In some embodiments, as shown in FIG. 1, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal) of the sense or antisense strand of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0310] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) of the sense or antisense strand of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0311] In some embodiments, at least one nucleotide-based enhancer unit is attached, either directly or via a linker unit, to an internal position (e.g., a nucleotide other than the 3' or 5' end) of the sense or antisense strand of the nucleic acid agent (e.g., siRNA), and is attached, either directly or via a linker unit, to the ligand.

[0312] In some embodiments, as shown in Complex No. 1 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0313] In some embodiments, as shown in Complex No. 2 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0314] In some embodiments, as shown in Complex No. 3 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0315] In some embodiments, as shown in Complex No. 4 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0316] In some embodiments, as shown in Complex No. 5 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0317] In some embodiments, as shown in Complex No. 6 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0318] In some embodiments, as shown in Complex No. 7 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0319] In some embodiments, as shown in Complex No. 8 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0320] In some embodiments, as shown in Complex No. 9 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0321] In some embodiments, as shown in Complex No. 10 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0322] In some embodiments, as shown in Complex No. 11 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0323] In some embodiments, as shown in Complex No. 12 in Figure 1, at least one nucleotide-based enhancer unit is attached to the nucleic acid agent (e.g., siRNA) directly or via a linker unit, and attached to the ligand directly or via a linker unit.

[0324] In some embodiments, at least one ligand is directly attached to the nucleic acid agent (eg, siRNA).

[0325] In some embodiments, at least one ligand is attached to the nucleic acid agent (eg, siRNA) via a linker unit.

[0326] In some embodiments, at least one ligand is attached to the nucleic acid agent (eg, siRNA) via a nucleotide-based enhancer unit.

[0327] In some embodiments, at least one ligand is directly attached to the nucleotide-based enhancer unit.

[0328] In some embodiments, at least one ligand is attached to the nucleotide-based enhancer unit via a linker unit.

[0329] In some embodiments, at least one ligand is attached to the nucleic acid agent (eg, siRNA) via a nucleotide-based enhancer unit and a linker unit.

[0330] In some embodiments, the complex comprises: (nucleic acid agent)-[(linker unit) 0-1 -(nucleotide-based enhancer unit)-(linker unit) 0-1 -(ligand)] 1-3 , (nucleic acid agent)-[(linker unit) 0-1 -(ligand)-(linker unit) 0-1 -(nucleotide-based enhancing unit)] 1-3 , or [(nucleotide-based enhancer unit)-(linker unit) 0-1 ] 1-3 -(nucleic acid agent)-[(linker unit) 0-1 -(ligand)] 1-3 Including, When attached to the nucleic acid agent (e.g., siRNA), the linker unit, nucleotide-based enhancer unit, and ligand are each independently attached to a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal nucleotide) of the nucleic acid agent (e.g., siRNA).

[0331] In some embodiments, the conjugate is selected from the conjugates shown in Table D, and the nucleic acid agent may optionally be further conjugated to one or more linker units, one or more nucleotide-based enhancer units, and / or one or more ligands at one or more of the internal positions of the nucleic acid agent, in accordance with the conjugations described herein. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0332] The one or more linkages to the nucleic acid agent may be at one or more internal positions of the nucleic acid agent (e.g., [ka] ), the 3' terminal position (e.g., [ka] ), or at the 5' terminal position (e.g., [ka] ) can be present.

[0333] In some embodiments, the complex is selected from the complexes shown in FIG. 1 and FIG.

[0334] In some embodiments, the complex is selected from the complexes shown in FIG.

[0335] In some embodiments, the complex is complex number 1 shown in FIG.

[0336] In some embodiments, the complex is Complex No. 1 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0337] In some embodiments, the conjugate is conjugate number 1 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0338] In some embodiments, the complex is complex number 1 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent, and is attached directly or via a linker unit to the ligand.

[0339] In some embodiments, the complex is complex number 1 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent, and is attached directly or via a linker unit to the ligand.

[0340] In some embodiments, the complex is complex number 2 shown in FIG.

[0341] In some embodiments, the complex is complex number 2 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0342] In some embodiments, the conjugate is conjugate number 2 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0343] In some embodiments, the complex is complex number 2 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent, and is attached directly or via a linker unit to the ligand.

[0344] In some embodiments, the complex is complex number 2 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent, and is attached directly or via a linker unit to the ligand.

[0345] In some embodiments, the complex is complex number 3 shown in FIG.

[0346] In some embodiments, the conjugate is conjugate number 3 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0347] In some embodiments, the conjugate is conjugate number 3 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0348] In some embodiments, the complex is complex number 3 shown in Figure 1, in which the ligand is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit.

[0349] In some embodiments, the complex is complex number 3 shown in Figure 1, in which the ligand is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit.

[0350] In some embodiments, the complex is complex number 4 shown in FIG.

[0351] In some embodiments, the conjugate is conjugate number 4 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0352] In some embodiments, the conjugate is conjugate number 4 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0353] In some embodiments, the complex is complex number 4 shown in Figure 1, in which the ligand is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit.

[0354] In some embodiments, the complex is complex number 4 shown in Figure 1, in which the ligand is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit.

[0355] In some embodiments, the complex is complex number 5 shown in FIG.

[0356] In some embodiments, the conjugate is conjugate number 5 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0357] In some embodiments, the conjugate is conjugate number 5 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0358] In some embodiments, the complex is complex number 5 shown in Figure 1, in which the nucleotide-based enhancer unit is attached to an internal position of the antisense strand of the nucleic acid agent, either directly or via a linker unit, and the ligand is attached to the internal position of the antisense strand of the nucleic acid agent, either directly or via a linker unit.

[0359] In some embodiments, the complex is complex number 5 shown in Figure 1, in which the nucleotide-based enhancer unit is attached to an internal position of the sense strand of the nucleic acid agent, either directly or via a linker unit, and the ligand is attached to the internal position of the sense strand of the nucleic acid agent, either directly or via a linker unit.

[0360] In some embodiments, the complex is complex number 6 shown in FIG.

[0361] In some embodiments, the conjugate is conjugate number 6 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0362] In some embodiments, the conjugate is conjugate number 6 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0363] In some embodiments, the complex is complex number 6 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent.

[0364] In some embodiments, the complex is complex number 6 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent.

[0365] In some embodiments, the complex is complex number 6 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent.

[0366] In some embodiments, the complex is complex number 6 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent.

[0367] In some embodiments, the complex is complex number 7 shown in FIG.

[0368] In some embodiments, the conjugate is conjugate number 7 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0369] In some embodiments, the conjugate is conjugate number 7 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0370] In some embodiments, the complex is complex number 7 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0371] In some embodiments, the complex is complex number 7 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0372] In some embodiments, the complex is complex number 7 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0373] In some embodiments, the complex is complex number 7 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0374] In some embodiments, the complex is complex number 8 shown in FIG.

[0375] In some embodiments, the conjugate is conjugate number 8 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0376] In some embodiments, the conjugate is conjugate number 8 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0377] In some embodiments, the complex is complex number 8 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent.

[0378] In some embodiments, the complex is complex number 8 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent.

[0379] In some embodiments, the complex is complex number 8 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent.

[0380] In some embodiments, the complex is complex number 8 shown in Figure 1, in which the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and the ligand is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent.

[0381] In some embodiments, the complex is complex number 9 shown in FIG.

[0382] In some embodiments, the conjugate is conjugate number 9 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0383] In some embodiments, the conjugate is conjugate number 9 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0384] In some embodiments, the complex is complex number 9 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent.

[0385] In some embodiments, the complex is complex number 9 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the antisense strand of the nucleic acid agent.

[0386] In some embodiments, the complex is complex number 9 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent.

[0387] In some embodiments, the complex is complex number 9 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to the 5' end of the sense strand of the nucleic acid agent.

[0388] In some embodiments, the complex is complex number 10 shown in FIG.

[0389] In some embodiments, the conjugate is conjugate number 10 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0390] In some embodiments, the conjugate is conjugate number 10 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0391] In some embodiments, the complex is complex number 10 shown in Figure 1, in which the ligand is attached to an internal position of the antisense strand of the nucleic acid agent, either directly or via a linker unit, and the nucleotide-based enhancer unit is attached to an internal position of the antisense strand of the nucleic acid agent, either directly or via a linker unit.

[0392] In some embodiments, the complex is complex number 10 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent.

[0393] In some embodiments, the complex is complex number 10 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent.

[0394] In some embodiments, the complex is complex number 10 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and the nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent.

[0395] In some embodiments, the complex is complex number 11 shown in FIG.

[0396] In some embodiments, the complex is complex number 11 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0397] In some embodiments, the conjugate is conjugate number 11 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0398] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0399] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0400] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0401] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0402] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0403] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0404] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0405] In some embodiments, the complex is complex number 11 shown in Figure 1, in which the ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0406] In some embodiments, the complex is complex number 12 shown in FIG.

[0407] In some embodiments, the conjugate is conjugate number 12 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the antisense strand of the nucleic acid agent.

[0408] In some embodiments, the conjugate is conjugate number 12 shown in Figure 1, wherein the nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent.

[0409] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or through a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or through a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or through a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or through a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0410] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0411] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0412] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0413] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0414] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0415] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0416] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0417] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0418] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0419] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0420] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the antisense strand of the nucleic acid agent.

[0421] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0422] In some embodiments, the complex is complex number 12 shown in Figure 1, in which one ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, another ligand is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, one nucleotide-based enhancer unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent, and another nucleotide-based enhancer unit is attached directly or via a linker unit to the 3' end of the sense strand of the nucleic acid agent.

[0423] In some embodiments, the complex is selected from the complexes shown in FIG.

[0424] In some embodiments, the conjugate is selected from the structures set forth in Table C: The linkage to [a nucleic acid agent] (e.g., the antisense or sense strand of the nucleic acid agent) can independently be at a terminal position (e.g., the 3'- or 5'-terminal nucleotide) or at an internal position (e.g., a nucleotide other than the 3'- or 5'-terminal) of the nucleic acid agent; The bonds to the [nucleic acid agent] and [ligand] can independently be direct or via a linker unit. [Table 3-1] [Table 3-2]

[0425] In some embodiments, the conjugate is selected from the structures set forth in Table C, and [nucleic acid agent] is the antisense strand of a nucleic acid agent.

[0426] In some embodiments, the conjugate is selected from the structures set forth in Table C, and [nucleic acid agent] is the sense strand of the nucleic acid agent.

[0427] Nucleotide-based enhancer units As used herein, the term "nucleotide-based enhancer unit" refers to a moiety in which the * of the 5'-terminal nucleotide and / or the ** of the 2'-terminal or 3'-terminal nucleotide corresponds to a nucleotide-based enhancer bound to a ligand, linker unit, and / or nucleic acid agent.

[0428] In some embodiments, a "nucleotide-based enhancer unit" refers to a nucleotide-based enhancer agent where the * of the 5'-terminal nucleotide corresponds to a ligand, linker unit, and / or nucleic acid agent attached to the nucleotide-based enhancer agent.

[0429] In some embodiments, a "nucleotide-based enhancer unit" corresponds to a nucleotide-based enhancer agent in which the 2'- or 3'-terminal nucleotide is attached to a ligand, linker unit, and / or nucleic acid agent.

[0430] In some embodiments, a "nucleotide-based enhancer unit" corresponds to a nucleotide-based enhancer agent in which the * of the 5'-terminal nucleotide is bound to a ligand, linker unit, and / or nucleic acid agent, and the ** of the 2'- or 3'-terminal nucleotide is bound to a ligand, linker unit, and / or nucleic acid agent.

[0431] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (I) or (II), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based builder unit, is H, or, if the nucleotide is at the 5'-terminus of the nucleotide-based builder unit, is attached to a ligand, linker unit, and / or nucleic acid agent; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid agent if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0432] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (I') or (II'), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0433] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (I'-A), (II'-A), (III'-A), or (IV'-A), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0434] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (I'-A), (II'-A), (III'-A), or (IV'-A), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0435] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (IB) or (II-B), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0436] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (I'-B) or (II'-B), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0437] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (IC), (II-C), (III-C), or (IV-C), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0438] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is represented by formula (I'-C), (II'-C), (III'-C), or (IV'-C), wherein: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0439] In some embodiments, at least one nucleotide of the nucleotide-based enhancer unit is selected from Table L: * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 5'-terminus of the nucleotide-based enhancer unit; and ** is attached to the 5' position of another nucleotide of the nucleotide-based enhancer unit, is H, or is attached to a ligand, linker unit, and / or nucleic acid if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit.

[0440] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides.

[0441] In some embodiments, the nucleotide-based enhancing unit comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides.

[0442] In some embodiments, the nucleotide-based enhancer unit comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.

[0443] In some embodiments, the nucleotide-based enhancer unit comprises 5 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 6 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 7 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 8 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 9 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 10 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 11 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 12 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 13 nucleotides.

[0444] In some embodiments, the nucleotide-based enhancing unit comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides.

[0445] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides.

[0446] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0447] In some embodiments, the nucleotide-based enhancer unit comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0448] In some embodiments, the nucleotide-based enhancer unit comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0449] In some embodiments, the nucleotide-based enhancer unit comprises 5 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 6 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 7 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 8 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 9 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 10 nucleotides, and further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 11 nucleotides, and further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 12 nucleotides, and further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 13 nucleotides, and further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0450] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0451] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0452] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0453] In some embodiments, the nucleotide-based enhancer unit comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0454] In some embodiments, the nucleotide-based enhancer unit comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0455] In some embodiments, the nucleotide-based enhancer unit comprises 5 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 6 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 7 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 8 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 9 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 10 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 11 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer unit comprises 12 nucleotides and further comprises one or more phosphodiester linkers, hi some embodiments, the nucleotide-based enhancer unit comprises 13 nucleotides and further comprises one or more phosphodiester linkers.

[0456] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0457] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0458] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 28 nucleotides, 2 to 26 nucleotides, 2 to 24 nucleotides, 2 to 22 nucleotides, 2 to 20 nucleotides, 2 to 18 nucleotides, 2 to 16 nucleotides, 2 to 14 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, or 2 to 3 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0459] In some embodiments, the nucleotide-based enhancer unit comprises 4 to 30 nucleotides, 6 to 30 nucleotides, 8 to 30 nucleotides, 10 to 30 nucleotides, 12 to 30 nucleotides, 14 to 30 nucleotides, 16 to 30 nucleotides, 18 to 30 nucleotides, 20 to 30 nucleotides, 21 to 30 nucleotides, 22 to 30 nucleotides, 23 to 30 nucleotides, 24 to 30 nucleotides, 25 to 30 nucleotides, 26 to 30 nucleotides, 27 to 30 nucleotides, 28 to 30 nucleotides, or 29 to 30 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0460] In some embodiments, the nucleotide-based enhancer unit comprises 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0461] In some embodiments, the nucleotide-based enhancer unit comprises 5 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 6 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 7 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 8 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 9 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 10 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 11 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based builder unit comprises 12 nucleotides and further comprises one or more phosphorothioate linkers, hi some embodiments, the nucleotide-based builder unit comprises 13 nucleotides and further comprises one or more phosphorothioate linkers.

[0462] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 21 nucleotides, 3 to 20 nucleotides, 4 to 19 nucleotides, 5 to 18 nucleotides, 6 to 17 nucleotides, 7 to 16 nucleotides, 8 to 15 nucleotides, 9 to 14 nucleotides, 10 to 13 nucleotides, or 11 to 12 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0463] In some embodiments, the nucleotide-based enhancer unit comprises 2 to 19 nucleotides, 3 to 18 nucleotides, 4 to 17 nucleotides, 5 to 16 nucleotides, 6 to 15 nucleotides, 7 to 14 nucleotides, 8 to 13 nucleotides, 9 to 12 nucleotides, or 10 to 11 nucleotides. In some embodiments, the nucleotide-based enhancer unit comprises 2 to 16 nucleotides, 3 to 15 nucleotides, 4 to 14 nucleotides, 5 to 13 nucleotides, 6 to 12 nucleotides, 7 to 11 nucleotides, 8 to 10 nucleotides, or 9 to 11 nucleotides, and the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0464] Linker Unit As used herein, "Linker unit" or "linker unit" refers to a moiety attached to a nucleotide-based enhancer unit, a ligand, and / or a nucleic acid agent.

[0465] In some embodiments, the linker unit comprises a linkage to a nucleotide-based enhancer unit and a ligand.

[0466] In some embodiments, the linker unit comprises a linkage to a ligand and a nucleic acid agent.

[0467] In some embodiments, the linker unit comprises a linkage to a nucleotide-based enhancer unit and a nucleic acid agent.

[0468] In some embodiments, the linker unit is a ribose derivative.

[0469] In some embodiments, the linker unit is a 1'-alkyl modified ribose derivative, for example, as described in International Application No. PCT / US2022 / 039517, which is incorporated herein by reference.

[0470] In some embodiments, the linker unit is a 2'-alkyl or 3'-alkyl modified ribose derivative, for example, as described in International Application No. PCT / US2022 / 044377, which is incorporated herein by reference.

[0471] In some embodiments, the linker unit is a polyhydroxylated cyclopentane derivative, for example, as described in International Application No. PCT / US2022 / 045748, which is incorporated herein by reference.

[0472] Ligand As used herein, the term "ligand" or "ligand" refers to a moiety that, when covalently attached to a nucleic acid agent (e.g., an oligonucleotide), can mediate its entry into or facilitate its delivery to a target site (e.g., a target cell or tissue).

[0473] In some embodiments, the ligand comprises a sugar ligand moiety (eg, N-acetylgalactosamine (GalNAc)) that directs hepatic uptake of the oligonucleotide.

[0474] In some embodiments, the ligand binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand binds to the liver, such as liver parenchymal cells (e.g., via ASGPR).

[0475] In some embodiments, the ligand is capable of binding to a glucagon-like peptide receptor, e.g., a glucagon-like peptide-1 receptor (GLP-1 receptor). In some embodiments, the ligand binds to a glucagon-like peptide-1 receptor (GLP-1 receptor). In some embodiments, the ligand binds to a pancreatic, e.g., beta beta cell (e.g., via the GLP-1 receptor).

[0476] In some embodiments, the ligand is a small molecule, carbohydrate, oligonucleotide, antibody, or peptide.

[0477] In some embodiments, the ligand is a peptide capable of binding to a glucagon-like peptide receptor, such as a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0478] Suitable ligands include, but are not limited to, those disclosed in Winkler (Ther. Deliv., 2013, 4(7): 791-809), Mayendraraj et al. (Peptides, 2022, 170749), Willard et al. (Exp. Diabetes Res., 2012; 2012: 709893), Sloop et al. (Diabetes, 2010, 59(12): 3099-3107), Knudsen et al. (PNAS, 2007, 104(3): 937-942), and Wang et al. (Acta Pharmacol. Sin. 2010, 31: 1026-1030), PCT patent application publications WO 2005 / 018536, WO 2008 / 086086, WO 2011 / 075393, WO 2011 / 056644, WO 2016 / 100401, WO 2012 / 089352, WO 2009 / 082607, and WO 2019 / 092618, U.S. ...18536, WO 2008 / 018536, WO 2008 / 018536, WO 2008 / 018536, WO 2008 / 018536, WO 2008 / 018536, WO Nos. 2006 / 0275288, 2007 / 124461, 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, 2014 / 0206607, and 2009 / 0247608, and U.S. Pat. Nos. 9,187,522, 8,329,419, and 8,389,689, each of which is incorporated by reference.

[0479] In some embodiments, the ligand comprises a carbohydrate moiety.

[0480] As used herein, a "carbohydrate moiety" refers to a moiety comprising one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. In some embodiments, the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide containing about 4-9 monosaccharide units. In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose, or a polysaccharide gum).

[0481] In some embodiments, the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0482] In some embodiments, the carbohydrate moiety comprises an oligosaccharide (eg, containing about 4-9 monosaccharide units).

[0483] In some embodiments, the carbohydrate moiety comprises a polysaccharide (eg, starch, glycogen, cellulose, or a polysaccharide gum).

[0484] In some embodiments, the ligand is capable of binding to a human asialoglycoprotein receptor (ASGPR), such as human asialoglycoprotein receptor 2 (ASGPR2).

[0485] In some embodiments, the carbohydrate moiety comprises a sugar (eg, 1, 2, or 3 sugars).

[0486] In some embodiments, the carbohydrate moiety comprises galactose or a derivative thereof (eg, 1, 2, or 3 galactoses or derivatives thereof).

[0487] In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (eg, 1, 2, or 3 N-acetylgalactosamines or derivatives thereof).

[0488] In some embodiments, the carbohydrate moiety comprises an N-acetyl-D-galactosylamine or derivative thereof (eg, one, two, or three N-acetyl-D-galactosylamines or derivatives thereof).

[0489] In some embodiments, the carbohydrate moiety comprises an N-acetylgalactosamine (eg, 1, 2, or 3 N-acetylgalactosamines).

[0490] In some embodiments, the carbohydrate moiety comprises an N-acetyl-D-galactosylamine (eg, one, two, or three N-acetyl-D-galactosylamines).

[0491] In some embodiments, the carbohydrate moiety comprises mannose or a derivative thereof (eg, mannose-6-phosphate).

[0492] In some embodiments, the carbohydrate moiety further comprises a linking moiety that connects one or more sugars (eg, N-acetyl-D-galactosylamine) to the linker unit.

[0493] In some embodiments, the linking moiety comprises a thioether (eg, thiosuccinimide or its hydrolyzed analog), a disulfide, a triazole, a phosphorothioate, a phosphodiester, an ester, an amide, or any combination thereof.

[0494] In some embodiments, the linking moiety is a three-branched linking moiety.

[0495] Suitable ligands include, but are not limited to, those disclosed in PCT Patent Application Publication Nos. WO 2015 / 006740, WO 2016 / 100401, WO 2017 / 214112, WO 2018 / 039364, and WO 2018 / 045317, each of which is incorporated herein by reference.

[0496] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0497] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0498] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0499] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0500] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0501] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0502] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0503] In some embodiments, the ligand is [ka] (e.g., 1, 2, or 3 [ka] ) is included.

[0504] In some embodiments, the ligand is [ka] Includes.

[0505] In some embodiments, the ligand is [ka] Includes.

[0506] In some embodiments, the ligand is [ka] Includes.

[0507] In some embodiments, the ligand is [ka] Includes.

[0508] In some embodiments, the ligand is [ka] Includes.

[0509] In some embodiments, the ligand is [ka] Includes.

[0510] In some embodiments, the ligand is [ka] Includes.

[0511] In some embodiments, the ligand is [ka] Includes.

[0512] In some embodiments, the ligand comprises a lipid moiety (eg, 1, 2, or 3 lipid moieties).

[0513] In some embodiments, the lipid moiety is C8-C 24 It includes fatty acids, cholesterol, vitamins, sterols, phospholipids, or any combination thereof (eg, one, two, or three).

[0514] In some embodiments, the ligand comprises a peptide moiety (e.g., 1, 2, or 3 peptide moieties), hi some embodiments, the ligand comprises a peptide moiety (e.g., 1, 2, or 3 peptide moieties) that can bind to a glucagon-like peptide receptor, e.g., the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0515] In some embodiments, the peptide moiety comprises (eg, one, two, or three) integrins, insulin, glucagon-like peptides (eg, GLP-1), or any combination thereof.

[0516] In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) glucagon-like peptides (e.g., GLP-1). In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) glucagon.

[0517] In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) dulaglutide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) lixisenatide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) exenatide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) semaglutide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) albiglutide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) liraglutide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) tirzepatide. In some embodiments, the peptide moiety comprises (e.g., 1, 2, or 3) avexitide. In some embodiments, the peptide portion comprises (e.g., 1, 2, or 3) HISHS-2001. In some embodiments, the peptide portion comprises (e.g., 1, 2, or 3) MAR709. In some embodiments, the peptide portion comprises (e.g., 1, 2, or 3) exendin-4.

[0518] In some embodiments, the ligand comprises an antibody portion (e.g., transferrin). In some embodiments, the ligand comprises an antibody portion capable of binding to a glucagon-like peptide receptor, e.g., the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0519] In some embodiments, the ligand comprises one, two, or three antibody moieties (e.g., transferrin). In some embodiments, the ligand comprises one, two, or three antibody moieties capable of binding to a glucagon-like peptide receptor, e.g., the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0520] In some embodiments, the ligand comprises an oligonucleotide (e.g., an aptamer or CpG), hi some embodiments, the ligand comprises an oligonucleotide (e.g., an aptamer or CpG) capable of binding to a glucagon-like peptide receptor, e.g., the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0521] In some embodiments, the ligand comprises one, two, or three oligonucleotides (e.g., aptamers or CpGs). In some embodiments, the ligand comprises one, two, or three oligonucleotides (e.g., aptamers or CpGs) capable of binding to a glucagon-like peptide receptor, such as the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0522] In some embodiments, the ligand comprises a small molecule moiety. In some embodiments, the ligand comprises a small molecule moiety that can bind to a glucagon-like peptide receptor, such as a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0523] In some embodiments, the ligand comprises one, two, or three small molecule moieties, hi some embodiments, the ligand comprises one, two, or three small molecule moieties capable of binding to a glucagon-like peptide receptor, e.g., the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0524] In some embodiments, the ligand is one, two, or three sugars (e.g., N-acetyl-D-galactosylamine); 1, 2, or 3 lipid moieties, 1, 2, or 3 peptide moieties; 1, 2, or 3 antibody moieties; 1, 2, or 3 oligonucleotides, or This includes any combination thereof.

[0525] In some embodiments, the ligand is 1, 2, or 3 carbohydrate moieties, 1, 2, or 3 peptide moieties; 1, 2, or 3 antibody moieties; one, two, or three small molecule moieties, or This includes any combination thereof.

[0526] Nucleic acid agents In some embodiments, the nucleic acid agent comprises an oligonucleotide.

[0527] In some embodiments, the nucleic acid agent (eg, the oligonucleotide) includes one or more phosphate groups or analogs of one or more phosphate groups.

[0528] In some embodiments, the linker unit is attached to the nucleic acid agent (eg, the oligonucleotide) via a phosphate group or an analog of a phosphate group in the nucleic acid agent.

[0529] In some embodiments, the oligonucleotides are 1 to 40 nucleotides, 10 to 40 nucleotides, 12 to 35 nucleotides, 15 to 30 nucleotides, 18 to 25 nucleotides, or 20 to 23 nucleotides in length. In some embodiments, the oligonucleotides are 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the oligonucleotides are 20, 21, 22, or 23 nucleotides in length.

[0530] In some embodiments, the nucleic acid agent comprises RNA, DNA, or a mixture thereof.

[0531] In some embodiments, the nucleic acid agent comprises RNA.

[0532] In some embodiments, the oligonucleotide is an siRNA (e.g., a single-stranded siRNA (e.g., a hairpin single-stranded siRNA) or a double-stranded siRNA), a microRNA, an anti-microRNA, a microRNA mimic, an antagomir, a dsRNA, a ssRNA, an aptamer, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a splice variant oligonucleotide, a triplex-forming oligonucleotide, a G-quadruplex, or an antisense oligonucleotide.

[0533] In some embodiments, the nucleic acid agent comprises double-stranded RNA (dsRNA), where the double-stranded RNA comprises a sense strand and an antisense strand, as described herein.

[0534] In some embodiments, the nucleic acid agent comprises a double-stranded siRNA (ds-siRNA), where the double-stranded siRNA comprises a sense strand and an antisense strand, as described herein.

[0535] It is understood that the sense strand is also known as the passenger strand, and the terms "sense strand" and "passenger strand" are used interchangeably herein.

[0536] The antisense strand is also known as the guide strand, and it is understood that the terms "antisense strand" and "guide strand" are used interchangeably herein.

[0537] In some embodiments, the oligonucleotide is an iRNA.

[0538] The term "iRNA" refers to an RNA agent (e.g., siRNA) that can downregulate the expression of a target gene, e.g., an endogenous or pathogen target RNA. Without being bound by theory, iRNAs may act by one or more mechanisms, including post-transcriptional cleavage of the target mRNA (referred to in the art as RNAi), or pre-transcriptional or pre-translational mechanisms. iRNAs can include a single strand or multiple strands (e.g., double-stranded iRNAs). If the iRNA is single-stranded, it can include a 5' modification that includes one or more phosphate groups or one or more phosphate group analogs. In some embodiments, the iRNA is double-stranded. In some embodiments, one or both strands of the double-stranded iRNA can be modified (e.g., 5' modified).

[0539] An iRNA typically contains a region of sufficient homology with a target gene and is of sufficient length, in terms of nucleotides, that the iRNA, or a fragment thereof, can mediate downregulation of the target gene. The iRNA is at least partially complementary to the target RNA, and in some embodiments, is or contains a region of full complementarity. While full complementarity between the iRNA and the target is not required, the correspondence may be sufficient to enable the iRNA or its cleavage products to induce sequence-specific silencing, for example, by RNAi cleavage of the target RNA (e.g., mRNA).

[0540] Nucleotides in the iRNA can be modified (e.g., one or more nucleotides contain a 2'-F or 2'-OCH3 group or can be a nucleotide substitute). Single- or double-stranded regions of the iRNA can be modified or contain nucleotide substitutes; for example, the unpaired region of a hairpin structure, e.g., the region connecting two complementary regions, can have modifications or nucleotide substitutes. For example, modifications to stabilize one or more of the 3' or 5' ends of the iRNA against exonucleases can be included. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), and special biotin or fluorescein reagents that occur as phosphoramidites and have an additional DMT-protected hydroxyl group, allowing for multiple couplings during RNA synthesis. Modifications can also include, for example, the use of modifications at the 2'OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, in place of ribonucleotides, and modifications at the phosphate group, e.g., phosphothioate modifications. In some embodiments, different strands include different modifications.

[0541] In some embodiments, the strands are selected so that the iRNA contains a single-stranded or unpaired region at one or both ends of the molecule. A double-stranded iRNA can include an overhang, such as one or two 5' or 3' overhangs (e.g., a 3' overhang of at least 2-3 nucleotides). In some embodiments, the iRNA has an overhang of 1, 2, or 3 nucleotides in length at each end, e.g., a 3' overhang. The overhangs can occur because one strand is longer than the other, or because two strands of the same length are alternated.

[0542] In some embodiments, the length of the duplex region between the strands of the iRNA is between 6 and 30 nucleotides. In some embodiments, the length of the duplex region is between 15 and 30 nucleotides, most preferably 18, 19, 20, 21, 22, and 23 nucleotides. In some embodiments, the duplex region is between 6 and 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11, and 12 nucleotides in length.

[0543] The oligonucleotides may be those described in U.S. Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is incorporated herein by reference.

[0544] In some embodiments, the oligonucleotide is an siRNA.

[0545] In some embodiments, the oligonucleotide is a single-stranded siRNA.

[0546] In some embodiments, the oligonucleotide is a double-stranded siRNA, eg, a double-stranded siRNA described herein.

[0547] As used herein, a "single-stranded siRNA" refers to an siRNA that is composed of a single strand and contains a duplex region formed by intrastrand pairing, and can be, for example, a hairpin structure or a panhandle structure. Single-stranded siRNAs can be antisense to a target molecule.

[0548] Single-stranded siRNA can be long enough to enter RISC and participate in the cleavage of target mRNA via RISC.The length of single-stranded siRNA is at least 14 nucleotides, and in some embodiments, it is at least 15, 20, 25, 29, 35, 40, or 50 nucleotides.In some embodiments, its length is less than 200, 100, 80, 60, 50, 40, or 30 nucleotides.

[0549] In some embodiments, the length of the single-stranded siRNA is 10 to 40 nucleotides, 12 to 35 nucleotides, 15 to 30 nucleotides, 18 to 25 nucleotides, or 20 to 23 nucleotides. In some embodiments, the length of the single-stranded siRNA is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the single-stranded siRNA is 20, 21, 22, or 23 nucleotides.

[0550] Hairpin siRNAs can have a duplex region equal to at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the duplex region can be up to 200, 100, or 50 nucleotide pairs. In some embodiments, the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length. The hairpin can include a single-stranded overhang or terminal unpaired region. In some embodiments, the overhang is 2 to 3 nucleotides in length. In some embodiments, the overhang is on the sense side of the hairpin, and in some embodiments, on the antisense side of the hairpin.

[0551] In some embodiments, the oligonucleotide is a double-stranded siRNA.

[0552] As used herein, a "double-stranded siRNA" is an siRNA that contains multiple, optionally two, strands that can form a region of duplex structure by interstrand hybridization.

[0553] In some embodiments, the length of the sense strand of the double-stranded siRNA may be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides or more. The length may be 200, 100, or 50 nucleotides or less. The length may be within the range of 17-25, 19-23, 19-21, 21-23, or 20-22 nucleotides.

[0554] In some embodiments, the length of the sense strand is 10 to 40 nucleotides, 12 to 35 nucleotides, 15 to 30 nucleotides, 18 to 25 nucleotides, or 20 to 23 nucleotides. In some embodiments, the length of the sense strand is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the sense strand is 20, 21, 22, or 23 nucleotides.

[0555] In some embodiments, the sense strand is 18, 19, 20, 21, or 22 nucleotides in length.

[0556] In some embodiments, the length of the antisense strand of the double-stranded siRNA may be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides or more. The length may be 200, 100, or 50 nucleotides or less. The length may be within the range of 17 to 25, 19 to 23, 19 to 21, 21 to 23, or 20 to 22 nucleotides.

[0557] In some embodiments, the length of the antisense strand is 10 to 40 nucleotides, 12 to 35 nucleotides, 15 to 30 nucleotides, 18 to 25 nucleotides, or 20 to 23 nucleotides. In some embodiments, the length of the antisense strand is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the antisense strand is 20, 21, 22, or 23 nucleotides.

[0558] In some embodiments, the antisense strand is 20, 21, 22, 23, or 24 nucleotides in length.

[0559] In some embodiments, the sense strand is 18, 19, 20, 21, or 22 nucleotides in length and the antisense strand is 20, 21, 22, 23, or 24 nucleotides in length.

[0560] In some embodiments, the sense strand is 18 nucleotides in length and the antisense strand is 20 nucleotides in length.

[0561] In some embodiments, the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length.

[0562] In some embodiments, the sense strand is 20 nucleotides in length and the antisense strand is 22 nucleotides in length.

[0563] In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0564] In some embodiments, the sense strand is 22 nucleotides in length and the antisense strand is 24 nucleotides in length.

[0565] The length of the double-stranded portion of the double-stranded siRNA may be at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs. The length may be up to 200, 100, or 50 nucleotide pairs. The length may be within the range of 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0566] In some embodiments, the siRNA is large enough that it is cleaved by an endogenous molecule, eg, Dicer, to generate smaller siRNAs, such as siRNA agents.

[0567] The sense and antisense strands can be selected so that the double-stranded siRNA contains a single-stranded or unpaired region at one or both ends of the molecule. Thus, the double-stranded siRNA contains a pair of sense and antisense strands and can contain an overhang (e.g., one or two 5' or 3' overhangs, or a 3' overhang of 1 to 3 nucleotides). The overhang can occur because one strand is longer than the other strand, or because two strands of the same length are alternately arranged. In some embodiments, at least one 3' overhang is present. In some embodiments, both ends of the siRNA molecule have a 3' overhang. In some embodiments, the overhang is 2 nucleotides.

[0568] In some embodiments, the length of the duplex region is 15 to 30, or 18, 19, 20, 21, 22, and 23 nucleotides, for example, in the ranges of ssiRNAs discussed above. ssiRNAs can be similar in length and structure to the natural Dicer-processed products of longer dsiRNAs. Also included are embodiments in which the two strands of the ssiRNA are linked, e.g., covalently linked. Hairpins or other single-stranded structures that provide the necessary duplex region and 3' overhang are also contemplated.

[0569] siRNA described herein, including double-stranded siRNA and single-stranded siRNA, can mediate the silencing of target RNA, for example, mRNA, for example, the transcript of the gene encoding protein.For convenience, this mRNA is also referred to herein as the mRNA to be silenced.This gene is also referred to as target gene.Generally, the RNA to be silenced is endogenous gene or pathogen gene.In addition, RNA other than mRNA, for example, tRNA and viral RNA can also be targeted.

[0570] As used herein, the phrase "mediating RNAi" refers to the ability to silence a target RNA in a sequence-specific manner. Without being bound by theory, it is believed that silencing utilizes the RNAi machinery or process and a guide RNA (e.g., a 21-23 nucleotide ssiRNA).

[0571] In some embodiments, the siRNA is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the siRNA silences production of the protein encoded by the target mRNA. In other embodiments, the siRNA is "fully complementary" to the target RNA, e.g., the target RNA and the siRNA anneal to form a hybrid consisting solely of Watson-Crick base pairs in the exact region of complementarity. A "sufficiently complementary" target RNA may include an internal region (e.g., at least 10 nucleotides) that is perfectly complementary to the target RNA. Furthermore, in some embodiments, the siRNA specifically discriminates between single nucleotide differences. In this case, the siRNA mediates RNAi only if perfect complementarity is found in the region (e.g., within 7 nucleotides) of the single nucleotide difference.

[0572] MicroRNA: MicroRNAs (miRNAs) are a class of highly conserved small RNA molecules that are transcribed from DNA in the genomes of plants and animals but are not translated into proteins. Processed miRNAs are 17-25 nucleotide (nt) single-stranded RNA molecules that are incorporated into the RNA-induced silencing complex (RISC) and have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in regulating gene expression by binding to the 3' untranslated region of specific mRNAs. RISC mediates downregulation of gene expression through translational inhibition, transcript cleavage, or both. RISC is also involved in transcriptional silencing in the nuclei of various eukaryotic organisms.

[0573] The number of miRNA sequences identified to date is vast and growing, as described, for example, in "miRBase: microRNA sequences, targets and gene nomenclature" Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34, Database Issue, D140-D144; "The microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111.

[0574] Antisense Oligonucleotide: In some embodiments, the nucleic acid is an antisense oligonucleotide to a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" is meant to include oligonucleotides complementary to a target polynucleotide sequence. Antisense oligonucleotides are single strands of DNA or RNA complementary to a selected sequence, such as the mRNA of a target gene. Antisense oligonucleotides are believed to inhibit gene expression by binding to complementary mRNA. Binding to the target mRNA can inhibit translation of the complementary mRNA strand or can inhibit gene expression by degradation of the target mRNA. Antisense DNA can be used to target specific complementary (coding or non-coding) RNA. Upon binding, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In some embodiments, antisense oligonucleotides contain about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also includes antisense oligonucleotides that may not be perfectly complementary to the intended target gene. Thus, it is contemplated that non-target specific activity may be observed with antisense, or that an antisense sequence containing one or more mismatches with the target sequence may be most preferable for a particular application.

[0575] Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and can therefore be used to specifically inhibit protein synthesis by target genes. The effect of antisense oligonucleotides in inhibiting protein synthesis has been well established. For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. Patent Nos. 5,739,119 and 5,759,829, each of which is incorporated herein by reference). Furthermore, examples of antisense inhibition have been demonstrated for the nuclear protein cyclin, the multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF (Jaskulski et al., Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989; 1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun. 15; 57(2):310-20; U.S. Pat. Nos. 5,801,154, 5,789,573, 5,718,709, and 5,610,288, each of which is incorporated herein by reference). Additionally, antisense constructs have been described that inhibit and can be used to treat various abnormal cell growth conditions, such as cancer (U.S. Pat. Nos. 5,747,470, 5,591,317, and 5,783,683, each of which is incorporated herein by reference).

[0576] Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. Selection of an antisense oligonucleotide sequence specific for a particular target sequence is based on analysis of the selected target sequence and determination of its secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or inhibit specific binding to the target mRNA in the host cell. Highly preferred target regions of mRNA include the region at or near the AUG translation initiation codon and sequences substantially complementary to the 5' region of the mRNA. These secondary structure analysis and target site selection studies can be performed, for example, using OLIGO primer analysis software (Molecular Biology Insights) version 4 and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0577] Antagomir: Antagomirs are RNA-like oligonucleotides with various modifications for pharmacological properties such as RNase protection and enhanced uptake into tissues and cells. They differ from conventional RNAs by, for example, complete 2'-O-methylation of the sugars, a phosphorothioate backbone, and a cholesterol moiety, for example, at the 3' end. Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes containing antagomir and endogenous miRNAs, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122 described in Krutzfeldt et al., Nature, 2005, 438: 685-689, the entire contents of which are expressly incorporated herein by reference. Antagomir RNAs can be synthesized using standard solid-phase oligonucleotide synthesis protocols. See U.S. Patent Application Publication Nos. 2007 / 0123482 and 2007 / 0213292, each of which is incorporated herein by reference.

[0578] Antagomir can include ligand-binding monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in U.S. Patent Application Publication No. 2005 / 0107325, the entire contents of which are incorporated herein by reference. Antagomir can have a ZXY structure as described in WO 2004 / 080406, the entire contents of which are incorporated herein by reference. Antagomir can be conjugated with an amphiphilic moiety. Exemplary amphiphilic moieties for use in oligonucleotide agents are described in WO 2004 / 080406, the entire contents of which are incorporated herein by reference.

[0579] Aptamers: Aptamers are nucleic acid or peptide molecules that bind to specific molecules with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990) and Ellington and Szostak, Nature 346:818 (1990), each of which is incorporated herein by reference in its entirety). DNA or RNA aptamers have been successfully produced to bind a variety of substances, from large proteins to small organic molecules. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997); Famulok, Curr. Opin. Struct. Biol. 9:324-9 (1999); and Hermann and Patel, Science 287:820-5 (2000), each of which is incorporated herein by reference in its entirety. Aptamers can be RNA- or DNA-based and include riboswitches. A riboswitch is a portion of an mRNA molecule that can directly bind to a small target molecule, and target binding affects gene activity. Thus, mRNA containing a riboswitch is directly involved in regulating its own activity depending on the presence or absence of the target molecule. Generally, aptamers are designed through repeated in vitro selection or the equivalent SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process to bind to various molecular targets, including small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers can be prepared by known methods, including synthetic, recombinant, and purified methods, and can be used alone or in combination with other aptamers specific to the same target. Furthermore, as described in more detail herein, the term "aptamer" specifically includes "secondary aptamers," which comprise consensus sequences obtained by comparing two or more known aptamers to a particular target.

[0580] Ribozymes: In another embodiment, the nucleic acid-lipid particles are conjugated to ribozymes, which are RNA molecular complexes containing specific catalytic domains with endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987 April 24; 49(2):211-20). For example, many ribozymes catalyze phosphoester transfer reactions with high specificity, often cleaving only one of multiple phosphates in an oligonucleotide substrate (Cech et al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 December 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14; 357(6374):173-6). This specificity results from the requirement that the substrate bind to the ribozyme's internal guide sequence ("IGS") through specific base-pairing interactions prior to chemical reaction.

[0581] Currently, at least six basic types of naturally occurring enzymatic RNAs are known. Each catalyzes the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions, thereby cleaving other RNA molecules. Generally, enzymatic nucleic acids first bind to and act on a target RNA. This binding occurs via the target-binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and binds to the target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. Strategic cleavage of such a target RNA disrupts its ability to direct synthesis of the encoded protein. After an enzymatic nucleic acid binds and cleaves an RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0582] Enzymatic nucleic acid molecules can be formed, for example, with hammerhead, hairpin, hepatitis delta virus, group I intron, or RNase P RNA (in conjunction with an RNA guide sequence), or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described in Rossi et al. Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Specific examples of hairpin motifs are described in Hampel et al. (EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13; 28(12):4929-33, Hampel et al., Nucleic Acids Res. 1990 Jan. 25; 18(2):299-304, and U.S. Pat. No. 5,631,359. An example of a hepatitis delta virus motif is described in Perrotta and Been, Biochemistry. 1992 Dec. 1; 31(47):11843-52. An example of an RNase P motif is described in Guerrier-Takada et al., Cell. 1983 Dec. 35(3 Pt 2):849-57. A Neurospora VS RNA ribozyme motif is described in Collins (Saville and Collins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct. 1; 88(19):8826-30; and Collins and Olive, Biochemistry. 1993 Mar. 23; 32(11):2795-9). An example of a group I intron is described in U.S. Pat. No. 4,987,071. An important feature of the enzymatic nucleic acid molecule used is that it has a specific substrate binding site complementary to one or more DNA or RNA regions of the target gene, and that it has a nucleotide sequence within or surrounding that substrate binding site that confers RNA cleavage activity to the molecule.Thus, ribozyme constructs need not be limited to the particular motifs described herein.

[0583] Methods for generating ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed as described in International Applications WO 93 / 23569 and WO 94 / 02595 (each incorporated herein by reference) and synthesized for in vitro and in vivo testing as described therein.

[0584] Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, modifications that prevent degradation by serum ribonucleases (see, e.g., International Applications WO 92 / 07065, WO 93 / 15187, and WO 91 / 03162, European Patent Application Publication No. 92110298.4, U.S. Pat. No. 5,334,711, and International Application WO 94 / 13688, which describe various chemical modifications that can be made to the sugar portion of enzymatic RNA molecules), modifications that increase efficiency within cells, and removal of stem II bases to shorten RNA synthesis time and reduce chemical requirements.

[0585] Immunostimulatory oligonucleotides: Nucleic acids associated with lipid particles can be immunostimulatory, including immunostimulatory oligonucleotides (ISS, single-stranded or double-stranded) that can induce an immune response when administered to a mammalian or other patient subject. ISSs include, for example, specific palindromes that lead to hairpin secondary structures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076, which is incorporated by reference in its entirety), or CpG motifs, and other known ISS features (such as multiple G domains; see WO 96 / 11266, which is incorporated by reference in its entirety).

[0586] The immune response can be an innate immune response or an adaptive immune response. The immune system is divided into a more innate immune system and an adaptive immune system in vertebrates, the latter of which is further divided into humoral and cellular components. In some embodiments, the immune response can be mucosal.

[0587] In some embodiments, the immunostimulatory nucleic acid is immunostimulatory only when administered in combination with a lipid particle, and is not immunostimulatory when administered in a "free form." Such oligonucleotides are considered immunostimulatory.

[0588] An immunostimulatory nucleic acid is considered non-sequence-specific if it does not need to specifically bind to and reduce the expression of a target polynucleotide in order to elicit an immune response. Thus, a particular immunostimulatory nucleic acid may contain a sequence that corresponds to a region of a naturally occurring gene or mRNA, yet still be considered a non-sequence-specific immunostimulatory nucleic acid.

[0589] In some embodiments, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide having a methylated cytosine. In some embodiments, the nucleic acid comprises a single CpG dinucleotide, wherein the cytosine within the CpG dinucleotide is methylated. In another embodiment, the nucleic acid comprises at least two CpG dinucleotides, wherein at least one cytosine within the CpG dinucleotide is methylated. In a further embodiment, each cytosine within the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises multiple CpG dinucleotides, wherein at least one of the CpG dinucleotides comprises a methylated cytosine.

[0590] Bonds between nucleotide-based enhancer units, linker units, nucleic acid agents, and ligands In some embodiments, the bond between the nucleotide-based enhancer unit and the linker unit is a bond.

[0591] In some embodiments, the bond between the nucleotide-based enhancer unit and the linker unit is a moiety (eg, a moiety that includes a cleavable group).

[0592] In some embodiments, the bond between the nucleotide-based enhancer unit and the linker unit comprises a -C(=O)- linked to the linker unit.

[0593] In some embodiments, the linkage between the nucleotide-based enhancer unit and the nucleic acid agent is a bond.

[0594] In some embodiments, the linkage between the nucleotide-based enhancer unit and the nucleic acid agent is a moiety (eg, a moiety that includes a cleavable group).

[0595] In some embodiments, the bond between the nucleotide-based enhancer unit and the ligand is a bond.

[0596] In some embodiments, the bond between the nucleotide-based enhancer unit and the ligand is a moiety (eg, a moiety that includes a cleavable group).

[0597] In some embodiments, the bond between the linker unit and the nucleic acid agent is a bond.

[0598] In some embodiments, the bond between the linker unit and the nucleic acid agent is a moiety (eg, a moiety that includes a cleavable group).

[0599] In some embodiments, the bond between the linker unit and the ligand is a bond.

[0600] In some embodiments, the bond between the linker unit and the ligand is a moiety (eg, a moiety that includes a cleavable group).

[0601] In some embodiments, the bond between the linker unit and the ligand comprises a -C(=O)- attached to the linker unit.

[0602] The bonds between the nucleotide-based enhancer unit, the linker unit, the nucleic acid agent, and the ligand can be cleavable or non-cleavable groups.Suitable groups include, for example, -NR-, -C(=O)-, -C(=O)NH-, -S(=O)-, -S(=O)2-, -S(=O)2NH-, or, but are not limited to, alkylene, alkenylene, alkynylene, arylalkylene, arylalkenylene, arylalkynylene, heteroarylalkylene, heteroarylalkenylene, heteroarylalkynylene, heterocyclylalkylene, heterocyclylalkenylene, heterocyclylalkynylene, arylene, heteroarylene, heterocyclylene, cycloalkyn ... alkylene, cycloalkenylene, alkylarylalkylene, alkylarylalkenylene, alkylarylalkynylene, alkenylarylalkylene, alkenylarylalkenylene, alkenylarylalkynylene, alkynylarylalkylene, alkynylarylalkenylene, alkynylarylalkynylene, alkylheteroarylalkylene, alkylheteroarylalkenylene, alkylheteroarylalkynylene, alkenylheteroarylalkylene, alkenylheteroarylalkenylene, Alkenylheteroarylalkynylene, alkynylheteroarylalkylene, alkynylheteroarylalkenylene, alkynylheteroarylalkynylene, alkylheterocyclylalkylene, alkylheterocyclylalkenylene, alkylheterocyclylalkynylene, alkenylheterocyclylalkylene, alkenylheterocyclylalkenylene, alkenylheterocyclylalkynylene, alkynylheterocyclylalkylene, alkynylheterocyclylalkenylene, alkynylheterocyclylalkynylene, alkyl and alkylarylene, alkenylarylene, alkynylarylene, alkylheteroarylene, alkenylheteroarylene, alkynylheteroarylene, and the like, each of which may be substituted or unsubstituted, and in which one or more methylenes may be interrupted or terminated by -O-, -S-, -S(=O)-, -S(=O)2-, -NR-, -C(=O)-, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle (wherein R is hydrogen, acyl, aliphatic, or substituted aliphatic).

[0603] A cleavable group is one that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties that hold the group together. In preferred embodiments, the cleavable group is cleaved at least 10 times faster, and preferably at least 100 times faster, in the target cell or under a first reference condition (e.g., which can be selected to mimic or represent conditions found within an cell) than in the subject's blood or under a second reference condition (e.g., which can be selected to mimic or represent conditions found in blood or serum).

[0604] Cleavable groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degrading molecules. Generally, cleaving agents are more abundant and present at higher levels or activity in cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a particular substrate or have no substrate specificity (e.g., intracellular oxidases or reductases or reducing agents such as mercaptans that can degrade redox-cleavable groups by reduction), esterases, endosomes, or substances that can create an acidic environment (e.g., pH 5 or less), enzymes that act as normal acids to hydrolyze or degrade acid-labile groups, peptidases (which may be substrate-specific), and phosphatases.

[0605] Cleavable groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. The pH of endosomes is more acidic, ranging from 5.5 to 6.0, and the pH of lysosomes is even more acidic, approximately 5.0. Some linkers have cleavable groups that cleave at a preferred pH, thereby releasing the cationic lipid from the ligand intracellularly or into the desired cellular compartment.

[0606] The conjugate may contain a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the conjugate varies depending on the target cell. For example, a liver-targeting ligand can be attached to a cationic lipid via a chemical moiety that contains an ester group. Liver cells are rich in esterases, so this group is cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0607] Coupling groups containing peptide bonds can be used to target cell types rich in peptidases, such as hepatocytes and synoviocytes.

[0608] In general, the suitability of a candidate cleavable group can be evaluated by testing the ability of a degradation agent (or condition) to cleave the candidate group. It may also be desirable to test the candidate cleavable group's ability to withstand cleavage when contacted with blood or other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions can be determined, with the first condition selected to be indicative of cleavage in target cells and the second condition selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and further confirm with whole animal evaluations. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0609] Redox-cleavable groups. One class of cleavable groups is redox-cleavable groups that are cleaved by reduction or oxidation. An example of a reductively cleavable group is a disulfide linkage (-SS-). To determine whether a candidate cleavable group is a suitable "reductively cleavable linkage," or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be referenced. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0610] Phosphate-based cleavable groups. Phosphate-based cleavable groups are cleaved by agents that degrade or hydrolyze phosphate groups. Examples of agents that cleave phosphate groups within cells include enzymes such as intracellular phosphatases. In some embodiments, the phosphate-based linking group is -OP(=O)(OR k )-O-, -OP(=S)(OR k )-O-, -OP(=S)(SR k )-O-, -SP(=O)(OR k )-O-, -OP(=O)(OR k )-S-, -SP(=O)(OR k )-S-, -OP(=S)(OR k )-s-, -SP(=S)(OR k )-O-, -OP(=O)(Rk )-O-, -OP(=S)(R k )-O-, -SP(=O)(R k )-O-, -SP(=S)(R k )-S- or -OP(=S)(R k )-S-. In some embodiments, the phosphate-based linking group is -OP(=O)(OH)-O-, -OP(=S)(OH)-O-, -OP(=S)(SH)-O-, -SP(=O)(OH)-O-, -OP(=O)(OH)-S-, -SP(=O)(OH)-S-, -SP(=S)(OH)-O-, -OP(=O)(H)-O-, -OP(=S)(H)-O-, -SP(=O)(H)-O-, -SP(=O)(H)-S-, or -OP(=S)(H)-S-. In some embodiments, the phosphate-based linking group is -OP(=O)(OH)-O-.

[0611] Acid-cleavable groups. Acid-cleavable groups are linking groups that are cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less) or by an agent such as an enzyme that can act as a normal acid. Within cells, certain low-pH organelles, such as endosomes and lysosomes, provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The general formula of an acid-cleavable group is represented by -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0612] Ester-based cleavable groups. Ester-based cleavable groups are cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The general formula for an ester-cleavable linking group is -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0613] Peptide-based cleavable groups. Peptide-based cleavable groups are cleaved by enzymes, such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to generate oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-), which are formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to generate peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to generate peptides and proteins, and do not include the entire amide functionality. The general formula for a peptide-based cleavable linking group is "-NHCHR A C(O)NHCHR B C(O)-" and R A and R Bare the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above. As used herein, "carbohydrate" refers to either a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having a carbohydrate moiety, consisting of one or more monosaccharide units, each with at least six carbon atoms (linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides, each containing approximately 4 to 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 or greater (preferably C5-C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5-C8).

[0614] Synthesis method In some aspects, the disclosure provides methods of preparing the compounds (eg, nucleotide-based enhancers) described herein.

[0615] In some aspects, the disclosure provides compounds obtainable or obtained by the methods for preparing compounds (e.g., nucleotide-based enhancers) described herein.

[0616] In some aspects, the present invention provides intermediates described herein that are suitable for use in methods of preparing compounds described herein (e.g., nucleotide-based enhancers).

[0617] The compounds of the present disclosure can be prepared by any suitable method known in the art. Specific processes for preparing these compounds are described in more detail in the accompanying examples.

[0618] In both the description of synthetic methods provided herein and referenced synthetic methods used to prepare starting materials, it is understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of experiment, and work-up procedures, can be selected by one skilled in the art.

[0619] One skilled in the art of organic synthesis understands that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions used.

[0620] It will be understood that during the synthesis of the compounds of the present invention in the processes defined herein, or during the synthesis of certain starting materials, it may be desirable to protect certain substituents to prevent their undesired reactions. A skilled chemist will understand when such protection is necessary and how to place and subsequently remove such protecting groups. For examples of protecting groups, see one of the many references on this subject, such as "Protective Groups in Organic Synthesis" by Theodora Green (Publisher: John Wiley & Sons, Inc.). Protecting groups can be removed by any convenient method described in the literature or known to skilled chemists that is appropriate for removing the protecting group in question, and such a method will be selected to achieve removal of the protecting group with minimal interference with other groups in the molecule. Thus, if a reactant contains a group such as amino, carboxy, or hydroxy, it may be desirable to protect that group in some of the reactions mentioned herein.

[0621] By way of example, suitable protecting groups for an amino or alkylamino group include, for example, an acyl group (e.g., an alkanoyl group such as acetyl), an alkoxycarbonyl group (e.g., methoxycarbonyl, ethoxycarbonyl, or t-butoxycarbonyl), an arylmethoxycarbonyl group (e.g., benzyloxycarbonyl), or an aroyl group (e.g., benzoyl). Suitable protecting groups for hydroxy or alkylhydroxy groups include, for example, acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), methyl ethers, or ethoxyethyl ethers (EE). Suitable protecting groups for 1,2-diols include, for example, acetals. Suitable protecting groups for 1,3-diols include, for example, tetraisopropyldisiloxanylidene (TIPDS).

[0622] The deprotection conditions for the above protecting groups necessarily vary depending on the choice of protecting group. Thus, for example, acyl groups such as alkanoyl or alkoxycarbonyl groups, or aroyl groups, can be removed by hydrolysis with a suitable base, such as an alkali metal hydroxide, for example, lithium hydroxide or sodium hydroxide. Alternatively, acyl groups such as tert-butoxycarbonyl groups can be removed by treatment with a suitable acid, for example, hydrochloric acid, sulfuric acid, phosphoric acid, or trifluoroacetic acid, and arylmethoxycarbonyl groups such as benzyloxycarbonyl groups can be removed by hydrogenation over a catalyst such as palladium on carbon or by treatment with a Lewis acid, for example, boron tris(trifluoroacetic acid). Suitable alternative protecting groups for primary amino groups include, for example, phthaloyl groups, which can be removed by treatment with an alkylamine (e.g., dimethylaminopropylamine) or hydrazine.

[0623] Suitable protecting groups for hydroxy groups include, for example, acyl groups (e.g., alkanoyl groups such as acetyl), aroyl groups (e.g., benzoyl), or arylmethyl groups (e.g., benzyl). The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or aroyl group can be removed by hydrolysis with a suitable base such as an alkali metal hydroxide, for example, lithium, sodium hydroxide, or ammonia. Alternatively, an arylmethyl group such as a benzyl group can be removed by, for example, hydrogenation over a catalyst such as palladium on carbon.

[0624] A suitable protecting group for a carboxy group is, for example, an esterifying group (for example a methyl or ethyl group which may be removed by hydrolysis with a base such as sodium hydroxide), a tert-butyl group which may be removed by treatment with an acid (for example an organic acid such as trifluoroacetic acid), or a benzyl group which may be removed by hydrogenation over a catalyst such as palladium on carbon.

[0625] Conveniently, the reaction of the compounds is carried out in the presence of a suitable solvent, preferably inert under the respective reaction conditions. Examples of suitable solvents include, but are not limited to, hydrocarbons such as hexane, petroleum ether, benzene, toluene, or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, tetrachloromethane, chloroform, or dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol, or tert-butanol; diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE), or diisopropyl ether. ethers such as hexane; glycol ethers such as ethylene glycol monomethyl ether, monoethyl ether, or ethylene glycol dimethyl ether (diglyme); ketones such as acetone, methyl isobutyl ketone (MIBK), or butanone; amides such as acetamide, dimethylacetamide, dimethylformamide (DMF), or N-methylpyrrolidinone (NMP); nitriles such as acetonitrile; sulfoxides such as dimethyl sulfoxide (DMSO); nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate or methyl acetate; or mixtures of these solvents or mixtures with water.

[0626] The reaction temperature is suitably about -100°C to 300°C depending on the reaction step and conditions used.

[0627] The reaction time generally ranges from a few minutes to several days, depending on the reactivity of each compound and the reaction conditions. The appropriate reaction time can be easily determined by methods known in the art, such as reaction monitoring. Based on the reaction temperatures listed above, the appropriate reaction time is generally within the range of 10 minutes to 48 hours.

[0628] Moreover, by utilizing the procedures described herein in combination with ordinary skills in the art, additional compounds of the present disclosure can be readily prepared. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds.

[0629] As will be appreciated by those skilled in the art of organic synthesis, the compounds of the present disclosure are readily accessible by a variety of synthetic routes, some of which are illustrated in the accompanying examples. Those skilled in the art will readily recognize, whenever necessary or useful, what kinds of reagents and reaction conditions should be used to obtain the compounds of the present disclosure, and how to apply and adapt them in a particular case. Furthermore, some of the compounds of the present disclosure can be readily synthesized by reacting other compounds of the present disclosure under appropriate conditions, for example, by applying standard synthetic methods such as reduction, oxidation, addition, or substitution reactions to convert specific functional groups present in the compounds of the present disclosure, or their appropriate precursor molecules, to other functional groups. These methods are well known to those skilled in the art. Similarly, those skilled in the art will apply synthetic protecting groups (or protecting groups) whenever necessary or useful. Suitable protecting groups and methods for their introduction and removal are well known to those skilled in the art of chemical synthesis and are described in detail, for example, in PGM Wuts, TW Greene, "Greene's Protective Groups in Organic Synthesis," 4th edition (2006) (John Wiley & Sons).

[0630] A general route for preparing the compounds of the present application is depicted in Scheme A herein. [ka]

[0631] Compounds (eg, nucleic acid agents, nucleic acid agents containing nucleotide-based enhancers, and conjugates) were prepared by solid phase synthesis according to standard synthetic protocols.

[0632] Briefly, oligonucleotide synthesis was performed on solid support, and each nucleoside phosphoramidite was incorporated from the 3' to 5' end to prepare a single-stranded oligo. ETT or BTT was used as an activating agent for the coupling reaction. The phosphite triester (P(III)) was oxidized to the phosphate backbone using iodine-containing water / pyridine / THF, and DDTT was used to prepare the phosphorothioate linkage. Aqueous ammonium chloride was used to cleave the oligo from the solid support and globally remove the protecting groups. The crude oligo was then concentrated and purified by strong anion exchange or reverse-phase HPLC. The purified fractions were combined and concentrated.

[0633] In some cases, the oligo single strands were conjugated with target ligands (e.g., peptides, antibodies) by post-synthesis conjugation to obtain conjugates. The conjugation reaction was carried out using standard conjugation methods. The crude conjugates were further purified by strong anion exchange or reverse-phase HPLC. The purified fractions were combined and concentrated.

[0634] The synthesized single strand was then dialyzed against water using a MidiTrap G-25 column, concentrated, and the OD was measured. Equimolar amounts of the sense and antisense strands were annealed at 95°C for 5 minutes and cooled to room temperature to obtain a complex duplex with a purity of over 90%. The duplex solution was lyophilized to obtain the desired complex. The amount was calculated based on the molar amount of single strand consumed in the annealing. Biological assays

[0635] Once compounds (e.g., nucleotide-based enhancers) or conjugates designed, selected, prepared, and / or optimized by the above methods are produced, they can be characterized using a variety of assays known to those of skill in the art to determine whether the compounds, scaffolds, or conjugates have biological activity. For example, compounds, scaffolds, or conjugates can be characterized by conventional assays, including but not limited to, the assays described below, to determine whether they have the desired activity, e.g., target binding activity and / or specificity and / or stability.

[0636] Furthermore, high-throughput screening can be used to speed up analysis using such assays. As a result, it may be possible to rapidly screen the activity of the molecules described herein using techniques known in the art. General methodologies for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker, and U.S. Pat. No. 5,763,263. High-throughput assays can use one or more different assay techniques, including, but not limited to, those described below.

[0637] A variety of in vitro or in vivo biological assays may be suitable for detecting the effects of the disclosed compounds, scaffolds, or complexes, including, but not limited to, enzyme activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and assays described herein.

[0638] In some embodiments, the biological assays are described in the Examples herein.

[0639] In some aspects, the present disclosure provides pharmaceutical compositions comprising a compound, scaffold, or complex of the present disclosure as an active ingredient.

[0640] As used herein, the term "composition" is intended to encompass a product containing the specified ingredients in the specified amounts, as well as any product resulting directly or indirectly from combining the specified ingredients in the specified amounts.

[0641] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, water for injection, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. The absorption of injectable compositions can be delayed by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0642] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the above-listed ingredients as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients as listed above.For sterile powder to prepare sterile injectable solution, preparation method is vacuum drying and freeze-drying, which obtains powder of the active ingredient and any additional ingredients from the solution previously sterile-filtered.

[0643] The formulation of the present disclosure may be in the form of an aqueous solution containing an aqueous vehicle. The aqueous vehicle component may include water and at least one pharmaceutically acceptable excipient. Suitable acceptable excipients include those selected from the group consisting of solubility enhancers, chelating agents, preservatives, isotonicity agents, viscosity / suspending agents, buffers, and pH adjusters, and mixtures thereof.

[0644] Any suitable solubility enhancer may be used. Examples of solubility enhancers include cyclodextrins, such as those selected from the group consisting of hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulfated-β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, branched-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin, and mixtures thereof.

[0645] Any suitable chelating agent can be used, examples of suitable chelating agents include those selected from the group consisting of ethylenediaminetetraacetic acid and its metal salts, edetate disodium, edetate trisodium, and edetate tetrasodium, and mixtures thereof.

[0646] Any suitable preservative can be used. Examples of preservatives include quaternary ammonium salts, such as those selected from the group consisting of benzalkonium halides (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetylpyridinium chloride, benzyl bromide, phenylmercuric nitrate, phenylmercuric acetate, phenylmercuric neodecanoate, merthiolate, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl parahydroxybenzoate, propylaminopropyl biguanide, and butyl parahydroxybenzoate, and sorbic acid, and mixtures thereof.

[0647] The aqueous vehicle may contain a tonicity agent to adjust the tonicity (osmotic pressure), which may be selected from the group consisting of glycols (such as propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, and mixtures thereof.

[0648] The formulation may contain a pH adjuster to adjust the formulation to an acceptable pH (typically within a pH range of about 5.0 to about 9.0, more preferably about 5.5 to about 8.5, particularly about 6.0 to about 8.5, about 7.0 to about 8.5, about 7.2 to about 7.7, about 7.1 to about 7.9, or about 7.5 to about 8.0). The pH adjuster is typically a mineral acid or metal hydroxide base selected from the group consisting of potassium hydroxide, sodium hydroxide, and hydrochloric acid, and mixtures thereof, preferably sodium hydroxide and / or hydrochloric acid. These acidic and / or basic pH adjusters are added to adjust the formulation to a target acceptable pH range. Therefore, it may not be necessary to use both an acid and a base; depending on the formulation, the addition of either an acid or a base may be sufficient to bring the mixture within the desired pH range.

[0649] The aqueous vehicle may also contain a buffering agent to stabilize the pH. If a buffer is used, the buffer is selected from the group consisting of phosphate buffer (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), borate buffer (such as boric acid or a salt thereof including disodium tetraborate), citrate buffer (such as citric acid or a salt thereof including sodium citrate), and ε-aminocaproic acid, and mixtures thereof.

[0650] According to a further aspect of the present disclosure, there is provided a pharmaceutical composition comprising a compound of the present disclosure as defined above, or a pharmaceutically acceptable salt, hydrate or solvate thereof, together with a pharmaceutically acceptable diluent or carrier.

[0651] Compositions of the present disclosure may be in a form suitable for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (e.g., as a finely divided powder or liquid aerosol), administration by insufflation (e.g., as a finely divided powder), or parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular administration, or as a suppository for rectal administration).

[0652] The compositions of the present disclosure may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more coloring agents, sweeteners, flavoring agents, and / or preservatives.

[0653] An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat or prevent, slow the progression of, and / or alleviate the symptoms associated with an inflammasome-associated condition referred to herein.

[0654] An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat, slow the progression of, and / or alleviate the symptoms associated with an inflammasome-associated condition referred to herein.

[0655] The size of the dose of a compound of formula (I) or (II) administered for therapeutic or prophylactic purposes will naturally vary according to well-known principles of medicine, depending on the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration.

[0656] How to use In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) expression of a target gene in a subject, comprising administering to the subject a conjugate of the present disclosure.

[0657] In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject, comprising administering to the subject a conjugate of the present disclosure.

[0658] In some aspects, the present disclosure provides a method of delivering a nucleic acid agent to a subject, comprising administering to the subject a complex of the present disclosure.

[0659] In some aspects, the present disclosure provides a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate of the present disclosure.

[0660] In some aspects, the present disclosure provides a conjugate of the present disclosure for modulating (e.g., reducing or eliminating) expression of a target gene in a subject.

[0661] In some aspects, the present disclosure provides a conjugate of the present disclosure for modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject.

[0662] In some aspects, the present disclosure provides a conjugate of the present disclosure for delivering a nucleic acid agent to a subject.

[0663] In some aspects, the present disclosure provides a conjugate of the present disclosure for treating or preventing a disease in a subject in need thereof.

[0664] In some aspects, the disclosure provides use of a conjugate of the disclosure in the manufacture of a medicament for modulating (e.g., reducing or eliminating) expression of a target gene in a subject.

[0665] In some aspects, the disclosure provides use of a conjugate of the disclosure in the manufacture of a medicament for modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject.

[0666] In some aspects, the present disclosure provides use of a conjugate of the present disclosure in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0667] In some aspects, the present disclosure provides use of a conjugate of the present disclosure in the manufacture of a medicament for treating or preventing a disease in a subject in need thereof.

[0668] In some embodiments, the subject is a cell.

[0669] In some embodiments, the target is a central nervous system cell, a peripheral nervous system cell, an adipocyte, a muscle cell, a cardiac cell, and / or a pancreatic cell. In some embodiments, the target is a pancreatic cell. In some embodiments, the target is a pancreatic beta cell.

[0670] In some embodiments, the subject is a tissue.

[0671] In some embodiments, the target tissue is central nervous system tissue, peripheral nervous system tissue, adipose tissue, muscle tissue, cardiac tissue, and / or pancreatic tissue, hi some embodiments, the target tissue is pancreatic tissue.

[0672] In some embodiments, the subject is a human.

[0673] In some embodiments, the target gene is Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, HBV, HCV, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA (p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21 (WAF1 / CIP1) gene, p27 (KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutation of a tumor suppressor gene, p53 tumor suppressor gene, LDHA, or any combination thereof.

[0674] In some embodiments, the disease is characterized by unwanted expression of the target gene.

[0675] In some embodiments, said administration results in reducing or eliminating expression of said target gene in said subject.

[0676] In some embodiments, the disease is a viral infection, such as an HCV, HBV, HPV, HSV, or HIV infection.

[0677] In some embodiments, the disease is cancer.

[0678] In some embodiments, the cancer is biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain tumor, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML). , chronic myelomonocytic leukemia (CMML), liver cancer, liver carcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal carcinoma, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary tumor, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma.

[0679] In some embodiments, the cancer is liver cancer, liver carcinoma, hepatocellular carcinoma, hepatocellular carcinoma, cholangiocarcinoma, or hepatoblastoma.

[0680] In some embodiments, the disease is proliferative, inflammatory, autoimmune, neurological, ophthalmic, respiratory, metabolic, cutaneous, auditory, hepatic, renal, or infectious, hi some embodiments, the disease is a disease of the liver.

[0681] In some embodiments, the condition is a disease of the central nervous system, peripheral nervous system, adipose tissue, muscle, heart, and / or pancreas. In some embodiments, the condition is a disease of the pancreas.

[0682] definition Unless otherwise stated, the following terms used in the specification and claims have the meanings indicated below.

[0683] Without intending to be limited by this description, it is understood that while various options for variables are described herein, the present disclosure is intended to encompass workable embodiments having combinations of options, and the present disclosure may be interpreted to exclude inoperable embodiments resulting from a particular combination of options.

[0684] As used herein, "alkyl," "C1, C2, C3, C4, C5, or C6 alkyl," or "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, or C6 straight-chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, or C6 branched saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl is intended to include C1, C2, C3, C4, C5, and C6 alkyl groups. Examples of alkyl include, but are not limited to, moieties having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl. In some embodiments, a straight-chain or branched alkyl has 6 or fewer carbon atoms (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and in other embodiments, a straight-chain or branched alkyl has 4 or fewer carbon atoms.

[0685] As used herein, the term "optionally substituted alkyl" refers to an unsubstituted alkyl or an alkyl having specified substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0686] Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both unsubstituted moieties and moieties having one or more of the specified substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethylpiperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

[0687] As used herein, the term "substituted" means that any one or more hydrogen atoms on the designated atom are replaced with one selected from the indicated group, provided that the replacement does not exceed the normal valence of the designated atom and that the replacement results in a stable compound. When the substituent is oxo or keto (i.e., =0), two hydrogen atoms on the atom are replaced. Keto substituents do not occur in aromatic moieties. As used herein, a ring double bond is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N). "Stable compound" and "stable structure" are meant to indicate a compound that has sufficient strength to be isolated to a useful degree of purity from a reaction mixture and formulated into an effective therapeutic agent.

[0688] When a bond to a substituent is shown to cross the bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom through which it is attached to the remainder of the compound of a given formula, then the substituent may be bonded through any atom in that formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0689] When any variable (e.g., R) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0 to 2 R moieties, that group may optionally be substituted with up to 2 R moieties, and R at each occurrence is selected independently of the definition of R. Also, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0690] As used herein, the term "hydroxy" or "hydroxyl" includes groups containing --OH or --O--.

[0691] The term "halo" or "halogen" as used herein refers to fluoro, chloro, bromo, and iodo.

[0692] The terms "haloalkyl" or "haloalkoxy" refer to an alkyl or alkoxy substituted with one or more halogen atoms.

[0693] As used herein, the term "optionally substituted haloalkyl" refers to an unsubstituted haloalkyl having specified substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0694] As used herein, the term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently bonded to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy group can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.

[0695] As used herein, the term "overhang" refers to a section of unpaired nucleotides at the end (e.g., the 3' end or the 5' end) of an oligonucleotide strand. An overhang at the 3' end of an oligonucleotide strand is a "3' overhang," and an overhang at the 5' end of an oligonucleotide strand is a "5' overhang."

[0696] As used herein, the expressions "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," "selected from the group consisting of A, B, and C," "selected from A, B, and C," and the like are used interchangeably and, unless otherwise specified, all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0697] It should be understood that the present disclosure provides methods for synthesizing the compounds, scaffolds, and conjugates described herein. The present disclosure also provides detailed methods for synthesizing the various disclosed compounds, scaffolds, and conjugates according to the schemes described herein and in the Examples.

[0698] Throughout the description, when a composition is described as having, including, or comprising certain components, it is understood that the composition is also intended to consist essentially of, or consist of, the recited components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process consists essentially of, or consists of, the recited process steps. Furthermore, it should be understood that the order of steps for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be performed simultaneously.

[0699] It will be understood that the synthetic processes of the present disclosure can tolerate a wide variety of functional groups, and thus can employ a variety of substituted starting materials. These processes generally provide the desired final compound at or near the completion of the overall process, although in some cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt.

[0700] It will be understood that the compounds, scaffolds, and complexes of the present disclosure can be prepared in a variety of ways by employing standard synthetic methods and procedures known to those skilled in the art or that will be apparent to those skilled in the art in light of the teachings herein, using commercially available starting materials, compounds known in the literature, or readily prepared intermediates. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field, including, but not limited to, classic textbooks (Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition, John Wiley & Sons: New York, 2001, Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) are useful and recognized reference textbooks of organic synthesis known to those skilled in the art.

[0701] Those skilled in the art will note that the order of certain steps, such as the introduction and removal of protecting groups, may be varied during the reaction sequences and synthetic schemes described herein. Those skilled in the art will recognize that certain groups may require protection from the reaction conditions through the use of protecting groups. Protecting groups can also be used to distinguish between similar functional groups within a molecule. For a list of protecting groups and methods for introducing and removing these groups, see Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons: New York, 1999.

[0702] Unless otherwise specified, any reference to a method of treatment or prevention is understood to include the use of the compounds, scaffolds, and conjugates to provide such treatment or prevention. Furthermore, unless otherwise specified, any reference to a method of treatment or prevention is understood to include the use of the compounds, scaffolds, and conjugates to prepare a medicament for treating or preventing such a condition. Said treatment or prevention includes treatment or prevention in humans or non-human animals, including rodents and other disease models.

[0703] Unless otherwise specified, any description of a method of treatment will be understood to include the use of the compounds, scaffolds, and conjugates to provide treatment as described herein. Furthermore, unless otherwise specified, any description of a method of treatment will be understood to include the use of the compounds, scaffolds, and conjugates to prepare a medicament for treating such a condition. Such treatment includes the treatment of humans or non-human animals, including rodents and other disease models.

[0704] As used herein, the term "subject" is interchangeable with the term "subject in need," and both refer to a subject having a disease or at high risk of developing a disease. "Subject" includes mammals. The mammal can be, for example, a human or a suitable non-human mammal, such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. The subject can also be a bird or poultry. In some embodiments, the mammal is a human. The subject in need can be a subject previously diagnosed or identified as having a disease or disorder disclosed herein. The subject in need can also be a subject suffering from a disease or disorder disclosed herein. Alternatively, the subject in need can be a subject at high risk relative to the general population for developing such a disease or disorder (i.e., a subject more susceptible to developing such a disorder relative to the general population). The subject in need can have a refractory or resistant disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant to treatment at the start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof has unsuccessfully undergone all known effective treatments for the disease or disorder disclosed herein. In some embodiments, the subject in need thereof has undergone at least one prior treatment.

[0705] As used herein, the term "treating" or "treat" refers to the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes administering a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph, or solvate thereof, to alleviate the symptoms or or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treating" may also include treatment of cells in vitro or animal models. It should be understood that reference to "treating" or "treatment" includes alleviation of established symptoms of the condition. Thus, "treating" or "treatment" of a condition, disorder, or symptom includes: (1) delaying the onset of clinical symptoms of the condition, disorder, or condition occurring in a person who may be suffering from or predisposed to the condition, disorder, or condition, but who has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, alleviating, or delaying the progression of the disease or its recurrence (in the case of maintenance therapy) or at least one clinical or subclinical symptom thereof; or (3) palliating or alleviating the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or subclinical symptom thereof.

[0706] It is understood that the compounds, scaffolds, and complexes of the present disclosure, or pharmaceutically acceptable salts, polymorphs, or solvates thereof, can or may be used to prevent the associated disease, condition, or disorder, or to identify suitable candidates for such purposes.

[0707] As used herein, the terms "preventing," "prevent," or "protecting against" refer to reducing or eliminating the onset of symptoms or complications of such a disease, condition, or disorder.

[0708] It should be understood that the present invention also provides pharmaceutical compositions comprising any compound, scaffold, or complex described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0709] As used herein, the term "pharmaceutical composition" refers to a formulation containing a compound, scaffold, or complex of the present disclosure in a form suitable for administration to a subject. In some embodiments, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form may be in any of a variety of forms, such as, for example, a capsule, an IV bag, a tablet, a single pump of an aerosol inhaler, or a vial. The amount of active ingredient (e.g., a disclosed compound or a salt, hydrate, solvate, or isomeric formulation thereof) in a unit dose of the composition is an effective amount and will vary depending on the specific treatment involved. Those skilled in the art will appreciate that dosages may need to be modified periodically depending on the age and condition of the patient. The dosage also varies depending on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intraspinal, intranasal, and the like. Dosage forms for topical or transdermal administration of a compound of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In some embodiments, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and any needed preservatives, buffers, or propellants.

[0710] As used herein, the term "pharmaceutically acceptable" refers to compounds, scaffolds, complexes, anions, cations, materials, compositions, carriers, and / or dosage forms that are suitable, within the scope of sound medical judgment, 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.

[0711] As used herein, the term "pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipient" includes both one and more than one such excipient.

[0712] It is understood that pharmaceutical compositions of the present disclosure are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, oral (e.g., ingestion), inhalation, transdermal (topical), and transmucosal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and a tonicity adjuster such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0713] It is understood that the compounds or pharmaceutical compositions of the present disclosure can be administered to a subject by many well-known methods currently used for chemotherapy treatment. For example, the compounds of the present disclosure can be injected into the bloodstream or body cavity, or taken orally, or applied through the skin using a patch. The selected dose should be sufficient to constitute effective treatment, but not so high as to cause unacceptable side effects. Preferably, the patient's disease state (e.g., the disease or disorder disclosed herein) status and health status should be closely monitored during and for a reasonable period after treatment.

[0714] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent a specified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected by any assay method known in the art. The precise effective amount for a subject will vary depending on the subject's weight, size, health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a particular situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0715] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent to treat or ameliorate a specified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. This effect can be detected by any assay method known in the art. The precise effective amount for a subject will vary depending on the subject's weight, size, health, the nature and extent of the condition, and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a particular situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0716] It is understood that for any compound, the therapeutically effective amount can be estimated initially either in cell culture assays, for example, of tumor cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine effective doses and routes of administration in humans. Therapeutic / prophylactic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 (the dose that is lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is called the therapeutic index, and the LD 50 / ED 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage can vary within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0717] Dosage and administration are adjusted to provide adequate levels of the active agent or to maintain the desired effect. Factors to consider include the severity of the disease, the subject's general health, the subject's age, weight, sex, and diet, the time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment. Long-acting pharmaceutical compositions can be administered every 3-4 days, every week, or every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0718] Pharmaceutical compositions containing the active compounds of the present disclosure can be generally prepared by known methods, for example, conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, including excipients and / or adjuvants that facilitate the processing of the active compounds into pharmaceutically usable preparations. Of course, the appropriate formulation depends on the selected route of administration.

[0719] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, water for injection, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. The absorption of injectable compositions can be delayed by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0720] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the above-listed ingredients as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients as listed above.For sterile powder to prepare sterile injectable solution, preparation method is vacuum drying and freeze-drying, which obtains powder of the active ingredient and any additional ingredients from the solution previously sterile-filtered.

[0721] Oral compositions generally contain an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied to the mouth and expectorated by rinsing, or swallowed. Pharmaceutically compatible binding agents and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, troches, and the like may contain the following ingredients, or compounds of similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, orange flavor, and the like.

[0722] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0723] For intranasal administration, the compound is administered in the form of a solution or solid formulation. In some embodiments, the compound is delivered in a solution as a spray, drop, or smear. In some embodiments, the compound is delivered as a powder. In some embodiments, the compound is included in a kit further comprising an intranasal applicator.

[0724] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams, as generally known in the art.

[0725] The active compounds can be prepared with pharmaceutically acceptable carriers that protect the compound against rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals. Liposomal suspensions (including liposomes that target infected cells using monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0726] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically discrete unit suitable as a unitary dosage for treatment subject, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined and directly depends on the inherent properties of active compound and the specific therapeutic effect to be achieved.

[0727] For therapeutic applications, dosages of pharmaceutical compositions used in accordance with the present disclosure will vary depending on the agent, the recipient patient's age, weight, and clinical condition, and the experience and judgment of the clinician or practitioner administering the treatment, among other factors that will influence the selected dosage. Generally, the dosage will be sufficient to delay, preferably cause regression of, and preferably complete regression of, the symptoms of a disease or disorder disclosed herein. Dosages may range from about 0.01 mg / kg to about 5000 mg / kg per day. An effective amount of a pharmaceutical agent is one that produces an objectively identifiable improvement as determined by a clinician or other qualified observer. Improved survival and growth indicate regression. As used herein, the term "dosage-effective regime" refers to the amount of active compound that produces the desired biological effect in a subject or cell.

[0728] It will be appreciated that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0729] The compounds, scaffolds, or complexes of the present disclosure may further form salts, and it is understood that all of these forms are also included within the scope of the claimed disclosure.

[0730] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present disclosure in which the parent compound has been modified by making acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic and organic acid salts of basic residues such as amines, alkali organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic and organic acids. For example, such conventional non-toxic salts include 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycollyarsanilic acid, hexylresorcylic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isopropyl alcohol, methyl alcohol, methyl methyl ketone, ... These include, but are not limited to, salts derived from inorganic and organic acids selected from sethionic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, napsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and common amino acids such as glycine, alanine, phenylalanine, and arginine.

[0731] In some embodiments, the pharmaceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, a magnesium salt, a diethylamine salt, a choline salt, a meglumine salt, a benzathine salt, a tromethamine salt, an ammonia salt, an arginine salt, or a lysine salt.

[0732] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, and the like. The present disclosure also encompasses salts formed by replacing an acidic proton present in the parent compound with a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or by coordination with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It is understood that in the salt form, the ratio of the compound to the cation or anion of the salt may be 1:1, or any ratio other than 1:1, for example, 3:1, 2:1, 1:2, or 1:3.

[0733] It is understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystalline forms (polymorphs) of the same salt as defined herein.

[0734] The compounds or pharmaceutically acceptable salts thereof may be administered by oral, nasal, transdermal, pulmonary, inhalation, buccal, sublingual, intraperitoneal, subcutaneous, intramuscular, intravenous, rectal, intrapleural, intraspinal, and parenteral routes. In some embodiments, the compounds are administered orally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0735] The dosing regimen using the compound is selected depending on various factors, such as the type, species, age, weight, sex, and health of the patient, the severity of the condition being treated, the route of administration, the patient's renal and hepatic function, and the particular compound or salt thereof being used. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to prevent, treat, or arrest the progression of the condition. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the drug required to treat or arrest the progression of the condition.

[0736] Techniques for formulating and administering the compounds of this disclosure are described in Remington: The Science and Practice of Pharmacy, 1999. th edition, Mack Publishing Co., Easton, PA (1995). In one embodiment, the compounds described herein, and their pharmaceutically acceptable salts, are used in pharmaceutical formulations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous organic solutions. The compound will be present in such pharmaceutical compositions in an amount sufficient to provide the desired dosage within the range described herein.

[0737] All percentages and ratios used herein are by weight unless otherwise specified. Other features and advantages of the present disclosure will be apparent from the various examples. The examples provided illustrate various components and methodologies useful in practicing the present disclosure. These examples do not limit the claimed disclosure. Based on this disclosure, one skilled in the art will be able to identify and use other components and methodologies useful in practicing the present disclosure.

[0738] In the synthetic schemes described herein, for simplicity, compounds may be depicted in one particular configuration. Such a particular configuration should not be construed as limiting the disclosure to one or another isomer, tautomer, positional isomer, or stereoisomer, nor does it exclude mixtures of isomers, tautomers, positional isomers, or stereoisomers. However, it is understood that a given isomer, tautomer, positional isomer, or stereoisomer may have a higher level of activity than another isomer, tautomer, positional isomer, or stereoisomer.

[0739] All publications and patent documents cited herein are incorporated by reference as if each individual publication or document was specifically and individually indicated to be incorporated by reference. Citation of publications and patent documents is not an admission of the relevant prior art, nor is it an admission of the contents or date thereof. While the invention has been described herein, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and the following examples are intended to be illustrative and not limiting of the scope of the claims.

[0740] Illustrative Embodiments Exemplary Embodiment 1. A nucleotide-based enhancer that is a compound or a pharmaceutically acceptable salt thereof, comprising 2 to 30 nucleotides, each nucleotide independently having the formula (I) or (II): [ka] is expressed as wherein each * independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer, or represents H when the nucleotide is at the 5' terminus of the nucleotide-based enhancer; each ** independently represents an attachment of the nucleotide-based enhancer to the 5' position of another nucleotide, or represents H if the nucleotide is at the 2' or 3' terminus of the nucleotide-based enhancer; B is H, C1-C6 alkyl, or a nucleobase moiety; V is -O-, -NR V - or -C(R V )2-, Each R V are independently H or C1-C6 alkyl; X is H, halogen, or -OR X That is, R X is H, C1-C 12 Alkyl, or -(C1-C6 alkyl)-(C6-C 10 aryl), and the C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) optionally includes one or more R Xa or replaced by R X and R 4 together form a C1-C6 alkylene, Each R Xa are independently halogen, C1-C6 alkyl, or —O—(C1-C6 alkyl), wherein said C1-C6 alkyl or —O—(C1-C6 alkyl) is optionally substituted with one or more halogens; Y is -P(R Y )-, -P(OR Y )-, -P(N(R Y )2)-, -P(=O)(OR Y )-, -P(=O)(R Y )-, -P(=S)(OR Y )-, -P(=S)(R Y )-, -P(=O)(SR Y )-, or -P(=S)(SR Y )-is, Each R Y are independently H or C1-C6 alkyl optionally substituted with one or more halogens or cyano; R 1 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 3is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens, or R 4 and R X together form a C1-C6 alkylene, and Each R 5 are independently H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens.

[0741] Exemplary embodiment 2. (i) one or more nucleic acid agents; (ii) one or more ligands, and (iii) one or more nucleotide-based enhancer units, each nucleotide-based enhancer unit independently comprising 2 to 30 nucleotides, each nucleotide independently comprising [ka] A complex or a pharmaceutically acceptable salt thereof, comprising a nucleotide-based enhancer unit represented by Variables B, V, X, Y, R 1 , R 2 , R 3 , R 4 , and R 5 is described herein, wherein each # independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 5' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the complex; and A conjugate or a pharmaceutically acceptable salt thereof, wherein each ## independently represents a bond to the 5' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the conjugate.

[0742] Exemplary Embodiment 3. The nucleotide-based enhancer or conjugate of Exemplary Embodiment 1 or 2, wherein B is H.

[0743] Exemplary Embodiment 4. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 3, wherein B is a nucleobase moiety.

[0744] Exemplary Embodiment 5. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 4, wherein the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0745] Exemplary Embodiment 6. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 5, wherein the nucleobase moiety is a modified nucleobase.

[0746] Exemplary Embodiment 7. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 6, wherein the nucleobase moiety is an artificial nucleobase.

[0747] Exemplary Embodiment 8. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 7, wherein V is -O-.

[0748] Exemplary Embodiment 9. V is -NR V 9. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 8, wherein

[0749] Exemplary Embodiment 10. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 9, wherein V is -NH-.

[0750] Exemplary Embodiment 11. V is -C(R V 11. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 10, wherein:

[0751] Exemplary Embodiment 12. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 11, wherein V is -CH2-.

[0752] Exemplary Embodiment 13. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 12, wherein X is H.

[0753] Exemplary Embodiment 14. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 13, wherein X is a halogen.

[0754] Exemplary embodiment 15. X is -OR X 15. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 14, wherein:

[0755] Exemplary Embodiment 16 The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 15, wherein X is -OH.

[0756] Exemplary Embodiment 17. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 16, wherein X is -O-(C1-C6 alkyl).

[0757] Exemplary Embodiment 18. The nucleotide-based enhancer or conjugate of any one of Exemplary Embodiments 1 to 17, wherein X is -O-(C1-C6 alkyl)-O-(C1-C6 alkyl).

[0758] Exemplary Embodiment 19. X is optionally one or more R Xa -O-(C1-C6 alkyl)-(C6-C 10 19. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 18, wherein the nucleotide-based enhancer or conjugate is aryl.

[0759] Exemplary embodiment 20. R X 20. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 19, wherein

[0760] Exemplary embodiment 21. R X optionally one or more R Xa 21. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 20, wherein the nucleotide-based enhancer or conjugate is C1-C6 alkyl substituted with

[0761] Exemplary embodiment 22. R X optionally one or more R Xa -(C1-C6 alkyl)-(C6-C 10 22. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 21, wherein the nucleotide-based enhancer or conjugate is aryl.

[0762] Exemplary embodiment 23. R X -(C1-C6 alkyl)-(C6-C 10 23. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 22, wherein the nucleotide-based enhancer or conjugate is aryl.

[0763] Exemplary embodiment 24. R X and R 4 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 23, wherein: are taken together to form a C1-C6 alkylene.

[0764] Exemplary embodiment 25. Y is -P(R Y 25. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 24, wherein

[0765] Exemplary embodiment 26. Y is -P(OR Y 26. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 25, wherein

[0766] Exemplary embodiment 27. Y is -P(N(R Y 27. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 26, wherein:

[0767] Exemplary embodiment 28. Y is -P(=O)(OR Y 28. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 27, wherein

[0768] Exemplary embodiment 29. Y is -P(=O)(R Y 29. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 28, wherein

[0769] Exemplary embodiment 30. Y is -P(=S)(OR Y 30. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 29, wherein

[0770] Exemplary embodiment 31. Y is -P(=S)(R Y 31. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 30, wherein

[0771] Exemplary embodiment 32. Y is -P(=O)(SR Y 32. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 31, wherein

[0772] Exemplary embodiment 33. Y is -P(=S)(SR Y 33. The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 32, wherein

[0773] Exemplary embodiment 34. Each R Y The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 33, wherein

[0774] Exemplary embodiment 35. At least one R Y The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 34, wherein is C1-C6 alkyl optionally substituted with one or more halogens or cyano.

[0775] Exemplary embodiment 36. Each RY The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 35, wherein is C1-C6 alkyl optionally substituted with one or more halogens or cyano.

[0776] Exemplary embodiment 37. R 1 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 36, wherein

[0777] Exemplary embodiment 38. R 1 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 37, wherein is a halogen.

[0778] Exemplary embodiment 39. R 1 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 38, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0779] Exemplary embodiment 40. R 2 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 39, wherein

[0780] Exemplary embodiment 41. R 2 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 40, wherein is a halogen.

[0781] Exemplary embodiment 42. R 2 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 41, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0782] Exemplary embodiment 43. R 3 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 42, wherein

[0783] Exemplary embodiment 44. R 3The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 43, wherein is a halogen.

[0784] Exemplary embodiment 45. R 3 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 44, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0785] Exemplary embodiment 46. R 4 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 45, wherein

[0786] Exemplary embodiment 47. R 4 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 46, wherein is a halogen.

[0787] Exemplary embodiment 48. R 4 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 47, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0788] Exemplary embodiment 49. R 4 and R X The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 48, wherein: are taken together to form a C1-C6 alkylene.

[0789] Exemplary embodiment 50. Each R 5 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 49, wherein

[0790] Exemplary embodiment 51. At least one R 5 The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 50, wherein is a halogen.

[0791] Exemplary embodiment 52. At least one R 5The nucleotide-based enhancer or conjugate of any one of exemplary embodiments 1 to 51, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0792] Exemplary embodiment 53. R a , R b , R 1 , R 2 , R 3 , R 4 , and R 5 and n is H.

[0793] Exemplary Embodiment 54. At least one nucleotide of the nucleotide-based enhancer has Formula (I') or (II'): [ka] 54. The nucleotide-based enhancer of any one of exemplary embodiments 1 to 53, wherein:

[0794] Exemplary Embodiment 55. At least one nucleotide of the nucleotide-based enhancer has Formula (IA), (II-A), (III-A), or (IV-A): [ka] 55. The nucleotide-based enhancer of any one of exemplary embodiments 1 to 54, wherein:

[0795] Exemplary Embodiment 56. At least one nucleotide of the nucleotide-based enhancer has Formula (I'-A), (II'-A), (III'-A), or (IV'-A): [ka] 56. The nucleotide-based enhancer of any one of exemplary embodiments 1 to 55, wherein:

[0796] Exemplary Embodiment 57 The nucleotide-based enhancer of any one of Exemplary Embodiments 1 to 56, wherein the nucleotide-based enhancer is selected from the compounds set forth in Table L and pharmaceutically acceptable salts thereof.

[0797] Exemplary Embodiment 58. At least one nucleotide of the nucleotide-based enhancer has Formula (IB) or (II-B): [ka] 58. The nucleotide-based enhancer of any one of exemplary embodiments 1 to 57, wherein:

[0798] Exemplary Embodiment 59. At least one nucleotide of the nucleotide-based enhancer has Formula (I'-B) or (II'-B): [ka] The nucleotide-based enhancer of any one of exemplary embodiments 1 to 58, wherein

[0799] Exemplary Embodiment 60. At least one nucleotide of the nucleotide-based enhancer has formula (IC), (II-C), (III-C), or (IV-C): [ka] 60. The nucleotide-based enhancer of any one of exemplary embodiments 1 to 59, wherein:

[0800] Exemplary Embodiment 61. At least one nucleotide of the nucleotide-based enhancer has formula (I'-C), (II'-C), (III'-C), or (IV'-C): [ka] 61. The nucleotide-based enhancer of any one of exemplary embodiments 1 to 60, wherein:

[0801] Exemplary Embodiment 62 The nucleotide-based enhancer of any one of Exemplary Embodiments 1 to 61, wherein at least one nucleotide of the nucleotide-based enhancer is selected from the nucleotides listed in Table L.

[0802] Exemplary Embodiment 63 An isotopic derivative of the nucleotide-based enhancer of any one of Exemplary Embodiments 1 to 62.

[0803] Exemplary Embodiment 64 The complex of any one of Exemplary Embodiments 1 to 63, comprising double-stranded RNA.

[0804] Exemplary Embodiment 65. The conjugate of any one of Exemplary Embodiments 1 to 64, selected from the conjugates depicted in FIG. 1.

[0805] Exemplary Embodiment 66 The conjugate of any one of Exemplary Embodiments 1 to 65, wherein the conjugate is selected from the structures set forth in Table C.

[0806] Exemplary Embodiment 67. The conjugate of any one of Exemplary Embodiments 1 to 66, wherein the nucleotide-based enhancer unit has an * at the 5'-end of the nucleotide corresponding to a nucleotide-based enhancer bound to a ligand, a linker unit, and / or a nucleic acid agent.

[0807] Exemplary Embodiment 68 The conjugate of any one of Exemplary Embodiments 1 to 67, wherein the ligand comprises a carbohydrate moiety.

[0808] Exemplary Embodiment 69 The conjugate of any one of Exemplary Embodiments 1 to 68, wherein the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0809] Exemplary Embodiment 70 The conjugate of any one of Exemplary Embodiments 1 to 69, wherein the carbohydrate moiety comprises galactose or a derivative thereof.

[0810] Exemplary Embodiment 71. The ligand: [ka] 71. The conjugate of any one of exemplary embodiments 1 to 70, comprising:

[0811] Exemplary Embodiment 72. The ligand: [ka] 72. The conjugate of any one of exemplary embodiments 1 to 71, comprising:

[0812] Exemplary Embodiment 73. The ligand: [ka] 73. The conjugate of any one of exemplary embodiments 1 to 72, comprising:

[0813] Exemplary Embodiment 74. The conjugate of any one of claims 1 to 73, wherein the ligand comprises a moiety capable of binding to the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0814] Exemplary Embodiment 75. The conjugate of any one of claims 1 to 74, wherein the ligand comprises glucagon-like peptide-1 (GLP-1) or a derivative thereof.

[0815] Exemplary Embodiment 76. The conjugate of any one of claims 1 to 75, wherein the ligand comprises glucagon or a derivative thereof.

[0816] Exemplary Embodiment 77. The ligand: one, two, or three sugars (e.g., N-acetyl-D-galactosylamine); 1, 2, or 3 lipid moieties, 1, 2, or 3 peptide moieties; 1, 2, or 3 antibody moieties; 1, 2, or 3 oligonucleotides, or The conjugate of any one of exemplary embodiments 1 to 76, including any combination thereof.

[0817] Exemplary Embodiment 78. A pharmaceutical composition comprising the nucleotide-based enhancer or complex of any one of Exemplary Embodiments 1 to 77.

[0818] Exemplary Embodiment 79. A method of modulating expression of a target gene in a subject, comprising administering to the subject a complex of any one of Exemplary Embodiments 1 to 78.

[0819] Exemplary Embodiment 80. A method of delivering a nucleic acid agent to a subject, comprising administering to the subject a complex of any one of Exemplary Embodiments 1 to 79.

[0820] Exemplary Embodiment 81. A method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate of any one of Exemplary Embodiments 1 to 80.

[0821] Exemplary Embodiment 82 A conjugate of any one of Exemplary Embodiments 1 to 81 for modulating expression of a target gene in a subject.

[0822] Exemplary Embodiment 83. A conjugate of any one of Exemplary Embodiments 1 to 82 for delivering a nucleic acid agent to a subject.

[0823] Exemplary Embodiment 84. A conjugate of any one of exemplary embodiments 1 to 83 for treating or preventing a disease in a subject in need thereof.

[0824] Exemplary Embodiment 85 Use of a conjugate of any one of Exemplary Embodiments 1 to 84 in the manufacture of a medicament for modulating expression of a target gene in a subject.

[0825] Exemplary Embodiment 86 Use of a conjugate of any one of Exemplary Embodiments 1 to 85 in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0826] Exemplary Embodiment 87. Use of a conjugate of any one of exemplary embodiments 1 to 86 in the manufacture of a medicament for treating or preventing a disease in a subject in need thereof.

[0827] Exemplary Embodiment 88 The method, conjugate, or use of any one of Exemplary Embodiments 1 to 87, wherein the subject is a human. [Example]

[0828] Example 1. Synthesis of duplex 1 to duplex 5 Scheme 1. Synthesis of conjugate duplex 1 [ka] Duplex 1 was synthesized according to Scheme 1. Sense strand 1a and antisense strand 1 were prepared by solid phase synthesis according to the manufacturer's protocol.

[0829] Sense strand 1b was produced by post-synthetic conjugation. Sense strand 1a (10.00 mg, 0.71 μmol) and water (HO) (0.50 mL) were added to Eppendorf tube 1 and mixed vigorously to obtain a clear solution. DMF (0.50 mL), N-β-maleimidopropyloxysuccinimide ester (0.38 mg, 1.42 μmol), and DIPEA (1.24 μL, 7.10 μmol) were added to Eppendorf tube 2 and mixed vigorously. The DMF solution from Eppendorf tube 2 was then added to Eppendorf tube 1, and the resulting mixture was stirred at room temperature. After LC-MS analysis showed the reaction was complete, the reaction was quenched with water (HO) (10 mL). The reaction mixture was dialyzed against water (3 × 15 mL) using an Amicon® Ultra-15 centrifugal filter with a 3K MW cutoff. The solution was lyophilized to give crude sense strand 1b (8.23 mg, 82% yield), which was used directly in the next step without further purification.

[0830] Sense strand 1 was prepared by post-synthetic conjugation. Sense strand 1b (8.23 mg, 0.58 μmol) and water (HO) (0.20 mL) were added to Eppendorf tube 1 and mixed vigorously to obtain a clear solution. DMA (0.60 mL), peptide (3.9 mg, 0.87 μmol), and DIPEA (1.02 μL, 5.80 μmol) were added to Eppendorf tube 2 and mixed vigorously. Next, the DMA solution in Eppendorf tube 2 was added to Eppendorf tube 1, and the resulting mixture was stirred at room temperature. After LC-MS analysis showed the reaction was complete, the reaction was quenched with water (HO) (2 mL). The reaction mixture was purified by reverse-phase HPLC (C18 column, 100 mM TEAA containing iodine (I) and water (HO), 10% to 95% gradient, 30 min). The fractions were combined and dialyzed against saline (15 mL) and water (3 × 15 mL) using an Amicon® Ultra-15 centrifugal filter (3K) with a MW cutoff of 3K, and the solution was then lyophilized to give sense strand 1 as a white solid (6.42 mg, 58% yield).

[0831] Duplex 1 was generated by annealing sense strand 1 and antisense strand 1. Sense strand 1 solution (10 mg / mL) and antisense strand 1 solution (10 mg / mL) were prepared according to the OD amount. Equimolar amounts of the sense and antisense strands were mixed and annealed at 95°C for 5 minutes, then cooled to room temperature to obtain a complex duplex with a purity of over 90%. The duplex solution was lyophilized to obtain the desired duplex 1 (96% yield).

[0832] Duplexes 2 through 5 (see Figure 2) were prepared in comparable yields and purity using procedures similar to those described for the preparation of duplex 1.

[0833] Example 2. In vivo KD activity of duplexes 1 to 5 CD-1 female mice were subcutaneously administered duplexes 1 to 5 at 30 mg / kg. The control group received phosphate-buffered saline (PBS). Animals were sacrificed 7 days after treatment. Specific tissues of interest were harvested and stored in 10% ethanol for 24 hours. The tissues were then transferred to 75% ethanol and embedded in paraffin blocks. 4 μm tissue sections were prepared from the paraffin blocks. Slides were deparaffinized twice with 100% xylene for 5 minutes each and twice with 100% ethanol for 5 minutes each. RNASCOPE assays for target genes were performed according to the instructions in the RNASCOPE kit (RNASCOPE 2.5 HD Assay-Red, Catalog No. 322350, Advanced Cell Diagnostics). Slides after RNASCOPE were imaged using a Keyence microscope, and images were quantified using Indica Halo software. All samples were normalized to a PBS-treated control sample and plotted using GraphPad Prism software (GraphPad Software, La Jolla, CA). For detailed results, see Figures 3-5.

[0834] Doctrine of Equivalents The details of one or more embodiments of the present disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In this specification and the appended claims, the singular forms "a," "an," and "the" encompass plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference.

[0835] The foregoing description has been presented for purposes of illustration only, and is not intended to be limited to the precise form disclosed, but rather by the claims appended hereto.

Claims

1. (i) one or more nucleic acid agents; (ii) one or more ligands, and (iii) one or more nucleotide-based enhancing units, each nucleotide-based enhancing unit independently comprising 2 to 30 nucleotides, each nucleotide independently comprising 【Chemistry 1】 a nucleotide-based enhancer unit, or a pharmaceutically acceptable salt thereof, the nucleic acid agent comprises an oligonucleotide; the ligand is a moiety that, when covalently attached to the nucleic acid agent, can mediate entry of the nucleic acid agent to a target site or facilitate delivery of the nucleic acid agent to a target site; the one or more nucleic acid agents, the one or more ligands, and the one or more nucleotide-based enhancer units are covalently linked to each other directly or via a linker unit; In the formula, B is H, C １ -C ６ alkyl, or nucleobase moiety, V is -O-, -NR Ｖ -, or -C(R Ｖ ) ２ -is, Each R Ｖ are independently H or C １ -C ６ is alkyl, X is H, halogen, or -OR Ｘ That is, R Ｘ is H, C １ -C １２ alkyl, or -(C １ -C ６ alkyl)-(C ６ -C １０ aryl), and １ -C ６ Alkyl or -(C １ -C ６ alkyl)-(C ６ -C １０ aryl) optionally contains one or more R Ｘａ or substituted with R Ｘ and R ４ Let's get together and C １ -C ６ forming an alkylene, Each R Ｘａ are independently halogen, C １ -C ６ Alkyl, or —O—(C １ -C ６ alkyl), and １ -C ６ Alkyl or —O—(C １ -C ６ alkyl) optionally substituted with one or more halogens; Y is -P(R Ｙ ), -P(OR Ｙ ), -P(N(R Ｙ )) ２ ), -P(=O)(OR Ｙ ), -P(=O)(R Ｙ ), -P(=S)(OR Ｙ ), -P(=S)(R Ｙ ), -P(=O)(SR Ｙ ), or -P(=S)(SR Ｙ ), Each R Ｙ are independently H or C optionally substituted with one or more halogens or cyanos. １ -C ６ is alkyl, R １ is H, halogen, or C optionally substituted with one or more halogens １ -C ６ is alkyl, R ２ is H, halogen, or C optionally substituted with one or more halogens １ -C ６ is alkyl, R ３ is H, halogen, or C optionally substituted with one or more halogens １ -C ６ is alkyl, R ４ is H, halogen, or C optionally substituted with one or more halogens １ -C ６ alkyl or R ４ and R Ｘ Let's get together and C １ -C ６ forming an alkylene, Each R ５ are independently H, halogen, or C optionally substituted with one or more halogens. １ -C ６ is alkyl, each # independently represents a bond to the 2' or 3' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 5' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the complex; and each ## independently represents a bond to the 5' position of another nucleotide of the nucleotide-based enhancer unit, or, if the nucleotide is at the 2' or 3' end of the nucleotide-based enhancer unit, a bond to H or the remainder of the complex; The complex or a pharmaceutically acceptable salt thereof.

2. 2. The conjugate of claim 1, wherein at least one nucleotide-based enhancer unit comprises 3 to 20 nucleotides.

3. 3. The conjugate of claim 1 or 2, wherein at least one nucleotide-based enhancer unit comprises 6 to 15 nucleotides.

4. 4. The complex of claim 1, wherein at least one nucleotide-based enhancer unit comprises 9 nucleotides.

5. The complex of claim 1 , wherein at least one nucleotide-based enhancer unit is attached to the nucleic acid agent directly or via a linker unit.

6. The complex of claim 1 , wherein at least one nucleotide-based enhancer unit is attached to the sense strand of the nucleic acid agent directly or via a linker unit.

7. The complex of claim 1 , wherein at least one nucleotide-based enhancer unit is attached to the 3′ end of the nucleic acid agent directly or via a linker unit.

8. The complex of claim 1 , wherein at least one nucleotide-based enhancer unit is not bound to any portion of the antisense strand of the nucleic acid agent.

9. 9. The conjugate of claim 1, wherein at least one nucleotide-based enhancer unit is attached to the ligand directly or via a linker unit.

10. At least one nucleotide-based enhancer unit comprises: (i) directly to the nucleic acid agent and the ligand; (ii) directly to the nucleic acid agent and to the ligand via a linker unit; (iii) directly to the ligand and to the nucleic acid agent via a linker unit; or (iv) connecting the nucleic acid agent and the ligand via a linker unit The complex of any one of claims 1 to 9, which is linked.

11. The complex of claim 1 , wherein the nucleic acid agent is double-stranded RNA.

12. 12. The complex of claim 1, wherein the complex has the structure shown in FIG.

13. 13. The conjugate of any one of claims 1 to 12, wherein the conjugate has a structure set forth in Table D.

14. 14. The conjugate of any one of claims 1 to 13, wherein the conjugate has a structure set forth in Table C.

15. 15. The complex of claim 1, wherein the ligand comprises a carbohydrate moiety.

16. 16. The conjugate of claim 1, wherein the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

17. 17. The conjugate of claim 1, wherein the carbohydrate moiety comprises galactose or a derivative thereof.

18. 18. The conjugate of any one of claims 1 to 17, wherein the ligand comprises a moiety capable of binding to the glucagon-like peptide-1 receptor (GLP-1 receptor).

19. 19. The conjugate of any one of claims 1 to 18, wherein the ligand comprises glucagon-like peptide-1 (GLP-1) or a derivative thereof.

20. 20. The conjugate of claim 1, wherein the ligand comprises glucagon or a derivative thereof.

21. 21. An isotopic derivative of the complex of any one of claims 1 to 20.

22. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 21.

23. 22. A method for regulating expression of a target gene in a subject, the method comprising administering to the subject a complex described in any one of claims 1 to 21.

24. 22. A method for delivering a nucleic acid agent to a subject, comprising administering to the subject a complex described in any one of claims 1 to 21.

25. 22. A method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate of any one of claims 1 to 21: 【Chemistry 2】

Images