Nucleotide-based enhancers for RNA delivery and therapy

Abstract

The present disclosure provides a nucleotide-based enhancer, a pharmaceutically acceptable salt thereof, and related conjugates, the nucleotide-based enhancer being a compound comprising from 2 to 30 nucleotides or a pharmaceutically acceptable salt thereof wherein each nucleotide independently has formula (I) or formula (II). The present disclosure also relates to uses of nucleotide-based enhancers and conjugates, for example in delivery of nucleic acids and / or treatment or prevention of disease. # imgabs0 #

Description

[0001] Related Applications

[0002] This application claims the priority and benefit of U.S. Application No. 63 / 421,799, filed on November 2, 2022, the entire content of which is incorporated herein by reference.

[0003] Incorporation of Sequence Listing

[0004] The content of the electronic sequence listing (SANB_011_001WO_SeqList_ST26.xml; size: 7,063 bytes; and creation date: October 31, 2023) is incorporated herein by reference in its entirety.

[0005] Background

[0006] 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 helps direct the nucleic acid to the site of interest. The targeting moiety can improve delivery via receptor-mediated endocytosis. This process is initiated by activating cell surface or membrane receptors after binding a specific ligand to the receptor. Many receptor-mediated endocytosis systems are known, including those that recognize sugars such as galactose, mannose, 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 and is highly abundant on hepatocytes. ASGP-R shows a high affinity for N-acetyl-D-galactosamine (GalNAc) compared to D-Gal. Similarly, the glucagon-like peptide-1 receptor (GLP-1 receptor) is present in the β-cells of the pancreas and can also be targeted with an appropriate ligand to direct delivery. Recently, certain carbohydrate conjugates have been shown to be valuable alternatives to liposomes for nucleic acid delivery. In addition, after successful delivery into cells, the stability of nucleic acids in the cellular environment is important for achieving a desired therapeutic effect.

[0007] Accordingly, there continues to be a need for novel modifications to enhance nucleic acid delivery. This disclosure addresses this need.

[0008] Summary

[0009] In some aspects, the present disclosure provides a nucleotide-based enhancer that is a compound comprising from 2 to 30 nucleotides or a pharmaceutically acceptable salt thereof, wherein each nucleotide independently has formula (I) or formula (II):

[0010]

[0011] Wherein:

[0012] Each * independently represents an attachment to the 2'- or 3'-position of another nucleotide of a nucleotide-based enhancer, or H when the nucleotide is at the 5'-end of the nucleotide-based enhancer;

[0013] Each ** independently represents an attachment to the 5'-position of another nucleotide of a nucleotide-based enhancer, or H when the nucleotide is at the 2'- or 3'-end of the nucleotide-based enhancer;

[0014] B is H, C 1 -C 6 alkyl or a nucleobase moiety;

[0015] V is -O-, -NR V -, or -C(R V ) 2 -;

[0016] Each R V independently is H or C 1 -C 6 alkyl;

[0017] X is H, halogen or -OR X ;

[0018] R X is H, C 1 -C 12 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl), wherein the C 1 -C 6 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl) is optionally substituted with one or more R Xa ; or R X and R 4 together form C 1 -C 6 alkylene;

[0019] Each R Xa independently is halogen, C 1 -C 6 alkyl or -O-(C 1 -C6 alkyl), wherein said C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl) is optionally substituted with one or more halogens;

[0020] 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 )-;

[0021] Each R Y is independently H or C 1 -C 6 alkyl optionally substituted with one or more halogens or cyano;

[0022] R 1 is H, halogen or C 1 -C 6 alkyl optionally substituted with one or more halogens;

[0023] R 2 is H, halogen or C 1 -C 6 alkyl optionally substituted with one or more halogens;

[0024] R 3 is H, halogen or C 1 -C 6 alkyl optionally substituted with one or more halogens;

[0025] R 4 is H, halogen or C 1 -C 6 alkyl; or R 4 and R X together form C 1 -C 6 alkylene; and

[0026] Each R 5 is independently H, halogen or C 1 -C 6 alkyl.

[0027] In some aspects, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof, comprising:

[0028] (i) one or more nucleic acid agents;

[0029] (ii) one or more ligands; and

[0030] (iii) one or more nucleotide-based enhancing units, wherein each nucleotide-based enhancing unit independently comprises from 2 to 30 nucleotides, and wherein each nucleotide independently has:

[0031]

[0032] wherein:

[0033] the variables B, V, X, Y, R 1 、R 2 、R 3 、R 4 and R 5 are described herein;

[0034] each # independently represents an attachment to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit, or when the nucleotide is at the 5'-terminus of the nucleotide-based enhancing unit, represents H or an attachment to the remainder of the conjugate; and

[0035] each ## independently represents an attachment to the 5'-position of another nucleotide of the nucleotide-based enhancing unit, or when the nucleotide is at the 2'-terminus or 3'-terminus of the nucleotide-based enhancing unit, represents H or an attachment to the remainder of the conjugate.

[0036] In some aspects, the present disclosure provides isotopically-derivatized forms of the nucleotide-based enhancers described herein.

[0037] In some aspects, the present disclosure provides a pharmaceutical composition comprising the nucleotide-based enhancer or conjugate described herein.

[0038] In some aspects, the present disclosure provides a method of modulating the expression of a target gene in a subject, the method comprising administering to the subject the conjugate described herein.

[0039] In some aspects, the present disclosure provides a method of delivering a nucleic acid agent to a subject, the method comprising administering to the subject the conjugate described herein.

[0040] In some aspects, the present disclosure provides a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate described herein.

[0041] In some aspects, the present disclosure provides a conjugate described herein for modulating the expression of a target gene in a subject.

[0042] In some aspects, the present disclosure provides a conjugate described herein for delivering a nucleic acid agent to a subject.

[0043] In some aspects, the present disclosure provides a conjugate described herein for treating or preventing a disease in a subject in need thereof.

[0044] In some aspects, the present disclosure provides the use of a conjugate described herein in the manufacture of a medicament for modulating the expression of a target gene in a subject.

[0045] In some aspects, the present disclosure provides the use of a conjugate described herein in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0046] In some aspects, the present disclosure provides 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.

[0047] 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. In this specification, unless the context clearly dictates otherwise, the singular forms also include the plural. 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. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. In case of conflict between the chemical structure and the name of the compounds disclosed herein, the chemical structure will control.

[0048] Other features and advantages of the present disclosure will be apparent from the following detailed description of the specification and claims. Brief Description of the Drawings

[0050] Figure 1 is a diagram showing various exemplary configurations of a conjugate described herein.

[0051] Figure 2Figure showing the chemical structures of exemplary conjugate duplexes 1 - duplex 5.

[0052] Figure 3 Figure showing the in vivo KD activity of duplexes 1 - duplex 5 in the tissue of interest.

[0053] Figure 4 Image of RNASCOPE - based mRNA silencing assessment of duplexes 1 - duplex 5 in the tissue of interest.

[0054] Figure 5 Image of miRNASCOPE - based imaging of siRNA molecules accumulated in the tissue of interest.

[0055] Detailed description

[0056] 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 can help direct the nucleic acid to the site of interest.

[0057] It should be understood that oligonucleotides may need to bypass several extracellular and intracellular barriers to achieve potent gene knockdown in diseases of specific tissues or cell types. Chemical modification and conjugation with targeting ligands can improve the pharmacological properties of oligonucleotides and enhance tissue - specific delivery. However, previously, oligonucleotides conjugated to a single targeting ligand still failed to achieve clinically relevant knockdown activity and specificity, despite testing of various conjugates with peptides, antibodies, small molecules, carbohydrates, and linkers. These sub - optimal delivery methods may be due to poor pharmacokinetics and ADME profiles of the conjugates, lack of efficient tissue accumulation and cellular uptake, as well as less efficient intracellular sorting and endosomal escape, and many other delivery barriers.

[0058] The present disclosure provides nucleotide - based enhancers that can improve targeted delivery efficiency and enhance the cellular activity of nucleic acids, for example, by improved PK, stability, cellular uptake and trafficking, or other mechanisms. The present disclosure also provides conjugates comprising the nucleotide - based enhancers. The present disclosure also relates to the use of the nucleotide - based enhancers and conjugates, for example, in the delivery of nucleic acids and / or the treatment or prevention of diseases.

[0059] In some embodiments, the conjugates comprising the nucleotide - based enhancers have higher targeted delivery efficiency compared to conjugates without the nucleotide - based enhancers.

[0060] In some aspects, the present disclosure provides a general delivery enhancer that can further improve the delivery efficiency, specificity, and knockdown activity of oligonucleotides in the presence or absence of a targeting ligand. The delivery enhancer can increase the binding affinity of the conjugate to serum proteins, enhance its nuclease resistance, and improve pharmacokinetic properties. The delivery enhancer can also facilitate the passage of oligonucleotides across capillary endothelium and into the tissue interstitium. The delivery enhancer can improve the binding of the oligonucleotide conjugate to the target receptor, thereby improving receptor-mediated endocytosis. The delivery enhancer can also facilitate the intracellular trafficking and endosomal escape of oligonucleotides, resulting in improved intracellular activity. The delivery enhancer can improve the delivery and gene knockdown activity of oligonucleotides via other mechanisms or pathways.

[0061] Nucleotide-based enhancer

[0062] In some aspects, the present disclosure provides a nucleotide-based enhancer that is a compound comprising from 2 to 30 nucleotides or a pharmaceutically acceptable salt thereof, wherein each nucleotide independently has formula (I) or formula (II):

[0063]

[0064] Wherein:

[0065] Each * independently represents an attachment to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer, or H when the nucleotide is at the 5'-terminus of the nucleotide-based enhancer;

[0066] Each ** independently represents an attachment to the 5'-position of another nucleotide of the nucleotide-based enhancer, or H when the nucleotide is at the 2'-terminus or 3'-terminus of the nucleotide-based enhancer;

[0067] B is H, C 1 -C 6 alkyl or a nucleobase moiety;

[0068] V is -O-, -NR V -, or -C(R V ) 2 -;

[0069] Each R V independently is H or C 1 -C 6 alkyl;

[0070] X is H, halogen, or -OR X ;

[0071] R X is H, C1 -C 12 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl), wherein the C 1 -C 6 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl) is optionally substituted with one or more R Xa ; or R X and R 4 together form C 1 -C 6 alkylene;

[0072] Each R Xa is independently halogen, C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl), wherein the C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl) is optionally substituted with one or more halogen atoms;

[0073] 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 )-;

[0074] Each R Y is independently H or C 1 -C 6 alkyl optionally substituted with one or more halogen or cyano groups;

[0075] R 1 is H, halogen or C 1 -C 6 alkyl optionally substituted with one or more halogen atoms;

[0076] R 2is H, a halogen, or C optionally substituted with one or more halogens 1 -C 6 alkyl;

[0077] R 3 is H, a halogen, or C optionally substituted with one or more halogens 1 -C 6 alkyl;

[0078] R 4 is H, a halogen, or C optionally substituted with one or more halogens 1 -C 6 alkyl; or R 4 and R X together form C 1 -C 6 alkylene; and

[0079] each R 5 is independently H, a halogen, or C optionally substituted with one or more halogens 1 -C 6 alkyl.

[0080] In some embodiments, the nucleotide-based enhancer comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides.

[0081] In some embodiments, the nucleotide-based enhancer comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides.

[0082] 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.

[0083] 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.

[0084] In some embodiments, the nucleotide-based enhancer comprises from 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.

[0085] In some embodiments, the nucleotide-based enhancer comprises from 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 from 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.

[0086] 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, every two adjacent nucleotides of the nucleotide-based enhancer are linked via a phosphodiester linker or a phosphorothioate linker.

[0087] In some embodiments, the nucleotide-based enhancer comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0088] In some embodiments, the nucleotide-based enhancer comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0089] 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0090] In some embodiments, the nucleotide-based enhancer comprises 5 nucleotides, wherein 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 6 nucleotides, wherein 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 7 nucleotides, wherein 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 8 nucleotides, wherein 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 9 nucleotides, wherein 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, wherein 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, wherein 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, wherein 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0091] In some embodiments, the nucleotide-based enhancer comprises from 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0092] In some embodiments, the nucleotide-based enhancer comprises from 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 from 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0093] In some embodiments, the nucleotide-based enhancer comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0094] In some embodiments, the nucleotide-based enhancer comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0095] 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0096] In some embodiments, the nucleotide-based enhancer comprises 5 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 6 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 7 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 8 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 9 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 10 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 11 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 12 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancer comprises 13 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0097] In some embodiments, the nucleotide-based enhancer comprises from 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0098] In some embodiments, the nucleotide-based enhancer comprises from 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 from 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, wherein the nucleotide-based enhancer further comprises one or more phosphodiester linkers.

[0099] In some embodiments, the nucleotide-based enhancer comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0100] In some embodiments, the nucleotide-based enhancer comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0101] In some embodiments, the nucleotide-based enhancer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0102] In some embodiments, the nucleotide-based enhancer comprises 5 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 6 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 7 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 8 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 9 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 10 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 11 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 12 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer comprises 13 nucleotides, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0103] In some embodiments, the nucleotide-based enhancer comprises from 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, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0104] In some embodiments, the nucleotide-based enhancer comprises from 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 from 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, wherein the nucleotide-based enhancer further comprises one or more phosphorothioate linkers.

[0105] It should be understood that for 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 can each independently be selected from the groups described herein, where applicable, and any group described herein for any one 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 can, where applicable, be combined with any group described herein for one or more of the remainder 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 .

[0106] The variable B

[0107] In some embodiments, B is H.

[0108] In some embodiments, B is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl).

[0109] In some embodiments, B is methyl, ethyl, or propyl.

[0110] In some embodiments, B is a nucleobase moiety.

[0111] As used herein, the term "nucleobase moiety" refers to a nucleobase that is attached, for example, via an atom of the nucleobase or a functional group thereof to the remainder of the compound.

[0112] In some embodiments, the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0113] In some embodiments, the nucleobase moiety is where indicates the point of attachment to the remainder of the nucleotide-based enhancer.

[0114] In some embodiments, the nucleobase moiety is a modified nucleobase.

[0115] In some embodiments, the modified nucleobase is 5-methylcytosine.

[0116] In some embodiments, the modified nucleobase is hypoxanthine, xanthine, or 7-methylguanine.

[0117] In some embodiments, the modified nucleobase is 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethylcytosine.

[0118] In some embodiments, the nucleobase moiety is an artificial nucleobase.

[0119] In some embodiments, the artificial nucleobase is isoguanine, isocytosine, 2-amino-6-(2-thienyl)purine, or pyrrole-2-carbaldehyde.

[0120] The variables V and R V

[0121] In some embodiments, V is -O-.

[0122] In some embodiments, V is -NR V -.

[0123] In some embodiments, V is -NH-.

[0124] In some embodiments, V is -C(R V ) 2 -.

[0125] In some embodiments, V is -CH 2 -.

[0126] In some embodiments, at least one R V is H.

[0127] In some embodiments, each R V is H.

[0128] In some embodiments, at least one R V is C 1 -C 6 -alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0129] In some embodiments, each R V is C 1 -C 6 -alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0130] The variables X, R X 、R Xa 、Y and R Y

[0131] In some embodiments, X is H.

[0132] In some embodiments, X is a halogen (e.g., F, Cl, Br or I).

[0133] In some embodiments, X is F or Cl.

[0134] In some embodiments, X is F.

[0135] In some embodiments, X is -OR X .

[0136] In some embodiments, X is -OH.

[0137] In some embodiments, X is -O-(C 1 -C 12 -alkyl).

[0138] In some embodiments, X is -O-(C 1 -C 6 -alkyl) (e.g., wherein the C 1 -C 6 -alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0139] In some embodiments, X is -OCH 3 .

[0140] In some embodiments, X is -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 alkyl) (e.g., where C 1 -C 6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl).

[0141] In some embodiments, X is -OCH 2 CH 2 OCH 3 .

[0142] In some embodiments, X is optionally substituted with one or more R Xa -O-(C 1 -C 6 alkyl)-(C 6 -C 10 aryl).

[0143] In some embodiments, X is -O-(C 1 -C 6 alkyl)-(C 6 -C 10 aryl).

[0144] In some embodiments, X is where indicates the attachment point to the remainder of the nucleotide-based enhancer.

[0145] In some embodiments, X is optionally substituted with one or more R Xa -substituted where indicates the attachment point to the remainder of the nucleotide-based enhancer.

[0146] In some embodiments, X is optionally substituted with one or more halogens where indicates the attachment point to the remainder of the nucleotide-based enhancer.

[0147] In some embodiments, X is optionally substituted with one or more C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl)-substituted wherein the C 1 -C 6 alkyl or -O-(C 1 -C 6alkyl) optionally substituted with one or more halogens, and indicates the attachment point to the remainder of the nucleotide-based enhancer.

[0148] In some embodiments, R X is H.

[0149] In some embodiments, R X is C Xa optionally substituted with one or more R 1 -C 12 alkyl.

[0150] In some embodiments, R X is C 1 -C 12 alkyl.

[0151] In some embodiments, R X is C Xa optionally substituted with one or more R 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0152] In some embodiments, R X is C 1 -C 6 alkyl optionally substituted with one or more halogens (e.g., F, Cl, Br or I) or -O-(C 1 -C 6 alkyl) optionally substituted with one or more halogens (e.g., where the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0153] In some embodiments, R X is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0154] In some embodiments, R X is methyl, ethyl or propyl.

[0155] In some embodiments, R X is methyl.

[0156] In some embodiments, RX is a C substituted by one or more halogens (e.g., F, Cl, Br, or I) 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl).

[0157] In some embodiments, R X is a C substituted by one or more -O-(C 1 -C 6 alkyl) (e.g., where the C 1 -C 6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl), where -O-(C 1 -C 6 alkyl) is optionally substituted by one or more halogens.

[0158] In some embodiments, R X is an optionally substituted by one or more R Xa substituted -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl).

[0159] In some embodiments, R X is an optionally substituted by one or more halogens (e.g., F, Cl, Br, or I), C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) or -O-(C 1 -C 6 alkyl) (e.g., where C 1 -C 6 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) 1 -C 6 alkyl)-(C 6 -C 10 aryl), where the C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl) is optionally substituted by one or more halogens.

[0160] In some embodiments, R X is -(C 1 -C 6 -alkyl)-(C 6 -C 10 -aryl).

[0161] In some embodiments, R X and R 4 together form C 1 -C 6 -alkylene (such as methylene, ethylene, propylene, butylene, pentylene or hexylene).

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

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

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

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

[0166] In some embodiments, Y is -P(R Y )-.

[0167] In some embodiments, Y is -PH-.

[0168] In some embodiments, Y is -P(OR Y )-.

[0169] In some embodiments, Y is -P(OH)-.

[0170] In some embodiments, Y is -P(N(R Y ) 2 )-.

[0171] In some embodiments, Y is -P(NH 2 )-.

[0172] In some embodiments, Y is -P(=O)(OR Y )-.

[0173] In some embodiments, Y is -P(=O)(OH)-.

[0174] In some embodiments, Y is -P(=O)(R Y )-.

[0175] In some embodiments, Y is -P(=O)H-.

[0176] In some embodiments, Y is -P(=S)(OR Y )-.

[0177] In some embodiments, Y is -P(=S)(OH)-.

[0178] In some embodiments, Y is -P(=S)(R Y )-.

[0179] In some embodiments, Y is -P(=S)H-.

[0180] In some embodiments, Y is -P(=O)(SR Y )-.

[0181] In some embodiments, Y is -P(=O)(SH)-.

[0182] In some embodiments, Y is -P(=S)(SR Y )-.

[0183] In some embodiments, Y is -P(=S)(SH)-.

[0184] In some embodiments, at least one R Y is H.

[0185] In some embodiments, each R Y is H.

[0186] In some embodiments, at least one R Y is C 1 -C 6 alkyl (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br or I) or cyano.

[0187] In some embodiments, each R Y is C 1 -C 6 alkyl (such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br or I) or cyano.

[0188] In some embodiments, at least one R Yis H, and at least one R Y is C optionally substituted by one or more halogens or cyano 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0189] The variable R 1 、R 2 、R 3 、R 4 and R 5

[0190] In some embodiments, R 1 is H.

[0191] In some embodiments, R 1 is halogen (e.g., F, Cl, Br or I).

[0192] In some embodiments, R 1 is F or Cl.

[0193] In some embodiments, R 1 is C optionally substituted by one or more halogens (e.g., F, Cl, Br or I) 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0194] In some embodiments, R 1 is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0195] In some embodiments, R 1 is C substituted by one or more halogens (e.g., F, Cl, Br or I) 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0196] In some embodiments, R 2 is H.

[0197] In some embodiments, R 2 is halogen (e.g., F, Cl, Br or I).

[0198] In some embodiments, R 2 is F or Cl.

[0199] In some embodiments, R 2 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br or I).

[0200] In some embodiments, R 2 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0201] In some embodiments, R 2 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl) substituted with one or more halogens (e.g., F, Cl, Br or I).

[0202] In some embodiments, R 3 is H.

[0203] In some embodiments, R 3 is a halogen (e.g., F, Cl, Br or I).

[0204] In some embodiments, R 3 is F or Cl.

[0205] In some embodiments, R 3 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br or I).

[0206] In some embodiments, R 3 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl).

[0207] In some embodiments, R 3 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl) substituted with one or more halogens (e.g., F, Cl, Br or I).

[0208] In some embodiments, R 4 is H.

[0209] In some embodiments, R 4 is a halogen (e.g., F, Cl, Br, or I).

[0210] In some embodiments, R 4 is F or Cl.

[0211] In some embodiments, R 4 is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0212] In some embodiments, R 4 is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl).

[0213] In some embodiments, R 4 is C 1 -C 6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0214] In some embodiments, R 4 and R X together form C 1 -C 6 alkylene (e.g., methylene, ethylene, propylene, butylene, pentylene, or hexylene).

[0215] In some embodiments, R 4 and R X together form methylene, ethylene, or propylene.

[0216] In some embodiments, R 4 and R X together form methylene.

[0217] In some embodiments, R 4 and R X together form ethylene.

[0218] In some embodiments, R 4 and RX Together form a propylene group.

[0219] In some embodiments, each R 5 is H.

[0220] In some embodiments, at least one R 5 is a halogen (e.g., F, Cl, Br, or I) or a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0221] In some embodiments, at least one R 5 is a halogen (e.g., F, Cl, Br, or I).

[0222] In some embodiments, at least one R 5 is F or Cl.

[0223] In some embodiments, at least one R 5 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0224] In some embodiments, at least one R 5 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl).

[0225] In some embodiments, at least one R 5 is a C 1 -C 6 alkyl group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0226] In some embodiments, each of R 1 , R 2 , R 3 , R 4 , and R 5 is H.

[0227] Exemplary embodiments of the nucleotide of the nucleotide-based enhancer

[0228] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I’) or formula (II’):

[0229]

[0230] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I-A), formula (II-A), formula (III-A), or formula (IV-A):

[0231]

[0232] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I’-A), formula (II’-A), formula (III’-A), or formula (IV’-A):

[0233]

[0234]

[0235] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I-B) or formula (II-B):

[0236]

[0237] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I’-B) or formula (II’-B):

[0238]

[0239] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I-C), formula (II-C), formula (III-C), or formula (IV-C):

[0240]

[0241]

[0242] In some embodiments, at least one nucleotide of the nucleotide-based enhancer has formula (I’-C), formula (II’-C), formula (III’-C), or formula (IV’-C):

[0243]

[0244] In some embodiments, at least one nucleotide of the nucleotide-based enhancer is selected from the nucleotides described in Table L.

[0245] Table L

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263] In some aspects, the present disclosure provides isotopically derivatized nucleotide-based boosters disclosed herein (e.g., isotopically labeled compounds).

[0264] It should be understood that isotopically derivatized compounds can be prepared using any of a variety of techniques well known in the art. For example, isotopically derivatized compounds can generally be prepared by implementing the procedures disclosed in the protocols and / or examples herein, by substituting non-isotopically labeled reagents with isotopically labeled reagents.

[0265] In some embodiments, the isotopically derivatized compound is a deuterium-labeled derivative.

[0266] As used herein, the term "isotope derivative" refers to a derivative of a chemical structure in which one or more atoms are isotope-enriched or labeled. For example, an isotope derivative of an agent is isotope-enriched with one or more isotopes or labeled with one or more isotopes compared to the corresponding agent. In some embodiments, the isotope derivative is enriched with one or more atoms selected from 2 H, 13 C, 14 C, 15 N, 18 O, 29 Si, 32 P, and 34 S or labeled with one or more atoms selected from 2 H, 13 C, 14 C, 15 N, 18 O, 29 Si, 32 P, and 34 S. In some embodiments, the isotope derivative is a deuterium-labeled derivative (i.e., enriched with 2 H with respect to one or more of its atoms). In some embodiments, the derivative is a 2 H-labeled derivative. In some embodiments, the derivative is a 13 C-labeled derivative or a 14 C-labeled derivative. In some embodiments, the derivative is a 18 F-labeled derivative. In some embodiments, the derivative is a 123 I-labeled derivative, 124 I-labeled derivative, 125 I-labeled derivative, 129 I-labeled derivative, 131 I-labeled derivative, 135 I-labeled derivative, or any combination thereof. In some embodiments, the derivative is a 31 P-labeled derivative or a 32 P-labeled derivative. In some embodiments, the derivative is a 33 S-labeled derivative, 34 S-labeled derivative, 35 S-labeled derivative, 36 S-labeled derivative, or any combination thereof.

[0267] It should also be understood that isotope substitution can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

[0268] For the avoidance of doubt, it should be understood that, in this specification, where a group is qualified by "described herein", the group encompasses the first and broadest definition as well as each and every specific definition of the group.

[0269] It will be understood that, although the compounds disclosed herein may be presented in a 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 a mixture 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 the compound, or any mixture thereof; and such presentation is also intended to refer to the specific configuration of the compound.

[0270] It will be understood that, although the compounds disclosed herein may be presented without a specified configuration (e.g., without specified stereochemistry). Such a presentation is intended to encompass all of the available isomers, tautomers, regioisomers and stereoisomers of the compound. In some embodiments, the presentation of a compound herein without a specified configuration is intended to refer to each of the available isomers, tautomers, regioisomers and stereoisomers of the compound, or any mixture thereof.

[0271] As used herein, the term "isomerism" means compounds having the same molecular formula but different in the order of their atomic bonding or in the arrangement of their atoms in space. Compounds having the same molecular formula but different in the nature or order of their atomic bonding or in the arrangement of their atoms in space are called "isomers". Isomers that are different in the arrangement of their atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereoisomers", and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers". When a compound has an asymmetric center (e.g., it is bonded to four different groups), pairs of enantiomers are possible. Enantiomers can be characterized by the absolute configuration of their asymmetric center and described by the R and S ranking rules of Cahn and Prelog or by the manner in which the molecule rotates the plane of polarized light and is designated as dextrorotatory or levorotatory (i.e., the (+) isomer or the (-) isomer, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".

[0272] Compounds of the present disclosure may have one or more asymmetric centers; thus, such compounds can be produced as individual (R)-stereoisomers or (S)-stereoisomers or as mixtures thereof. Unless otherwise indicated, the description or naming of a particular compound in the specification and claims is intended to include both the individual enantiomers and mixtures thereof (racemic or otherwise). Methods for determining stereochemistry and separating 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 by resolution of the racemic form. Some of the compounds of the present disclosure may have geometric isomer centers (E-isomers and Z-isomers). It is to be understood that the present disclosure encompasses all optical isomers, diastereomers, and geometric isomers and mixtures thereof having inflammasome inhibitory activity.

[0273] As used herein, the term “chiral center” refers to a carbon atom bonded to four different substituents.

[0274] As used herein, the term “chiral isomer” means a compound having at least one chiral center. Compounds having more than one chiral center may exist as individual diastereomers or as a mixture of diastereomers (referred to as a “mixture of diastereomers”). When there is one chiral center, the stereoisomers can be characterized by the absolute configuration (R or S) of that chiral center. The absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are arranged according to the sequence rules of Cahn, Ingold, and Prelog. (Cahn et al., Angew. Chem. Inter. Ed. 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).

[0275] As used herein, the term “geometric isomer” means a diastereomer whose existence is attributed to restricted rotation about a double bond or a cycloalkylidene linker (e.g., 1,3-cyclobutylene). These configurations are distinguished in their names by the prefixes cis and trans or Z and E, which indicate whether the groups are on the same side or opposite sides of the double bond in the molecule according to the Cahn-Ingold-Prelog rules.

[0276] It should be understood that the compounds of the present disclosure can be depicted as different chiral or geometric isomers. It should also be understood that when a compound has a chiral or geometric isomer form, all isomer forms are intended to be included within the scope of the present disclosure, and the naming of the compound does not exclude any isomer form. It should be understood that not all isomers may have the same level of activity.

[0277] It should be understood that the structures and other compounds discussed in the present disclosure include all of their atropic isomers. It should also be understood that not all atropic isomers may have the same level of activity.

[0278] As used herein, the term "atropic isomer" is a class of stereoisomers in which the atoms of the two isomers are arranged differently in space. Atropic isomers owe their existence to restricted rotation due to hindered rotation of large groups around a central bond. Such atropic isomers usually exist as mixtures. However, due to recent advances in chromatographic techniques, it has become possible to separate mixtures of the two atropic isomers in selected cases.

[0279] As used herein, the term "tautomer" is one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom, accompanied by the switching of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In a solution where tautomerization is possible, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that can be interconverted by tautomerization is called tautomerism. Among the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism, a simultaneous transfer of electrons and hydrogen atoms occurs. Ring-chain tautomerism results from the reaction of an aldehyde group (-CHO) in a sugar-chain molecule with one of the hydroxyl groups (-OH) in the same molecule to give its cyclic (ring) form, as presented by glucose.

[0280] It should be understood that the compounds of the present disclosure can be described as different tautomers. It should also be understood that when a compound has a tautomeric form, all tautomeric forms will be included within the scope of the present disclosure, and the naming of these compounds does not exclude any tautomer form. It should be understood that certain tautomers may have a higher level of activity compared to other tautomers.

[0281] It should be understood that compounds of any formula described herein include the compounds themselves, as well as their salts and their solvates, if applicable. For example, salts can form between an anion and a positively charged group (such as an amino group) on the substituted compounds disclosed herein. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, mesylate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (such as trifluoroacetate).

[0282] As used herein, the term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also form between a cation and a negatively charged group (such as a carboxylate group) on the substituted compounds disclosed herein. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations, such as tetramethylammonium ion or diethylamine ion. The substituted compounds disclosed herein also include those salts containing a quaternary nitrogen atom.

[0283] It should be understood that the compounds of the present disclosure, such as salts of the compounds, can exist in hydrated or non-hydrated (anhydrous) forms, 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.

[0284] As used herein, the term "solvate" means a solvate addition form containing a stoichiometric or non-stoichiometric amount of a solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water, the solvate formed is a hydrate, and if the solvent is an alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more water molecules with one molecule of a substance, where water retains its molecular state as H 2 O.

[0285] As used herein, the term "analogue" refers to a compound that is structurally similar to another compound but has a slightly different composition (such as an atom being replaced by an atom of a different element or the presence of a specific functional group, or one functional group being replaced by another functional group). Thus, an analogue is a compound that is similar or equivalent to a reference compound in function and appearance, but is not similar or equivalent to the reference compound in structure or origin.

[0286] As used herein, the term "derivative" refers to a compound having a common core structure and substituted by various groups as described herein.

[0287] As used herein, the term "bioisostere" refers to a compound produced by the exchange of one atom or group of atoms for another, generally similar atom or group of atoms. The purpose of bioisosteric replacement is to create a new compound with biological properties similar to those of the parent compound. Bioisosteric replacements can be based on physicochemical or topological properties. 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.

[0288] It should also be understood that certain compounds of any of the formulas disclosed herein can exist in solvated as well as unsolvated forms, such as, for example, hydrated forms. Suitable pharmaceutically acceptable solvates are, for example, hydrates such as hemihydrates, monohydrates, dihydrates, or trihydrates. It is understood that the present disclosure encompasses all such solvated forms having inflammasome inhibitory activity.

[0289] It should also be understood that certain compounds of any of the formulas disclosed herein can exhibit polymorphism, and the present disclosure encompasses all such forms or mixtures thereof having 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, thermogravimetric analysis, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, near-infrared (NIR) spectroscopy, solution and / or solid-state nuclear magnetic resonance spectroscopy. The water content of such crystalline materials can be determined by Karl Fischer analysis.

[0290] Compounds of any of the formulas disclosed herein can exist in a variety of different tautomeric forms, and reference to a compound of any of the formulas includes all such forms. To avoid doubt, in cases where a compound can exist in one of several tautomeric forms and only one is specifically described or shown, however, all other forms are included by the formulas disclosed herein. Examples of tautomeric forms include keto, enol, and enolate forms, such as in, for example, the following tautomeric pairs: keto / enol (illustrated below), imine / enamine, amide / imino alcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, and nitro / aci-nitro.

[0291]

[0292] Compounds of any of the formulas disclosed herein that contain an amine functionality may also form N - oxides. Compounds of any of the formulas herein that contain an amine functionality as referred to herein also include N - oxides. In cases where a compound contains several amine functionalities, one or more than one nitrogen atom may be oxidized to form an N - oxide. Specific examples of N - oxides are N - oxides of the nitrogen atoms of tertiary amines or nitrogen - containing heterocycles. N - oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid), see, for example, Advanced Organic Chemistry, Jerry March, 4th Edition, Wiley Interscience. More particularly, N - oxides can be prepared by the procedure of L.W. Deady (Syn. Comm. 1977, 7, 509 - 514), in which the amine compound is reacted with meta - chloroperoxybenzoic acid (mCPBA) in an inert solvent such as dichloromethane.

[0293] Compounds of any of the formulas disclosed herein may be administered in the form of a prodrug, which decomposes in the human or animal body to release the compounds of the present disclosure. Prodrugs can be used to alter the physical properties 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 modifying group can be attached.

[0294] Accordingly, the present disclosure includes those compounds of any of the formulas disclosed herein as defined above when obtainable by organic synthesis and when obtainable by cleavage of their prodrugs in the human or animal body. Thus, the present disclosure includes those compounds of any of the formulas disclosed herein produced by organic synthetic means, as well as also such compounds produced in the human or animal body by metabolism of a precursor compound, i.e., a compound of any of the formulas disclosed herein can be a synthetically produced compound or a metabolically produced compound.

[0295] Suitable pharmaceutically acceptable prodrugs of the compounds of any of the formulas disclosed herein are prodrugs that are suitable, based on reasonable medical judgment, for administration to the human or animal body without undue pharmacological activity and without excessive toxicity. For example, various forms of prodrugs have been described in the following documents: a) Methods in Enzymology, Volume 42, pages 309 - 396, edited by K. Widder et al. (Academic Press, 1985); b) Design of Pro-drugs, 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 Pro-drugs", H. Bundgaard, pages 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); g) T. Higuchi and V. Stella, "Pro-Drugs as Novel Delivery Systems", A.C.S. Symposium Series, Volume 14; and h) E. Roche (editor), "Bioreversible Carriers in Drug Design", Pergamon Press, 1987.

[0296] The in vivo effects of the compounds of any of the formulas disclosed herein can be exerted in part by one or more metabolites formed in the human or animal body after administration of the compounds of any of the formulas disclosed herein. As stated above, the in vivo effects of the compounds of any of the formulas disclosed herein can also be exerted by the metabolism of precursor compounds (prodrugs).

[0297] Suitably, the present disclosure excludes any individual compound that does not have the biological activity as defined herein.

[0298] Conjugates containing nucleotide-based enhancing units

[0299] As used herein, the term "conjugate" refers to a compound or complex that comprises a nucleic acid agent covalently attached to a ligand. In some embodiments, the conjugate further comprises a nucleotide-based enhancing unit as described herein.

[0300] In some aspects, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof, comprising:

[0301] (i) one or more nucleic acid agents;

[0302] (ii) one or more ligands; and

[0303] (iii) one or more nucleotide-based enhancing units, wherein each nucleotide-based enhancing unit independently comprises from 2 to 30 nucleotides, and wherein each nucleotide independently has:

[0304]

[0305] wherein:

[0306] the variables B, V, X, Y, R 1 , R 2 , R 3 , R 4 and R 5 are described herein;

[0307] each # independently represents an attachment to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit, or represents H or an attachment to the remainder of the conjugate when the nucleotide is at the 5'-terminus of the nucleotide-based enhancing unit; and

[0308] each ## independently represents an attachment to the 5'-position of another nucleotide of the nucleotide-based enhancing unit, or represents H or an attachment to the remainder of the conjugate when the nucleotide is at the 2'-terminus or 3'-terminus of the nucleotide-based enhancing unit.

[0309] It should be understood that one or more ligands and one or more nucleotide-based enhancing units are each independently attached to a terminal position or an internal position of one or more nucleic acid agents. Further, it should be understood that when the nucleic acid agent comprises more than one strand (e.g., a sense strand and an antisense strand), one or more ligands and one or more nucleotide-based enhancing units may be attached to the same strand or different strands of the nucleic acid agent.

[0310] In some embodiments, the conjugate further comprises one or more linker units.

[0311] In some embodiments, the conjugate comprises double-stranded RNA (e.g., double-stranded siRNA), one or more ligands, and one or more nucleotide-based enhancing units.

[0312] In some embodiments, at least one nucleotide-based enhancing unit is directly attached to the nucleic acid agent (e.g., siRNA).

[0313] In some embodiments, at least one nucleotide-based enhancing unit is attached to the nucleic acid agent (e.g., siRNA) via a linker unit.

[0314] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the nucleic acid agent (e.g., siRNA).

[0315] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the terminal position (e.g., the nucleotide at the 3'-end or 5'-end) or internal position (e.g., the nucleotide not at the 3'-end or 5'-end) of the nucleic acid agent (e.g., siRNA).

[0316] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the terminal position (e.g., the nucleotide at the 3'-end or 5'-end) of the nucleic acid agent (e.g., siRNA).

[0317] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the nucleotide at the 3'-end of the nucleic acid agent (e.g., siRNA).

[0318] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the nucleotide at the 5'-end of the nucleic acid agent (e.g., siRNA).

[0319] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the internal position (e.g., the nucleotide not at the 3'-end or 5'-end) of the nucleic acid agent (e.g., siRNA).

[0320] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the sense strand or the antisense strand of the nucleic acid agent (e.g., siRNA).

[0321] In some embodiments, at least one nucleotide-based enhancing unit is directly or via a linker unit attached to the sense strand of the nucleic acid agent (e.g., siRNA).

[0322] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the sense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0323] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the antisense strand of a nucleic acid agent (e.g., siRNA).

[0324] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the antisense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0325] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA).

[0326] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0327] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the nucleotide at the 3'-end of the sense strand of a nucleic acid agent (e.g., siRNA).

[0328] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the nucleotide at the 3'-end of the sense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0329] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the nucleotide at the 5'-end of the sense strand of a nucleic acid agent (e.g., siRNA).

[0330] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of the sense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0331] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide that is not at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA).

[0332] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide that is not at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0333] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0334] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0335] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 3'-end of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0336] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 3'-end of the antisense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0337] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0338] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of the antisense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0339] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide that is not at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0340] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide that is not at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA), and the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0341] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an overhang (e.g., a 3'-overhang or 5'-overhang) of a nucleic acid agent (e.g., siRNA).

[0342] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 3'-overhang of a nucleic acid agent (e.g., siRNA).

[0343] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 5'-overhang of a nucleic acid agent (e.g., siRNA).

[0344] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an overhang (e.g., a 3'-overhang or 5'-overhang) of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0345] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 3'-overhang of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0346] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 5'-overhang of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0347] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an overhang (e.g., a 3'-overhang or 5'-overhang) of the sense strand of a nucleic acid agent (e.g., siRNA).

[0348] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 3'-overhang of the sense strand of a nucleic acid agent (e.g., siRNA).

[0349] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 5'-overhang of the sense strand of a nucleic acid agent (e.g., siRNA).

[0350] In some embodiments, at least one nucleotide-based enhancing unit is attached to the sense strand of a nucleic acid agent (e.g., siRNA) and does not bind to any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0351] In some embodiments, at least one nucleotide-based enhancing unit is attached to the antisense strand of a nucleic acid agent (e.g., siRNA) and does not bind to any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0352] In some embodiments, at least one nucleotide-based enhancing unit does not bind to any portion of the antisense strand of a nucleic acid agent (e.g., siRNA).

[0353] In some embodiments, at least one nucleotide-based enhancing unit does not bind to any portion of the sense strand of a nucleic acid agent (e.g., siRNA).

[0354] In some embodiments, at least one nucleotide-based enhancing unit binds to the antisense strand of a nucleic acid agent (e.g., siRNA).

[0355] In some embodiments, at least one nucleotide-based enhancing unit binds to the sense strand of a nucleic acid agent (e.g., siRNA).

[0356] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) or an internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the sense or antisense strand of a nucleic acid agent (e.g., siRNA).

[0357] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0358] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'- or 5'-end) or an internal position (e.g., a nucleotide not at the 3'- or 5'-end) of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0359] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'- or 5'-end) of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0360] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 3'-end of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0361] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0362] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide not at the 3'- or 5'-end) of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0363] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the sense strand or the antisense strand of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0364] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the sense strand of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0365] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the sense strand of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0366] In some embodiments, at least one nucleotide-based enhancing unit is attached to the antisense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0367] In some embodiments, at least one nucleotide-based enhancing unit is attached to the antisense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0368] In some embodiments, at least one nucleotide-based enhancing unit is attached to the terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0369] In some embodiments, at least one nucleotide-based enhancing unit is attached to the terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0370] In some embodiments, at least one nucleotide-based enhancing unit is attached to the nucleotide at the 3'-end of the sense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0371] In some embodiments, at least one nucleotide-based enhancing unit is attached to the nucleotide at the 3'-end of the sense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0372] In some embodiments, at least one nucleotide-based enhancing unit is attached to the nucleotide at the 5'-end of the sense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0373] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of the sense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0374] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0375] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the sense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the antisense strand of the nucleic acid agent (e.g., siRNA).

[0376] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0377] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0378] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 3'-end of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0379] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 3'-end of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0380] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0381] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleotide at the 5'-end of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0382] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide that is not at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0383] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position (e.g., a nucleotide that is not at the 3'-end or 5'-end) of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand, wherein the nucleotide-based enhancing unit does not interact (e.g., bind) with any portion of the sense strand of the nucleic acid agent (e.g., siRNA).

[0384] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an overhang (e.g., a 3'-overhang or 5'-overhang) of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0385] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 3'-overhang of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0386] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 5'-overhang of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0387] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to an overhang (e.g., a 3'-overhang or 5'-overhang) of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0388] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 3'-overhang of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0389] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 5'-overhang of the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0390] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the overhang (e.g., 3'-overhang or 5'-overhang) of the sense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0391] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 3'-overhang of the sense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0392] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to the 5'-overhang of the sense strand of a nucleic acid agent (e.g., siRNA) and is attached directly or via a linker unit to a ligand.

[0393] In some embodiments, at least one nucleotide-based enhancing unit is attached to the sense strand of a nucleic acid agent (e.g., siRNA) and does not bind to any portion of the antisense strand of the nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0394] In some embodiments, at least one nucleotide-based enhancing unit is attached to the antisense strand of a nucleic acid agent (e.g., siRNA) and does not bind to any portion of the sense strand of the nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0395] In some embodiments, at least one nucleotide-based enhancing unit does not bind to any portion of the antisense strand of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0396] In some embodiments, at least one nucleotide-based enhancing unit does not bind to any portion of the sense strand of a nucleic acid agent (e.g., siRNA), and is attached directly or via a linker unit to a ligand.

[0397] In some embodiments, at least one nucleotide-based enhancing unit binds to the antisense strand of a nucleic acid agent (e.g., siRNA) and is attached to a ligand directly or via a linker unit.

[0398] In some embodiments, at least one nucleotide-based enhancing unit binds to the sense strand of a nucleic acid agent (e.g., siRNA) and is attached to a ligand directly or via a linker unit.

[0399] In some embodiments, at least one nucleotide-based enhancing unit is attached to the terminal position (e.g., a nucleotide at the 3'-end or 5'-end) or internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the sense or antisense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0400] In some embodiments, at least one nucleotide-based enhancing unit is attached to the terminal position (e.g., a nucleotide at the 3'-end or 5'-end) or internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the sense or antisense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit, as Figure 1 described in.

[0401] In some embodiments, at least one nucleotide-based enhancing unit is attached to the terminal position (e.g., a nucleotide at the 3'-end or 5'-end) of the sense or antisense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0402] In some embodiments, at least one nucleotide-based enhancing unit is attached to the internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the sense or antisense strand of a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit.

[0403] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit, as Figure 1 described in Conjugate No. 1 of.

[0404] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit, and is attached to a ligand directly or via a linker unit, as Figure 1 described in Conjugate No. 2 of.

[0405] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 3 of

[0406] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 4 of

[0407] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 5 of

[0408] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 6 of

[0409] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 7 of

[0410] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 8 of

[0411] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 9 of

[0412] In some embodiments, at least one nucleotide-based enhancing unit is attached to a nucleic acid agent (e.g., siRNA) directly or via a linker unit and is attached to a ligand directly or via a linker unit, such as Figure 1 described in Conjugate No. 10 of

[0413] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleic acid agent (e.g., siRNA) and directly or via a linker unit to a ligand, as Figure 1 described in conjugate number 11 of

[0414] In some embodiments, at least one nucleotide-based enhancing unit is attached directly or via a linker unit to a nucleic acid agent (e.g., siRNA) and directly or via a linker unit to a ligand, as Figure 1 described in conjugate number 12 of

[0415] In some embodiments, at least one ligand is attached directly to a nucleic acid agent (e.g., siRNA).

[0416] In some embodiments, at least one ligand is attached to a nucleic acid agent (e.g., siRNA) via a linker unit.

[0417] In some embodiments, at least one ligand is attached to a nucleic acid agent (e.g., siRNA) via a nucleotide-based enhancing unit.

[0418] In some embodiments, at least one ligand is attached directly to a nucleotide-based enhancing unit.

[0419] In some embodiments, at least one ligand is attached to a nucleotide-based enhancing unit via a linker unit.

[0420] In some embodiments, at least one ligand is attached to a nucleic acid agent (e.g., siRNA) via a nucleotide-based enhancing unit and a linker unit.

[0421] In some embodiments, the conjugate comprises:

[0422] (nucleic acid agent)-[(linker unit) 0-1 -(nucleotide-based enhancing unit)-(linker unit) 0-1 -(ligand)] 1-3 ;

[0423] (nucleic acid agent)-[(linker unit) 0-1 -(ligand)-(linker unit) 0-1 -(nucleotide-based enhancing unit)] 1-3 ; or

[0424] [(nucleotide-based enhancing unit)-(linker unit) 0-1 1-3 -(nucleic acid agent)-[(linker unit) 0-1 -(ligand)] 1-3 ;

[0425] ​Each of the linker unit, the nucleotide-based enhancing unit, and the ligand, when attached to a nucleic acid agent (e.g., siRNA), is independently attached to a terminal position (e.g., a nucleotide at the 3'-end or 5'-end) or an internal position (e.g., a nucleotide not at the 3'-end or 5'-end) of the nucleic acid agent (e.g., siRNA).

[0426] In some embodiments, the conjugate is selected from the conjugates described in Table D, wherein the nucleic acid agent can optionally be further attached to one or more linker units, one or more nucleotide-based enhancing units, and / or one or more ligands at one or more internal positions of the nucleic acid agent according to the attachment moieties described herein.

[0427] Table D

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439] It should be understood that one or more attachment moieties to the nucleic acid agent can be located at one or more internal positions (e.g., ), the 3'-terminal position (e.g., ), or the 5'-terminal position (e.g., ) of the nucleic acid agent.

[0440] In some embodiments, the conjugate is selected from Figure 1 and Figure 2 the conjugates described therein.

[0441] In some embodiments, the conjugate is selected from Figure 1 the conjugates described therein.

[0442] In some embodiments, the conjugate is Figure 1 conjugate number 1 as described in

[0443] In some embodiments, the conjugate is Figure 1 conjugate number 1 as described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0444] In some embodiments, the conjugate is Figure 1 conjugate number 1 as described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0445] In some embodiments, the conjugate is Figure 1 conjugate number 1 as described in, wherein the nucleotide-based enhancing 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.

[0446] In some embodiments, the conjugate is Figure 1 conjugate number 1 as described in, wherein the nucleotide-based enhancing 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.

[0447] In some embodiments, the conjugate is Figure 1 conjugate number 2 as described in

[0448] In some embodiments, the conjugate is Figure 1 conjugate number 2 as described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0449] In some embodiments, the conjugate is Figure 1 conjugate number 2 as described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0450] In some embodiments, the conjugate is Figure 1 conjugate number 2 as described in, wherein the nucleotide-based enhancing 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.

[0451] In some embodiments, the conjugate is Figure 1 conjugate number 2 as described in, wherein the nucleotide-based enhancing 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.

[0452] In some embodiments, the conjugate is Figure 1 conjugate number 3 as described in

[0453] In some embodiments, the conjugate is Figure 1 conjugate number 3 as described in

[0454] In some embodiments, the conjugate is Figure 1 conjugate number 3 as described in

[0455] In some embodiments, the conjugate is Figure 1 conjugate number 3 as described in

[0456] In some embodiments, the conjugate is Figure 1 conjugate number 3 as described in

[0457] In some embodiments, the conjugate is Figure 1 conjugate number 4 as described in

[0458] In some embodiments, the conjugate is Figure 1 conjugate number 4 as described in

[0459] In some embodiments, the conjugate is Figure 1 conjugate number 4 as described in

[0460] In some embodiments, the conjugate is Figure 1 conjugate number 4 as described in

[0461] In some embodiments, the conjugate is Figure 1 conjugate number 4 as described in

[0462] In some embodiments, the conjugate is Figure 1 conjugate number 5 as described in

[0463] In some embodiments, the conjugate is Figure 1Conjugate No. 5 as described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0464] In some embodiments, the conjugate is Figure 1 Conjugate No. 5 as described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0465] In some embodiments, the conjugate is Figure 1 Conjugate No. 5 as described in, wherein the nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent and is attached directly or via a linker unit to a ligand.

[0466] In some embodiments, the conjugate is Figure 1 Conjugate No. 5 as described in, wherein the nucleotide-based enhancing unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent and is attached directly or via a linker unit to a ligand.

[0467] In some embodiments, the conjugate is Figure 1 Conjugate No. 6 as described in.

[0468] In some embodiments, the conjugate is Figure 1 Conjugate No. 6 as described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0469] In some embodiments, the conjugate is Figure 1 Conjugate No. 6 as described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0470] In some embodiments, the conjugate is Figure 1 Conjugate No. 6 as described in, wherein the nucleotide-based enhancing 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.

[0471] In some embodiments, the conjugate is Figure 1 Conjugate No. 6 as described in, wherein the nucleotide-based enhancing 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.

[0472] In some embodiments, the conjugate is Figure 1 Conjugate No. 6 as described in, wherein the nucleotide-based enhancing 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.

[0473] In some embodiments, the conjugate is Figure 1 conjugate number 6 as described in

[0474] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0475] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0476] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0477] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0478] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0479] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0480] In some embodiments, the conjugate is Figure 1 conjugate number 7 as described in

[0481] In some embodiments, the conjugate is Figure 1 conjugate number 8 as described in

[0482] In some embodiments, the conjugate is Figure 1Conjugate No. 8 described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0483] In some embodiments, the conjugate is Figure 1 Conjugate No. 8 described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0484] In some embodiments, the conjugate is Figure 1 Conjugate No. 8 described in, wherein the nucleotide-based enhancing 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.

[0485] In some embodiments, the conjugate is Figure 1 Conjugate No. 8 described in, wherein the nucleotide-based enhancing 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.

[0486] In some embodiments, the conjugate is Figure 1 Conjugate No. 8 described in, wherein the nucleotide-based enhancing 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.

[0487] In some embodiments, the conjugate is Figure 1 Conjugate No. 8 described in, wherein the nucleotide-based enhancing 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.

[0488] In some embodiments, the conjugate is Figure 1 Conjugate No. 9 described in.

[0489] In some embodiments, the conjugate is Figure 1 Conjugate No. 9 described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0490] In some embodiments, the conjugate is Figure 1 Conjugate No. 9 described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0491] In some embodiments, the conjugate is Figure 1Conjugate No. 9 as described in, wherein 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 enhancing unit is attached directly or via a linker unit to the 5'-end of the antisense strand of the nucleic acid agent.

[0492] In some embodiments, the conjugate is Figure 1 Conjugate No. 9 as described in, wherein 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 enhancing unit is attached directly or via a linker unit to the 5'-end of the antisense strand of the nucleic acid agent.

[0493] In some embodiments, the conjugate is Figure 1 Conjugate No. 9 as described in, wherein 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 enhancing unit is attached directly or via a linker unit to the 5'-end of the sense strand of the nucleic acid agent.

[0494] In some embodiments, the conjugate is Figure 1 Conjugate No. 9 as described in, wherein 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 enhancing unit is attached directly or via a linker unit to the 5'-end of the sense strand of the nucleic acid agent.

[0495] In some embodiments, the conjugate is Figure 1 Conjugate No. 10 as described in.

[0496] In some embodiments, the conjugate is Figure 1 Conjugate No. 10 as described in, wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0497] In some embodiments, the conjugate is Figure 1 Conjugate No. 10 as described in, wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0498] In some embodiments, the conjugate is Figure 1 Conjugate No. 10 as described in, wherein 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 enhancing unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent.

[0499] In some embodiments, the conjugate is Figure 1 Conjugate No. 10 as described in, wherein 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 enhancing unit is attached directly or via a linker unit to an internal position of the antisense strand of the nucleic acid agent.

[0500] In some embodiments, the conjugate is Figure 1 conjugate number 10 as described in Figure 1 , wherein 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 enhancing unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent.

[0501] In some embodiments, the conjugate is Figure 1 conjugate number 10 as described in Figure 1 , wherein 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 enhancing unit is attached directly or via a linker unit to an internal position of the sense strand of the nucleic acid agent.

[0502] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in Figure 1 .

[0503] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in Figure 1 , wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0504] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in Figure 1 , wherein the nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent.

[0505] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0506] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0507] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0508] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in

[0509] wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent. Figure 1 In some embodiments, the conjugate is

[0510] conjugate number 11 as described in Figure 1 wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0511] In some embodiments, the conjugate is Figure 1 conjugate number 11 as described in

[0512] wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent. Figure 1

[0513] Figure 1 In some embodiments, the conjugate is conjugate number 12 as described in

[0514] Figure 1 In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in wherein the nucleotide-based enhancing unit is attached to the antisense strand of the nucleic acid agent.

[0515] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in

[0516] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in

[0517] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in

[0518] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in

[0519] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in

[0520] In some embodiments, the conjugate is Figure 1Conjugate No. 12 described therein, wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent.

[0521] In some embodiments, the conjugate is Figure 1 Conjugate No. 12 described therein, wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0522] In some embodiments, the conjugate is Figure 1 Conjugate No. 12 described therein, wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0523] In some embodiments, the conjugate is Figure 1 Conjugate No. 12 described therein, wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent.

[0524] In some embodiments, the conjugate is Figure 1 Conjugate No. 12 described therein, wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent.

[0525] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0526] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0527] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the antisense strand of the nucleic acid agent.

[0528] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent.

[0529] In some embodiments, the conjugate is Figure 1 conjugate number 12 as described in Figure 1 , wherein 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 enhancing 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 enhancing unit is attached directly or via a linker unit to the 3'-end of the sense strand of the nucleic acid agent.

[0530] In some embodiments, the conjugate is selected from Figure 2 the conjugates described in

[0531] In some embodiments, the conjugate is selected from the structures described in Table C, wherein

[0532] the attachment to the [nucleic acid agent] (e.g., the antisense or sense strand of the nucleic acid agent) can independently be at a terminal position (e.g., a nucleotide at the 3'- or 5'-end) or an internal position (e.g., a nucleotide not at the 3'- or 5'-end) of the nucleic acid agent; and

[0533] the attachment to the [nucleic acid agent] and the [ligand] can independently be direct or via a linker unit.

[0534] Table C

[0535]

[0536]

[0537] In some embodiments, the conjugate is selected from the structures described in Table C, wherein the [nucleic acid agent] is the antisense strand of the nucleic acid agent.

[0538] In some embodiments, the conjugate is selected from the structures described in Table C, wherein the [nucleic acid agent] is the sense strand of the nucleic acid agent.

[0539] Nucleotide-based enhancing unit

[0540] As used herein, "nucleotide-based enhancing unit" refers to the moiety corresponding to a nucleotide-based enhancer, wherein the * in the nucleotide at the 5'-end and / or the ** in the nucleotide at the 2'- or 3'-end is attached to a ligand, a linker unit, and / or a nucleic acid agent.

[0541] In some embodiments, the nucleotide-based enhancing unit corresponds to a nucleotide-based enhancer, wherein the * in the nucleotide at the 5'-end is attached to a ligand, a linker unit, and / or a nucleic acid agent.

[0542] In some embodiments, the nucleotide-based enhancing unit corresponds to a nucleotide-based enhancer, wherein the ** in the nucleotide at the 2'- or 3'-end is attached to a ligand, a linker unit, and / or a nucleic acid agent.

[0543] In some embodiments, the nucleotide-based enhancing unit corresponds to a nucleotide-based enhancer, wherein the * in the nucleotide at the 5'-end is attached to a ligand, a linker unit, and / or a nucleic acid agent, and the ** in the nucleotide at the 2'- or 3'-end is attached to a ligand, a linker unit, and / or a nucleic acid agent.

[0544] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I) or formula (II), wherein:

[0545] * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, * is H or is attached to a ligand, a linker unit, and / or a nucleic acid agent; and

[0546] ** is attached to the 5'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, ** is H or is attached to a ligand, a linker unit, and / or a nucleic acid agent.

[0547] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I') or formula (II'), wherein:

[0548] * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, * is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0549] ** is attached to the 5'-position of another nucleotide of the nucleotide-based enhancing unit, or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, ** is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0550] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I-A), formula (II-A), formula (III-A), or formula (IV-A), wherein:

[0551] * is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, * is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0552] ** is attached to the 5'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, ** is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0553] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I'-A), formula (II'-A), formula (III'-A), or formula (IV'-A), wherein:

[0554] *is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, *is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0555] **is attached to the 5'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, **is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0556] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I-B) or formula (II-B), wherein:

[0557] *is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, *is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0558] **is attached to the 5'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, **is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0559] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I'-B) or formula (II'-B), wherein:

[0560] *is attached to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, *is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0561] **is attached to the 5'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, **is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0562] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I-C), formula (II-C), formula (III-C) or formula (IV-C), wherein:

[0563] *is attached to the 2'-position or 3'-position of another nucleotide of a nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, *is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0564] **is attached to the 5'-position of another nucleotide of a nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, **is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0565] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit has formula (I'-C), formula (II'-C), formula (III'-C), or formula (IV'-C), wherein:

[0566] *is attached to the 2'-position or 3'-position of another nucleotide of a nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, *is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0567] **is attached to the 5'-position of another nucleotide of a nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, **is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0568] In some embodiments, at least one nucleotide in the nucleotide-based enhancing unit is selected from Table L, wherein:

[0569] *is attached to the 2'-position or 3'-position of another nucleotide of a nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, *is H or is attached to a ligand, a linker unit, and / or a nucleic acid; and

[0570] **is attached to the 5'-position of another nucleotide of a nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, **is H or is attached to a ligand, a linker unit, and / or a nucleic acid.

[0571] In some embodiments, the nucleotide-based enhancing unit comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides.

[0572] In some embodiments, the nucleotide-based enhancing unit comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides.

[0573] In some embodiments, the nucleotide-based enhancing 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.

[0574] In some embodiments, the nucleotide-based enhancing unit comprises 5 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 6 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 7 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 8 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 9 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 10 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 11 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 12 nucleotides. In some embodiments, the nucleotide-based enhancing unit comprises 13 nucleotides.

[0575] In some embodiments, the nucleotide-based enhancer unit comprises from 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.

[0576] In some embodiments, the nucleotide-based enhancer unit comprises from 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 from 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.

[0577] In some embodiments, the nucleotide-based enhancer unit comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0578] In some embodiments, the nucleotide-based enhancer unit comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0579] In some embodiments, the nucleotide-based enhancing unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0580] In some embodiments, the nucleotide-based enhancing unit comprises 5 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 6 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 7 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 8 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 9 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 10 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 11 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 12 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancing unit comprises 13 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0581] In some embodiments, the nucleotide-based enhancer unit comprises from 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, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0582] In some embodiments, the nucleotide-based enhancer unit comprises from 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 from 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, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers and / or one or more phosphorothioate linkers.

[0583] In some embodiments, the nucleotide-based enhancer unit comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0584] In some embodiments, the nucleotide-based enhancer unit comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0585] In some embodiments, the nucleotide-based enhancing unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers.

[0586] In some embodiments, the nucleotide-based enhancing unit comprises 5 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 6 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 7 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 8 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 9 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 10 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 11 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 12 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers. In some embodiments, the nucleotide-based enhancing unit comprises 13 nucleotides, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers.

[0587] In some embodiments, the nucleotide-based enhancing unit comprises from 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, wherein the nucleotide-based enhancing unit further comprises one or more phosphodiester linkers.

[0588] In some embodiments, the nucleotide-based enhancer unit comprises from 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 from 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, wherein the nucleotide-based enhancer unit further comprises one or more phosphodiester linkers.

[0589] In some embodiments, the nucleotide-based enhancer unit comprises from 2 to 28 nucleotides, from 2 to 26 nucleotides, from 2 to 24 nucleotides, from 2 to 22 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 16 nucleotides, from 2 to 14 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides, from 2 to 9 nucleotides, from 2 to 8 nucleotides, from 2 to 7 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, from 2 to 4 nucleotides, or from 2 to 3 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0590] In some embodiments, the nucleotide-based enhancer unit comprises from 4 to 30 nucleotides, from 6 to 30 nucleotides, from 8 to 30 nucleotides, from 10 to 30 nucleotides, from 12 to 30 nucleotides, from 14 to 30 nucleotides, from 16 to 30 nucleotides, from 18 to 30 nucleotides, from 20 to 30 nucleotides, from 21 to 30 nucleotides, from 22 to 30 nucleotides, from 23 to 30 nucleotides, from 24 to 30 nucleotides, from 25 to 30 nucleotides, from 26 to 30 nucleotides, from 27 to 30 nucleotides, from 28 to 30 nucleotides, or from 29 to 30 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0591] In some embodiments, the nucleotide-based enhancer unit comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0592] In some embodiments, the nucleotide-based enhancer unit comprises 5 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 6 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 7 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 8 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 9 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 10 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 11 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 12 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers. In some embodiments, the nucleotide-based enhancer unit comprises 13 nucleotides, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0593] In some embodiments, the nucleotide-based enhancer unit comprises from 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, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0594] In some embodiments, the nucleotide-based enhancer unit comprises from 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 from 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, wherein the nucleotide-based enhancer unit further comprises one or more phosphorothioate linkers.

[0595] Linker Unit

[0596] As used herein, "Linker Unit" or "linker unit" refers to a moiety having an attachment to a nucleotide-based enhancer unit, ligand, and / or nucleic acid agent.

[0597] In some embodiments, the linker unit has an attachment to a nucleotide-based enhancer unit and a ligand.

[0598] In some embodiments, the linker unit has an attachment to a ligand and a nucleic acid agent.

[0599] In some embodiments, the linker unit has an attachment to a nucleotide-based enhancer unit and a nucleic acid agent.

[0600] In some embodiments, the linker unit is a ribose derivative.

[0601] In some embodiments, the linker unit is a 1'-alkyl modified ribose derivative, e.g., as described in PCT Application No. PCT / US2022 / 039517, which is incorporated herein by reference.

[0602] In some embodiments, the linker unit is a 2'-alkyl modified ribose derivative or a 3'-alkyl modified ribose derivative, e.g., as described in PCT Application No. PCT / US2022 / 044377, which is incorporated herein by reference.

[0603] In some embodiments, the linker unit is a polyhydroxylated cyclopentane derivative, e.g., as described in PCT Application No. PCT / US2022 / 045748, which is incorporated herein by reference.

[0604] Ligand

[0605] 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), is capable of mediating its entry into a target site (e.g., a target cell or target tissue) or facilitating its delivery to a target site (e.g., a target cell or target tissue).

[0606] In some embodiments, the ligand comprises a glycoconjugate moiety (e.g., N-acetylgalactosamine (GalNAc)), which can directly uptake oligonucleotides into the liver.

[0607] In some embodiments, the ligand binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand binds to (e.g., via the ASGPR) the liver, such as the parenchymal cells of the liver.

[0608] In some embodiments, the ligand is capable of binding to a glucagon-like peptide receptor, such as the glucagon-like peptide-1 receptor (GLP-1 receptor). In some embodiments, the ligand binds to the glucagon-like peptide-1 receptor (GLP-1 receptor). In some embodiments, the ligand binds to (e.g., via the GLP-1 receptor) the pancreas, such as the β-cells of the pancreas.

[0609] In some embodiments, the ligand is a small molecule, carbohydrate, oligonucleotide, antibody, or peptide.

[0610] In some embodiments, the ligand is a peptide that is capable of binding to a glucagon-like peptide receptor, such as the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0611] Suitable ligands include, but are not limited to, ligands disclosed in the following: 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, US patent application publications 2006 / 0275288, 2007 / 124461, 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, 2014 / 0206607, and 2009 / 0247608, and US patents 9,187,522, 8,329,419, and 8,389,689, each of which is incorporated by reference.

[0612] In some embodiments, the ligand comprises a carbohydrate moiety.

[0613] As used herein, "carbohydrate moiety" refers to a moiety comprising one or more monosaccharide units, each of the one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each carbon atom bonded to an oxygen, nitrogen, or sulfur atom. In some embodiments, the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide. In some embodiments, the carbohydrate moiety comprises an oligosaccharide containing from about 4-9 monosaccharide units. In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose, or mucopolysaccharide).

[0614] In some embodiments, the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide.

[0615] In some embodiments, the carbohydrate moiety comprises an oligosaccharide (e.g., containing from about four to about nine monosaccharide units).

[0616] In some embodiments, the carbohydrate moiety comprises a polysaccharide (e.g., starch, glycogen, cellulose, or a polysaccharide gum).

[0617] In some embodiments, the ligand is capable of binding to the human asialoglycoprotein receptor (ASGPR), such as the human asialoglycoprotein receptor 2 (ASGPR2).

[0618] In some embodiments, the carbohydrate moiety comprises a sugar (e.g., one, two, or three sugars).

[0619] In some embodiments, the carbohydrate moiety comprises galactose or a derivative thereof (e.g., one, two, or three galactose or a derivative thereof).

[0620] In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (e.g., one, two, or three N-acetylgalactosamine or a derivative thereof).

[0621] In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosamine or a derivative thereof (e.g., one, two, or three N-acetyl-D-galactosamine or a derivative thereof).

[0622] In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine (e.g., one, two, or three N-acetylgalactosamine).

[0623] In some embodiments, the carbohydrate moiety comprises N-acetyl-D-galactosamine (e.g., one, two, or three N-acetyl-D-galactosamine).

[0624] In some embodiments, the carbohydrate moiety comprises mannose or a derivative thereof (e.g., mannose-6-phosphate).

[0625] In some embodiments, the carbohydrate moiety further comprises a linking moiety that links one or more sugars (e.g., N-acetyl-D-galactosamine) to the linker unit.

[0626] In some embodiments, the linking moiety comprises a thioether (e.g., succinimidyl thioester or a hydrolyzed analogue thereof), disulfide, triazole, thiophosphate, phosphodiester, ester, amide, or any combination thereof.

[0627] In some embodiments, the linking moiety is a triantennary linking moiety.

[0628] Suitable ligands include, but are not limited to, ligands disclosed in the following: PCT 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.

[0629] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0630] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0631] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0632] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0633] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0634] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0635] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0636] In some embodiments, the ligand comprises (e.g., one, two, or three ).

[0637] In some embodiments, the ligand comprises

[0638] In some embodiments, the ligand comprises

[0639] In some embodiments, the ligand comprises

[0640] In some embodiments, the ligand comprises

[0641] In some embodiments, the ligand comprises

[0642] In some embodiments, the ligand comprises

[0643] In some embodiments, the ligand comprises

[0644] In some embodiments, the ligand comprises

[0645] In some embodiments, the ligand comprises a lipid moiety (e.g., one, two, or three lipid moieties).

[0646] In some embodiments, the lipid moiety comprises C 8 -C 24 fatty acids, cholesterol, vitamins, sterols, phospholipids, or any combination thereof (e.g., one, two, or three of them).

[0647] In some embodiments, the ligand comprises a peptide moiety (e.g., one, two, or three peptide moieties). In some embodiments, the ligand comprises a peptide moiety (e.g., one, two, or three peptide moieties) that is capable of binding to a glucagon-like peptide receptor, e.g., a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0648] In some embodiments, the peptide moiety comprises integrin, insulin, glucagon-like peptide (e.g., GLP-1) (e.g., one, two, or three of them), or any combination thereof.

[0649] In some embodiments, the peptide moiety comprises (e.g., one, two, or three) glucagon-like peptide (e.g., GLP-1). In some embodiments, the peptide moiety comprises (e.g., one, two, or three) glucagon.

[0650] In some embodiments, the peptide moiety comprises (e.g., one, two, or three) dulaglutide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) lixisenatide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) exenatide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) semaglutide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) albiglutide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) liraglutide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) tirzepatide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) aexintide. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) HISHS-2001. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) MAR709. In some embodiments, the peptide moiety comprises (e.g., one, two, or three) exendin-4.

[0651] In some embodiments, the ligand comprises an antibody moiety (e.g., transferrin). In some embodiments, the ligand comprises an antibody moiety capable of binding to a glucagon-like peptide receptor, such as a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0652] 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, such as a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0653] In some embodiments, the ligand comprises an oligonucleotide (e.g., an aptamer or CpG). In some embodiments, the ligand comprises an oligonucleotide (e.g., an aptamer or CpG) capable of binding to a glucagon-like peptide receptor, such as a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0654] In some embodiments, the ligand comprises one, two, or three oligonucleotides (e.g., an aptamer or CpG). In some embodiments, the ligand comprises one, two, or three oligonucleotides (e.g., an aptamer or CpG) capable of binding to a glucagon-like peptide receptor, such as a glucagon-like peptide-1 receptor (GLP-1 receptor).

[0655] In some embodiments, the ligand comprises a small molecule moiety. In some embodiments, the ligand comprises a small molecule moiety capable of binding to a glucagon-like peptide receptor, such as the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0656] In some embodiments, the ligand comprises one, two, or three small molecule moieties. In some embodiments, the ligand comprises one, two, or three small molecule moieties capable of binding to a glucagon-like peptide receptor, such as the glucagon-like peptide-1 receptor (GLP-1 receptor).

[0657] In some embodiments, the ligand comprises:

[0658] one, two, or three sugars (e.g., N-acetyl-D-galactosamine);

[0659] one, two, or three lipid moieties;

[0660] one, two, or three peptide moieties;

[0661] one, two, or three antibody moieties;

[0662] one, two, or three oligonucleotides; or

[0663] any combination thereof.

[0664] In some embodiments, the ligand comprises:

[0665] one, two, or three carbohydrate moieties;

[0666] one, two, or three peptide moieties;

[0667] one, two, or three antibody moieties;

[0668] one, two, or three small molecule moieties; or

[0669] any combination thereof.

[0670] Nucleic acid agent

[0671] In some embodiments, the nucleic acid agent comprises an oligonucleotide.

[0672] In some embodiments, the nucleic acid agent (e.g., oligonucleotide) comprises one or more phosphate groups or one or more phosphate group analogs.

[0673] In some embodiments, the linker unit is attached to the nucleic acid agent (e.g., oligonucleotide) via a phosphate group or a phosphate group analog in the nucleic acid agent.

[0674] In some embodiments, the oligonucleotide has a length of from 1 to 40 nucleotides, from 10 to 40 nucleotides, from 12 to 35 nucleotides, from 15 to 30 nucleotides, from 18 to 25 nucleotides, or from 20 to 23 nucleotides. In some embodiments, the oligonucleotide has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the oligonucleotide has a length of 20, 21, 22, or 23 nucleotides.

[0675] In some embodiments, the nucleic acid agent comprises RNA, DNA, or a mixture thereof.

[0676] In some embodiments, the nucleic acid agent comprises RNA.

[0677] In some embodiments, the oligonucleotide is siRNA (e.g., single-stranded siRNA (e.g., hairpin single-stranded siRNA) or double-stranded siRNA), microRNA, anti-microRNA, microRNA mimic, antagomir, dsRNA, ssRNA, aptamer, immunostimulatory oligonucleotide, decoy oligonucleotide, splicing-altering oligonucleotide, triplex-forming oligonucleotide, G-quadruplex, or antisense oligonucleotide.

[0678] In some embodiments, the nucleic acid agent comprises double-stranded RNA (dsRNA), wherein the double-stranded RNA comprises a sense strand and an antisense strand, as described herein.

[0679] In some embodiments, the nucleic acid agent comprises double-stranded siRNA (ds-siRNA), wherein the double-stranded siRNA comprises a sense strand and an antisense strand, as described herein.

[0680] It should be understood that the sense strand is also referred to as the passenger strand, and the terms “sense strand” and “passenger strand” are used interchangeably herein.

[0681] It should be understood that the antisense strand is also referred to as the guide strand, and the terms “antisense strand” and “guide strand” are used interchangeably herein.

[0682] In some embodiments, the oligonucleotide is iRNA.

[0683] The term "iRNA" refers to an RNA agent that can down-regulate the expression of a target gene (such as siRNA), such as an endogenous or pathogen target RNA. Without wishing to be bound by theory, iRNA can act through one or more of a variety of mechanisms, including post-transcriptional cleavage of the target mRNA (known in the art as RNAi), or pre-transcriptional or pre-translational mechanisms. iRNA can be single-stranded or can include more than one strand, for example, it can be double-stranded iRNA. If the iRNA is single-stranded, it can include a 5' modification, which 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, such as a 5' modification.

[0684] iRNA generally includes a region having sufficient homology to the target gene and having sufficient length in terms of nucleotides such that the iRNA or a fragment thereof can mediate down-regulation of the target gene. The iRNA is or includes a region that is at least partially complementary to the target RNA and in some embodiments completely complementary to the target RNA. There does not necessarily need to be complete complementarity between the iRNA and the target, but this correspondence may be sufficient for the iRNA or its cleavage product to be able to direct sequence-specific silencing, such as by RNAi cleavage of the target RNA (such as mRNA).

[0685] The nucleotides in the iRNA can be modified (for example, one or more nucleotides can include a 2'-F group or a 2'-OCH 3 group, or be a nucleotide substitute). The single-stranded or double-stranded regions of the iRNA can be modified or include nucleotide substitutes, for example, one or more unpaired regions of a hairpin structure, for example, the region connecting two complementary regions, can have a modification or nucleotide substitute. Modifications that stabilize one or more 3'-ends or 5'-ends of the iRNA, for example, against exonucleases. 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), special biotin or fluorescein reagents, which appear as phosphoramidites and have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis. Modifications can also include, for example, the use of modifications at the 2'OH group of the ribose, such as the use of deoxyribonucleotides such as deoxythymidine in place of ribonucleotides, and the use of modifications of phosphate groups, such as phosphorothioate modifications. In some embodiments, different strands will include different modifications.

[0686] In some embodiments, the strands are selected such that the iRNA comprises single-stranded or unpaired regions at one or both ends of the molecule. Double-stranded iRNA can have overhangs, such as one or two 5' overhangs or 3' overhangs (e.g., at least 3' overhangs of 2-3 nucleotides). In some embodiments, the iRNA has overhangs of 1, 2, or 3 nucleotides in length at each end, such as 3' overhangs. The overhangs can be the result of one strand being longer than the other, or the result of the staggering of two strands of the same length.

[0687] In some embodiments, the length of the double-stranded region between the strands of the iRNA is between 6 nucleotides and 30 nucleotides. In some embodiments, the length of the double-stranded region is between 15 nucleotides and 30 nucleotides, most preferably 18, 19, 20, 21, 22, and 23 nucleotides. In some embodiments, the length of the double-stranded region is between 6 nucleotides and 20 nucleotides, most preferably 6, 7, 8, 9, 10, 11, and 12 nucleotides.

[0688] The oligonucleotide can be the oligonucleotide described in U.S. Patent Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, each of which is hereby incorporated by reference.

[0689] In some embodiments, the oligonucleotide is siRNA.

[0690] In some embodiments, the oligonucleotide is single-stranded siRNA.

[0691] In some embodiments, the oligonucleotide is double-stranded siRNA, such as the double-stranded siRNA described herein.

[0692] As used herein, "single-stranded siRNA" is an siRNA consisting of a single strand that includes a double-stranded region formed by intrastrand pairing, e.g., it can be a hairpin structure or a stem-loop structure or include a hairpin structure or a stem-loop structure. The single-stranded siRNA can be antisense with respect to the target molecule.

[0693] The single-stranded siRNA can be long enough such that it can enter RISC and participate in RISC-mediated cleavage of the target mRNA. The single-stranded siRNA is at least 14 nucleotides in length, and in some embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides. In some embodiments, the single-stranded siRNA is less than 200, 100, 80, 60, 50, 40, or 30 nucleotides in length.

[0694] In some embodiments, the single-stranded siRNA has a length of from 10 to 40 nucleotides, from 12 to 35 nucleotides, from 15 to 30 nucleotides, from 18 to 25 nucleotides, or from 20 to 23 nucleotides. In some embodiments, the single-stranded siRNA has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the single-stranded siRNA has a length of 20, 21, 22, or 23 nucleotides.

[0695] The hairpin siRNA may have a duplex region equal to or at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs. The length of the duplex region may be equal to or less than 200, 100, or 50 nucleotide pairs. In some embodiments, the length of the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs. The hairpin may have a single-stranded overhang or a terminal unpaired region. In some embodiments, the length of the overhang is 2-3 nucleotides. In some embodiments, the overhang is located on the sense side of the hairpin and in some embodiments on the antisense side of the hairpin.

[0696] In some embodiments, the oligonucleotide is a double-stranded siRNA.

[0697] As used herein, "double-stranded siRNA" is an siRNA that includes more than one strand and in some cases two strands, wherein regions of interstrand hybridization can form a duplex structure.

[0698] In some embodiments, the length of the sense strand of the double-stranded siRNA can be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides. The length of the sense strand of the double-stranded siRNA can be equal to or less than 200, 100, or 50 nucleotides. The length range can be from 17 to 25, 19 to 23, 19 to 21, 21 to 23, or 20 to 22 nucleotides.

[0699] In some embodiments, the sense strand has a length of from 10 to 40 nucleotides, from 12 to 35 nucleotides, from 15 to 30 nucleotides, from 18 to 25 nucleotides, or from 20 to 23 nucleotides. In some embodiments, the sense strand has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the sense strand has a length of 20, 21, 22, or 23 nucleotides.

[0700] In some embodiments, the sense strand has a length of 18, 19, 20, 21, or 22 nucleotides.

[0701] In some embodiments, the length of the antisense strand of the double-stranded siRNA can be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides. The length of the antisense strand of the double-stranded siRNA can be equal to or less than 200, 100, or 50 nucleotides. The length range can be from 17 to 25, from 19 to 23, from 19 to 21, from 21 to 23, or from 20 to 22 nucleotides.

[0702] In some embodiments, the antisense strand has a length from 10 to 40 nucleotides, from 12 to 35 nucleotides, from 15 to 30 nucleotides, from 18 to 25 nucleotides, or from 20 to 23 nucleotides. In some embodiments, the antisense strand has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the antisense strand has a length of 20, 21, 22, or 23 nucleotides.

[0703] In some embodiments, the antisense strand has a length of 20, 21, 22, 23, or 24 nucleotides.

[0704] In some embodiments, the sense strand has a length of 18, 19, 20, 21, or 22 nucleotides, and the antisense strand has a length of 20, 21, 22, 23, or 24 nucleotides.

[0705] In some embodiments, the sense strand has a length of 18 nucleotides, and the antisense strand has a length of 20 nucleotides.

[0706] In some embodiments, the sense strand has a length of 19 nucleotides, and the antisense strand has a length of 21 nucleotides.

[0707] In some embodiments, the sense strand has a length of 20 nucleotides, and the antisense strand has a length of 22 nucleotides.

[0708] In some embodiments, the sense strand has a length of 21 nucleotides, and the antisense strand has a length of 23 nucleotides.

[0709] In some embodiments, the sense strand has a length of 22 nucleotides, and the antisense strand has a length of 24 nucleotides.

[0710] The length of the double-stranded portion of double-stranded siRNA can be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs. The length of the double-stranded portion of double-stranded siRNA can be equal to or less than 200, 100, or 50 nucleotide pairs. The length ranges can be 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0711] In some embodiments, the siRNA is large enough such that it can be cleaved by endogenous molecules, such as by Dicer, to produce smaller siRNAs, such as siRNA agents.

[0712] The sense and antisense strands can be selected such that the double-stranded siRNA includes single-stranded regions or unpaired regions at one or both ends of the molecule. Thus, the double-stranded siRNA can comprise a sense strand and an antisense strand that are paired to include overhangs, such as one or two 5' overhangs or 3' overhangs or a 3' overhang of 1-3 nucleotides. The overhangs can be the result of one strand being longer than the other or the result of the staggering of two strands of the same length. Some embodiments will have at least one 3' overhang. In some embodiments, both ends of the siRNA molecule will have 3' overhangs. In some embodiments, the overhang is 2 nucleotides.

[0713] In some embodiments, the length of the double-stranded region is between 15 nucleotides and 30 nucleotides, or is 18, 19, 20, 21, 22, and 23 nucleotides, such as within the ssiRNA ranges discussed above. The ssiRNA can be similar in length and structure to the products of natural Dicer processing from long dsiRNA. Embodiments are also included where the two strands of the ssiRNA are attached, such as covalently attached. 3' overhangs as well as hairpins or other single-stranded structures that provide the desired double-stranded region are also contemplated.

[0714] The siRNAs described herein, including double-stranded siRNAs and single-stranded siRNAs, can mediate the silencing of target RNAs, such as mRNAs, such as transcripts of genes encoding proteins. For convenience, such mRNAs are also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Generally, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNAs can also be targeted, such as tRNAs and viral RNAs.

[0715] As used herein, the phrase "mediating RNAi" refers to the ability to silence a target RNA in a sequence-specific manner. Without wishing to be bound by theory, it is believed that silencing uses the RNAi mechanism or process and a guide RNA, such as a 21- to 23-nucleotide ssiRNA.

[0716] In some embodiments, the siRNA is "sufficiently complementary" to the target RNA (e.g., target mRNA) such that the siRNA silences the production of the protein encoded by the target mRNA. In another embodiment, the siRNA is "perfectly complementary" to the target RNA, e.g., the target RNA and the siRNA anneal, e.g., to form a hybrid made exclusively of Watson-Crick base pairs in the region of perfect complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., an internal region of at least 10 nucleotides) that is perfectly complementary to the target RNA. Additionally, in some embodiments, the siRNA specifically discriminates single nucleotide differences. In such cases, the siRNA mediates RNAi only when there is perfect complementarity in the region of the single nucleotide difference (e.g., within 7 nucleotides).

[0717] 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 not translated into proteins. The processed miRNA is a single-stranded ~17 - 25 nucleotide (nt) RNA molecule that becomes incorporated into the RNA-induced silencing complex (RISC) and has been identified as key regulators of development, cell proliferation, apoptosis, and differentiation. They are thought to play a role in the regulation of gene expression by binding to the 3'-untranslated region of specific mRNAs. RISC mediates the downregulation of gene expression through translational inhibition, transcript cleavage, or both. RISC is also associated with transcriptional silencing in the nuclei of a variety of eukaryotes.

[0718] The number of miRNA sequences identified to date is large and growing, and illustrative examples can be found, for example, in: "miRBase: microRNA sequences, targets and gene nomenclature" Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. NAR, 2006, 34, Database Issue, D140 - D144; "The microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109 - D111.

[0719] Antisense oligonucleotides: In some embodiments, the nucleic acid is an antisense oligonucleotide directed against a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" means an oligonucleotide that includes an oligonucleotide complementary to a target polynucleotide sequence. Antisense oligonucleotides are single-stranded DNA or RNA that are complementary to a selected sequence (such as target gene mRNA). Antisense oligonucleotides are thought to inhibit gene expression by binding to the complementary mRNA. Binding to the target mRNA can result in inhibition of gene expression by preventing translation of the complementary mRNA strand via binding to the complementary mRNA strand or by causing degradation of the target mRNA. Antisense DNA can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, the DNA / RNA hybrid can be degraded by the enzyme RNase H. In some embodiments, the antisense oligonucleotide comprises from about 10 to about 50 nucleotides, more preferably from about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be precisely complementary to the desired target gene. Thus, situations are envisioned where non-target-specific activity is found with antisense, or where an antisense sequence containing one or more mismatches to the target sequence is most preferred for a particular use.

[0720] Antisense oligonucleotides have been shown to be effective and targeted inhibitors of protein synthesis and, thus, can be used to specifically inhibit protein synthesis of a targeted gene. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalacturonase and the muscarinic 2 acetylcholine receptor has been inhibited by antisense oligonucleotides directed against their respective mRNA sequences (U.S. Patent Nos. 5,739,119 and 5,759,829, each incorporated by reference). In addition, examples of antisense inhibition have been demonstrated with nucleoprotein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor, and human EGF (Jaskulski et al., Science. June 10, 1988; 240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989; 1(4):225-32; Peris et al., Brain Res Mol Brain Res. June 15, 1998; 57(2):310-20; U.S. Patent Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288, each incorporated by reference). In addition, antisense constructs have been described that inhibit and can be used to treat various abnormal cell proliferations, such as cancer (U.S. Patent Nos. 5,747,470; 5,591,317 and 5,783,683, each incorporated by reference).

[0721] Methods for generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides that target any polynucleotide sequence. The selection of an antisense oligonucleotide sequence specific for a given target sequence is based on the analysis of the selected target sequence and the determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that would reduce or preclude specific binding to the target mRNA in the host cell. Highly preferred target regions of the mRNA include those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to the 5' region of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using version 4 of the OLIGO primer analysis software (Molecular Biology Insights) and / or the BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17): 3389-402).

[0722] Antagomir: An Antagomir is an RNA-like oligonucleotide that has multiple modifications for ribonuclease protection and pharmacological properties, such as enhanced tissue and cell uptake. They differ from normal RNA in, for example, sugars, complete 2'-O-methylation of the phosphorothioate backbone, and, for example, a cholesterol moiety at the 3'-terminus. Antagomirs can be used to effectively silence endogenous miRNAs by forming duplexes that contain an antagomir and an endogenous miRNA, 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, which is hereby incorporated by reference in its entirety. 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 by reference herein).

[0723] An Antagomir can include ligand-conjugated monomeric subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in U.S. Patent Application Publication No. 2005 / 0107325, which is incorporated by reference in its entirety. An Antagomir can have a ZXY structure, such as the ZXY structure described in WO 2004 / 080406, which is incorporated by reference in its entirety. An Antagomir can complex with an amphiphilic moiety. Exemplary amphiphilic moieties for use with oligonucleotide agents are described in WO 2004 / 080406, which is incorporated by reference in its entirety.

[0724] Aptamer: An aptamer is a nucleic acid or peptide molecule that binds to a specific molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990), each of which is incorporated by reference in its entirety). DNA aptamers or RNA aptamers have been successfully generated that bind many different entities 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 by reference in its entirety. Aptamers can be RNA- or DNA-based and can include riboswitches. A riboswitch is a part of an mRNA molecule that can directly bind a small target molecule and whose binding to the target affects the activity of the gene. Thus, an mRNA containing a riboswitch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule. Generally, aptamers are engineered by repeated rounds of in vitro selection or equivalent SELEX (systematic evolution of ligands by exponential enrichment) to bind a variety of molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods, and can be used alone or in combination with other aptamers specific for the same target. In addition, as described more fully herein, the term "aptamer" specifically includes "secondary aptamer", which contains a consensus sequence obtained by comparing two or more known aptamers to a given target.

[0725] Ribozymes: According to another embodiment, the nucleic acid-lipid particles are associated with ribozymes. Ribozymes are RNA molecular complexes having a specific catalytic domain with endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. December 1987; 84(24):8788-92; Forster and Symons, Cell. April 24, 1987; 49(2):211-20). For example, a large number of ribozymes accelerate phosphoester transfer reactions with high specificity, usually cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., Cell. December 1981; 27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. December 5, 1990; 216(3):585-610; Reinhold-Hurek and Shub, Nature. May 14, 1992; 357(6374):173-6). This specificity has been attributed to the requirement for the substrate to bind to the internal guide sequence ("IGS") of the ribozyme via specific base-pairing interactions prior to the chemical reaction.

[0726] At least six fundamental classes of naturally occurring catalytic RNAs are currently known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions (and can thus cleave other RNA molecules). In general, catalytic nucleic acids act by first binding to the target RNA. Such binding occurs through the target-binding portion of the catalytic nucleic acid, which is adjacent to the catalytic portion of the molecule that acts to cleave the target RNA. Thus, the catalytic nucleic acid first recognizes the target RNA, then binds to the target RNA by complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. Strategic cleavage of such target RNAs will disrupt their ability to direct the synthesis of encoded proteins. After the catalytic nucleic acid binds and cleaves its RNA target, it is released from the RNA to seek another target and can repeatedly bind and cleave new targets.

[0727] For example, an enzymatic nucleic acid molecule can be formed as a hammerhead, hairpin, hepatitis delta virus, group I intron or RNase P RNA (associated with an RNA guide sequence) or a Neurospora VS RNA motif. Specific examples of the hammerhead motif are described by Rossi et al., Nucleic Acids Res., Sep. 11, 1992; 20(17):4559-65. Examples of the hairpin motif are described by: Hampel et al. (European Patent Application Publication No. EP 0360257); Hampel and Tritz, Biochemistry, Jun. 13, 1989; 28(12):4929-33; Hampel et al., Nucleic Acids Res., Jan. 25, 1990; 18(2):299-304 and U.S. Patent No. 5,631,359. Examples of the hepatitis delta virus motif are described by Perrotta and Been, Biochemistry, Dec. 1, 1992; 31(47):11843-52; examples of the RNase P motif are described by Guerrier-Takada et al., Cell, Dec. 1983; 35(3Pt 2):849-57; the Neurospora VS RNA ribozyme motif is described by: Collins (Saville and Collins, Cell, May 18, 1990; 61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA, Oct. 1, 1991; 88(19):8826-30; Collins and Olive, Biochemistry, Mar. 23, 1993; 32(11):2795-9); and examples of group I introns are described in U.S. Patent No. 4,987,071. An important feature of the enzymatic nucleic acid molecules used is that they have specific substrate binding sites that are complementary to one or more target gene DNA or RNA regions, and they have a nucleotide sequence within or around the substrate binding site that confers RNA cleavage activity on the molecule. Thus, ribozyme constructs need not be limited to the specific motifs mentioned herein.

[0728] Methods for generating ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed as described in International Patent Application Publication Nos. WO 93 / 23569 and WO 94 / 02595, which are hereby expressly incorporated by reference in their entireties, and synthesized as described therein for in vitro and in vivo testing.

[0729] Ribozyme activity can be optimized by altering the length of the ribozyme binding arms or by chemically synthesizing ribozymes having modifications that prevent their degradation by serum ribonucleases (see, e.g., International Patent Application Publication Nos. WO 92 / 07065, WO 93 / 15187, and WO 91 / 03162; European Patent Application Publication No. 92110298.4; U.S. Patent No. 5,334,711; and International Patent Application Publication No. WO 94 / 13688, which describe various chemical modifications that can be made to the sugar moiety of enzymatically active RNA molecules), modifications that enhance their potency in cells, and removal of the stem II bases to shorten RNA synthesis time and reduce chemical requirements.

[0730] Immunostimulatory oligonucleotides: Nucleic acids associated with lipid particles can be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that are capable of inducing an immune response when administered to a subject, which can be a mammal or other patient. ISS include, for example, certain palindromes that result in 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, as well as other known ISS features (such as poly-G domains, see WO 96 / 11266, which is incorporated by reference in its entirety).

[0731] The immune response can be an innate immune response or an adaptive immune response. The immune system of vertebrates is further divided into the innate immune system and the acquired adaptive immune system, and the acquired adaptive immune system is further divided into humoral and cellular components. In some embodiments, the immune response can be mucosal.

[0732] In some embodiments, the immunostimulatory nucleic acid is only immunostimulatory when administered in combination with lipid particles and is not immunostimulatory when administered in its "free form". Such oligonucleotides are considered immunostimulatory.

[0733] An immunostimulatory nucleic acid is considered non-sequence-specific when it is not required to specifically bind to a target polynucleotide and reduce the expression of the target polynucleotide in order to elicit an immune response. Thus, certain immunostimulatory nucleic acids can contain sequences corresponding to regions of naturally occurring genes or mRNAs, but they can still be considered non-sequence-specific immunostimulatory nucleic acids.

[0734] In some embodiments, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide can 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 in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, wherein at least one cytosine in the CpG dinucleotides is methylated. In another embodiment, each cytosine in the CpG dinucleotides present in the sequence is methylated. In another embodiment, the nucleic acid comprises more than one CpG dinucleotide, wherein at least one of the CpG dinucleotides comprises a methylated cytosine.

[0735] Attachment between nucleotide-based enhancing unit, linker unit, nucleic acid agent, and ligand

[0736] In some embodiments, the attachment between the nucleotide-based enhancing unit and the linker unit is a bond.

[0737] In some embodiments, the attachment between the nucleotide-based enhancing unit and the linker unit is a moiety (e.g., a moiety comprising a cleavable group).

[0738] In some embodiments, the attachment between the nucleotide-based enhancing unit and the linker unit comprises -C(=O)- attached to the linker unit.

[0739] In some embodiments, the attachment between the nucleotide-based enhancing unit and the nucleic acid agent is a bond.

[0740] In some embodiments, the attachment between the nucleotide-based enhancing unit and the nucleic acid agent is a moiety (e.g., a moiety comprising a cleavable group).

[0741] In some embodiments, the attachment between the nucleotide-based enhancing unit and the ligand is a bond.

[0742] In some embodiments, the attachment between the nucleotide-based enhancing unit and the ligand is a moiety (e.g., a moiety comprising a cleavable group).

[0743] In some embodiments, the attachment between the linker unit and the nucleic acid agent is a bond.

[0744] In some embodiments, the attachment between the linker unit and the nucleic acid agent is a moiety (e.g., a moiety comprising a cleavable group).

[0745] In some embodiments, the attachment between the linker unit and the ligand is a bond.

[0746] In some embodiments, the attachment between the linker unit and the ligand is a moiety (e.g., a moiety comprising a cleavable group).

[0747] In some embodiments, the attachment between the linker unit and the ligand comprises -C(=O)- attached to the linker unit.

[0748] The attachment between the nucleotide-based enhancing unit, the linker unit, the nucleic acid agent, and the ligand can be a cleavable group or a non-cleavable group. Suitable groups include, for example, -NR-, -C(=O)-, -C(=O)NH-, -S(=O)-, -S(=O) 2 -, -S(=O) 2 NH- or an atomic chain such as, but not limited to, alkylene, alkenylene, alkynylene, arylalkylene, arylalkenylene, arylalkynylene, heteroarylalkylene, heteroarylalkenylene, heteroarylalkynylene, heterocycloalkylene, heterocycloalkenylene, heterocycloalkynylene, arylene, heteroarylene, heterocycloylene, cycloalkylene, cycloalkenylene, alkylarylalkylene, alkylarylalkenylene, alkylarylalkynylene, alkenylarylalkylene, alkenylarylalkenylene, alkenylarylalkynylene, alkynylarylalkylene, alkynylarylalkenylene, alkynylarylalkynylene, alkylheteroarylalkylene, alkylheteroarylalkenylene, alkylheteroarylalkynylene, alkenylheteroarylalkylene, alkenylheteroarylalkenylene, alkenylheteroarylalkynylene, alkynylheteroarylalkylene, alkynylheteroarylalkenylene, alkynylheteroarylalkynylene, alkylheterocycloalkylene, alkylheterocycloalkenylene, alkylheterocycloalkynylene, alkenylheterocycloalkylene, alkenylheterocycloalkenylene, alkenylheterocycloalkynylene, alkynylheterocycloalkylene, alkynylheterocycloalkenylene, alkynylheterocycloalkynylene, alkylarylene, alkenylarylene, alkynylarylene, alkylheteroarylene, alkenylheteroarylene, alkynylheteroarylene, each of which may be substituted or unsubstituted, and one or more methylenes thereof 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.

[0749] A cleavable group is a group that is stable enough extracellularly, but is cleaved after entering a target cell to release two moieties that were held together by the group. In a preferred embodiment, the cleavable group is cleaved at least 10-fold or more, preferably at least 100-fold faster in a target cell or under a first reference condition (which can be selected, for example, to mimic or represent intracellular conditions) than in the blood of a subject or under a second reference condition (which can be selected, for example, to mimic or represent conditions present in blood or serum).

[0750] Cleavable groups are susceptible to cleavage agents such as the presence of pH, redox potential, or degrading molecules. Generally, cleavage agents are more prevalent or present at higher levels or activities intracellularly than in serum or blood. Examples of such degrading agents include: redox agents, which are selected for a particular substrate or which are substrate non-specific, including for example oxidases or reductases or reducing agents such as thiols present in cells, which can degrade redox-cleavable groups by reduction; esterases; endosomes or agents that can create an acidic environment, such as those that result in a pH of 5 or lower; enzymes that can hydrolyze or degrade acid-cleavable groups by acting as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0751] Cleavable groups, such as disulfide bonds, can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range from about 7.1 - 7.3. Endosomes have a more acidic pH in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a cleavable group that is cleaved at a preferred pH to release a cationic lipid from a ligand intracellularly or into a desired compartment of the cell.

[0752] Conjugates can include cleavable groups that are cleavable by specific enzymes. The type of cleavable group incorporated into the conjugate can depend on the cell to be targeted. For example, a liver-targeting ligand can be attached to a cationic lipid through a chemical moiety that includes an ester group. Hepatocytes are rich in esterases, and thus the group is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung cells, renal cortical cells, and testicular cells.

[0753] When targeting cell types rich in peptidases such as hepatocytes and synoviocytes, a coupling group containing a peptide bond can be used.

[0754] Generally, the suitability of a candidate cleavable group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate group. It would also be desirable to test the ability of the candidate cleavable group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, where the first condition is chosen to indicate cleavage in target cells and the second condition is chosen to indicate cleavage in other tissues or biological fluids such as blood or serum. The evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform a preliminary evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In a preferred embodiment, a useful candidate compound cleaves at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or in in vitro conditions chosen to mimic intracellular conditions) than in blood or serum (or in in vitro conditions chosen to mimic extracellular conditions).

[0755] Redox cleavable groups. One class of cleavable groups are redox cleavable groups that are cleaved upon reduction or oxidation. An example of a reduction-cleavable group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable group is a suitable "reduction-cleavable linker", or for example whether it is suitable for use with a particular iRNA moiety and a particular targeting agent, one of ordinary skill in the art can refer to the methods described herein. For example, a candidate can be evaluated by incubating it with a reagent known in the art together with dithiothreitol (DTT) or other reducing agents, which mimics the cleavage rate observed in cells such as target cells. The candidate can also be evaluated under conditions chosen to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved by at most 10% in blood. In a preferred embodiment, a useful candidate compound degrades at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or in in vitro conditions chosen to mimic intracellular conditions) than in blood (or in in vitro conditions chosen to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetics assays under conditions chosen to mimic the intracellular medium and compared to conditions chosen to mimic the extracellular medium.

[0756] 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 in cells are enzymes in cells such as phosphatases. In some embodiments, the phosphate-based linking group is -O-P(=O)(OR k )-O-, -O-P(=S)(OR k )-O-, -O-P(=S)(SR k )-O-, -S-P(=O)(ORk )-O-, -O-P(=O)(OR k )-S-, -S-P(=O)(OR k )-S-, -O-P(=S)(OR k )-S-, -S-P(=S)(OR k )-O-, -O-P(=O)(R k )-O-, -O-P(=S)(R k )-O-, -S-P(=O)(R k )-O-, -S-P(=S)(R k )-O-, -S-P(=O)(R k )-S- or -O-P(=S)(R k )-S-. In some embodiments, the phosphate-based linking group is -O-P(=O)(OH)-O-, -O-P(=S)(OH)-O-, -O-P(=S)(SH)-O-, -S-P(=O)(OH)-O-, -O-P(=O)(OH)-S-, -S-P(=O)(OH)-S-, -O-P(=S)(OH)-S-, -S-P(=S)(OH)-O-, -O-P(=O)(H)-O-, -O-P(=S)(H)-O-, -S-P(=O)(H)-O-, -S-P(=S)(H)-O-, -S-P(=O)(H)-S- or -O-P(=S)(H)-S-. In some embodiments, the phosphate-based linking group is -O-P(=O)(OH)-O-.

[0757] Acid-cleavable group. An acid-cleavable group is a linking group that cleaves under acidic conditions. In preferred embodiments, the acid-cleavable group cleaves in an acidic environment having a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or cleaves by an agent such as an enzyme that can act as a general acid. In cells, specific low-pH organelles, such as endosomes and lysosomes, can 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. Acid-cleavable groups can have the general formula -C=NN-, C(O)O or -OC(O). Preferred embodiments are when the carbon attached 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 tert-butyl. Methods similar to those described above can be used to evaluate these candidates.

[0758] Ester-based cleavable groups. Ester-based cleavable groups are cleaved by enzymes in cells such as esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0759] Peptide-based cleavable groups. Peptide-based cleavable groups are cleaved by enzymes in cells such as peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene groups. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins and do not include the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHR A C(O)NHCHR B C(O)-, where R A and R B are 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 a compound that is a carbohydrate itself composed of one or more monosaccharide units, the one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched, or cyclic), each carbon atom bonded to an oxygen, nitrogen, or sulfur atom; or a compound that has as a part thereof a carbohydrate moiety composed of one or more monosaccharide units, the one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched, or cyclic), each carbon atom bonded to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing from about 4 - 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and mucopolysaccharides. Specific monosaccharides include C 5 and above (preferably C 5 -C 8 ) sugars; disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C 5 -C 8 ).

[0760] Synthetic methods

[0761] In some aspects, the present disclosure provides methods for preparing the compounds described herein (e.g., nucleotide-based enhancers).

[0762] In some aspects, the present disclosure provides compounds obtainable by, or obtained by, a method for preparing a compound described herein (e.g., a nucleotide-based enhancer).

[0763] In some aspects, the present disclosure provides an intermediate as described herein that is suitable for use in a method for preparing a compound described herein (e.g., a nucleotide-based enhancer).

[0764] The compounds of the present disclosure can be prepared by any suitable technique known in the art. Specific processes for preparing these compounds are additionally described in the accompanying examples.

[0765] In the description of the synthetic methods described herein and in any reference synthetic methods used to prepare starting materials, it is understood that all of the proposed reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and work-up procedures, can be selected by one of ordinary skill in the art.

[0766] One of ordinary skill in the art of organic synthesis understands that the functionality present on multiple portions of a molecule must be compatible with the reagents and reaction conditions being utilized.

[0767] It will be understood that during the synthesis of the compounds of the present disclosure, or during the synthesis of certain starting materials, in the processes defined herein, it may be desirable to protect certain substituent groups to prevent their unwanted reactions. A skilled chemist will understand when such protection is needed and how such protecting groups can be placed in the appropriate position and later removed. For examples of protecting groups, see one of the many general textbooks on the subject, e.g., ‘Protective Groups in Organic Synthesis’ by Theodora Green (publisher: John Wiley & Sons). The protecting groups can be removed by any convenient method described in the literature or known to a skilled chemist that is suitable for removing the protecting group under discussion, choosing such a method so as to effect removal of the protecting group with minimal perturbation of groups elsewhere in the molecule. Thus, if a reactant contains groups such as, for example, an amino, carboxyl, or hydroxyl group, it may be desirable to protect that group in some of the reactions mentioned herein.

[0768] By way of example, suitable protecting groups for an amino group or an alkylamino group are, for example, acyl groups such as: alkanoyl groups such as acetyl; alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl; arylmethoxycarbonyl groups such as benzyloxycarbonyl; or aroyl groups such as benzoyl. Suitable protecting groups for a hydroxyl group or an alkylhydroxyl group can be, 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 (such as trimethylsilyl (TMS) ether, tert-butyldimethylsilyl (TBDMS) ether, triisopropylsilyloxymethyl (TOM) ether and triisopropylsilyl (TIPS) ether), methyl ether or ethoxyethyl ether (EE). Suitable protecting groups for 1,2-diols can be, for example, acetals. Suitable protecting groups for 1,3-diols can be, for example, tetraisopropyl-disiloxanylidene (TIPDS).

[0769] The deprotection conditions for the protecting groups described above necessarily vary with the choice of protecting group. Thus, for example, acyl groups such as alkanoyl groups or alkoxycarbonyl groups or aroyl groups can be removed by hydrolysis, for example, with a suitable base such as an alkali metal hydroxide such as lithium hydroxide or sodium hydroxide. Alternatively, acyl groups such as tert-butoxycarbonyl groups can be removed, for example, by treatment with a suitable acid such as hydrochloric acid, sulfuric acid or phosphoric acid or trifluoroacetic acid, and arylmethoxycarbonyl groups such as benzyloxycarbonyl groups can be removed, for example, by hydrogenation over a catalyst such as palladium on carbon or by treatment with a Lewis acid such as borane tris(trifluoroacetate). Suitable alternative protecting groups for a primary amino group are, for example, phthaloyl groups which can be removed by treatment with an alkylamine such as dimethylaminopropylamine or with hydrazine.

[0770] Suitable protecting groups for a hydroxy group are, for example: acyl groups, such as alkanoyl groups like acetyl, aroyl groups such as benzoyl; or arylmethyl groups, such as benzyl. The deprotection conditions for the protecting groups described above will necessarily vary with the choice of the protecting group. Thus, for example, an acyl group such as an alkanoyl group or an aroyl group can be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide (e.g., lithium hydroxide, sodium hydroxide) or ammonia. Alternatively, an arylmethyl group such as a benzyl group can be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.

[0771] Suitable protecting groups for a carboxy group are, for example, esterifying groups, such as a methyl group or an ethyl group which can be removed, for example, by hydrolysis with a base such as sodium hydroxide, or a tert-butyl group which can be removed, for example, by treatment with an acid such as trifluoroacetic acid, or a benzyl group which can be removed, for example, by hydrogenation over a catalyst such as palladium on carbon.

[0772] Conveniently, the reaction of the compound is carried out in the presence of a suitable solvent, which is preferably inert under the corresponding 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, carbon tetrachloride, chloroform or dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE) or dioxane; ethylene glycol ethers such as ethylene glycol monomethyl or 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-methylpyrrolidone (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 said solvents or mixtures with water.

[0773] Depending on the reaction steps and conditions used, the reaction temperature is suitably between about -100 °C and 300 °C.

[0774] Depending on the reactivity of the corresponding compound and the corresponding reaction conditions, the reaction time is generally in the range of a fraction of a minute to several days. A suitable reaction time can be readily determined by methods known in the art, such as reaction monitoring. Based on the reaction temperature provided above, the suitable reaction time is generally in the range of 10 minutes and 48 hours.

[0775] In addition, by utilizing the procedures described herein and in combination with ordinary skill 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 procedures of the following preparation procedures can be used to prepare these compounds.

[0776] 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 which reagents and reaction conditions to use and how to apply and adjust them in any particular case (when necessary or useful) in order to obtain the compounds of the present disclosure. In addition, some of the compounds of the present disclosure can be readily synthesized by reacting other compounds of the present disclosure under suitable conditions, for example, by converting a specific functional group present in a compound of the present disclosure or a suitable precursor molecule thereof into another functional group via application of standard synthetic methods such as reduction, oxidation, addition, or substitution reactions; those methods are well known to those skilled in the art. Similarly, those skilled in the art will apply synthetic protecting (or protective) groups when necessary or useful; suitable protecting groups and methods for introducing and removing them are well known to those skilled in the art of chemical synthesis and are described in more detail, for example, in P.G.M. Wuts, T.W. Greene, “Greene’s Protective Groups in Organic Synthesis”, 4th Edition (2006) (John Wiley & Sons).

[0777] General routes for preparing the compounds of the present application are described in Scheme A herein.

[0778] Scheme A

[0779]

[0780] Compounds (e.g., nucleic acid agents, nucleic acid agents containing nucleotide-based enhancers, and conjugates) are prepared by solid-phase synthesis according to standard synthetic schemes.

[0781] Briefly, oligonucleotide synthesis is carried out on a solid support to incorporate each nucleoside phosphoramidite from the 3'-end to the 5'-end to prepare a single-stranded oligonucleotide. ETT or BTT is used as the activator for the coupling reaction. Iodine in water / pyridine / THF is used to oxidize the phosphite triester (P(III)) to provide the phosphate backbone, and DDTT is used for the preparation of phosphorothioate linkages. The oligonucleotide is cleaved from the solid support and the protecting groups are globally removed using aqueous ammonium solution. The crude oligonucleotide is then concentrated and purified by strong anion exchange or reverse-phase HPLC. The purified fractions are combined and concentrated.

[0782] In some cases, the single-stranded oligonucleotides are then conjugated to a targeting ligand (e.g., a peptide, an antibody) by a synthetic linker to provide a conjugate. The conjugation reaction is carried out using standard conjugation methods. The crude conjugate is further purified by strong anion exchange or reverse-phase HPLC. The purified fractions are combined and concentrated.

[0783] The synthesized single strands are then dialyzed against water using a MidiTrap G-25 column, concentrated, and their OD amounts are measured. Based on equimolar amounts, the sense and antisense strands are annealed at 95 °C for 5 min and cooled to room temperature to provide a conjugate duplex with >90% purity. The solution of the duplex is lyophilized to provide the desired conjugate, and its amount is calculated based on the molar amount of the single strands consumed in the annealing.

[0784] Biological assays

[0785] Compounds (e.g., nucleotide-based enhancers) or conjugates designed, selected, prepared, and / or optimized by the methods described above can be characterized using a variety of assays known to those of skill in the art after production to determine whether the compound, scaffold, or conjugate has biological activity. For example, the compound, scaffold, or conjugate can be characterized by conventional assays (including but not limited to those described below) to determine whether they have the desired activities, such as target binding activity and / or specificity and / or stability.

[0786] In addition, high-throughput screening can be used to accelerate the analysis using such assays. Thus, it may be possible to rapidly screen the molecules described herein for activity using techniques known in the art. General methods for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker; and U.S. Patent No. 5,763,263. High-throughput assays can use one or more different assay techniques, including but not limited to those described below.

[0787] A variety of in vitro biological assays or in vivo biological assays can be applicable to detect the effects of the compounds, scaffolds, or conjugates of the present disclosure. These in vitro biological assays or in vivo biological assays can include but not be limited to enzymatic activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and the assays described herein.

[0788] In some embodiments, the biological assays are described in the examples herein.

[0789] In some aspects, the present disclosure provides a pharmaceutical composition comprising a compound, scaffold, or conjugate of the present disclosure as an active ingredient.

[0790] As used herein, the term "composition" is intended to encompass a product comprising a specific amount of specific ingredients, as well as any product directly or indirectly resulting from the combination of specific amounts of specific ingredients.

[0791] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be 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, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. For example, the proper fluidity can be maintained by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. The preventing action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0792] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent (optionally with a combination of one or more of the ingredients listed above), followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0793] The formulations of the present disclosure can be in the form of an aqueous solution containing an aqueous vehicle. The aqueous vehicle component can contain water and at least one pharmaceutically acceptable excipient. Suitable acceptable excipients include excipients selected from the group consisting of solubilizers, chelating agents, preservatives, tonicity agents, viscosity / suspending agents, buffers, and pH regulators, and mixtures thereof.

[0794] Any suitable solubilizer can be used. Examples of solubilizers include cyclodextrins, such as cyclodextrins selected from the group consisting of: hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, ethylated-β-cyclodextrin, triacetyl-β-cyclodextrin, fully acetylated-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-chain-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, randomly methylated-γ-cyclodextrin, and trimethyl-γ-cyclodextrin, and mixtures thereof.

[0795] Any suitable chelating agent can be used. Examples of suitable chelating agents include chelating agents selected from the group consisting of: ethylenediaminetetraacetic acid and its metal salts, disodium edetate, trisodium edetate, and tetrasodium edetate, and mixtures thereof.

[0796] Any suitable preservative can be used. Examples of preservatives include preservatives selected from the group consisting of: quaternary ammonium salts such as benzalkonium halide (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetylpyridinium chloride, benzyl bromide, phenylmercuric nitrate, phenylmercuric acetate, phenylmercuric neodecanoate, thimerosal, methyl paraben, propyl paraben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl paraben, propylaminopropyl biguanide, and butyl paraben, and mixtures thereof.

[0797] The aqueous vehicle may also include a tonicity agent for adjusting the tonicity (osmotic pressure). The tonicity agent can be selected from the group consisting of: diols (e.g., propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, and mixtures thereof.

[0798] To adjust the formulation to an acceptable pH (usually in the 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 formulation may contain a pH regulator. The pH regulator is typically an inorganic acid or metal hydroxide base selected from the group consisting of: potassium hydroxide, sodium hydroxide, and hydrochloric acid, and mixtures thereof, and preferably sodium hydroxide and / or hydrochloric acid. These acidic and / or basic pH regulators are added to adjust the formulation to the target acceptable pH range. Thus, it may not be necessary to use both an acid and a base - depending on the formulation, adding either an acid or a base may be sufficient to bring the mixture to the desired pH range.

[0799] The aqueous medium may also contain a buffering agent to stabilize the pH. When used, the buffer is selected from the group consisting of: phosphate buffers (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), borate buffers (such as boric acid or its salts, including disodium tetraborate), citrate buffers (such as citric acid or its salts, including sodium citrate), and ε-aminocaproic acid and mixtures thereof.

[0800] 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 in combination with a pharmaceutically acceptable diluent or carrier.

[0801] The compositions of the present disclosure may be in a form suitable for: oral use (e.g., as tablets, lozenges, hard or soft gelatin 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 subdivided powder or a liquid aerosol), administration by insufflation (e.g., as a subdivided powder) or parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intraperitoneal administration or as a suppository for rectal administration).

[0802] 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 colorants, sweeteners, flavoring agents and / or preservatives.

[0803] An effective amount of the compound of the present disclosure for use in therapy is an amount sufficient to treat or prevent the inflammasome-related conditions mentioned herein, slow its progression and / or reduce the symptoms associated with the condition.

[0804] An effective amount of the compound of the present disclosure for use in therapy is an amount sufficient to treat the inflammasome-related conditions mentioned herein, slow its progression and / or reduce the symptoms associated with the condition.

[0805] In accordance with well-known medical principles, the size of the dose of a compound of formula (I) or formula (II) for therapeutic or prophylactic purposes will vary naturally according to the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration.

[0806] Method of Use

[0807] In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) the expression of a target gene in a subject, the method comprising administering to the subject a conjugate of the present disclosure.

[0808] In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) the expression of a target gene in a cell or tissue of a subject, the method comprising administering to the subject a conjugate of the present disclosure.

[0809] In some aspects, the present disclosure provides a method of delivering a nucleic acid agent to a subject, the method comprising administering to the subject a conjugate of the present disclosure.

[0810] In some aspects, the present disclosure provides a method of treating or preventing a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate of the present disclosure.

[0811] In some aspects, the present disclosure provides a conjugate of the present disclosure for modulating (e.g., reducing or eliminating) the expression of a target gene in a subject.

[0812] In some aspects, the present disclosure provides a conjugate of the present disclosure for modulating (e.g., reducing or eliminating) the expression of a target gene in a cell or tissue of a subject.

[0813] In some aspects, the present disclosure provides a conjugate of the present disclosure for delivering a nucleic acid agent to a subject.

[0814] 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.

[0815] In some aspects, the present disclosure provides the use of a conjugate of the present disclosure in the manufacture of a medicament for modulating (e.g., reducing or eliminating) the expression of a target gene in a subject.

[0816] In some aspects, the present disclosure provides the use of a conjugate of the present disclosure in the manufacture of a medicament for modulating (e.g., reducing or eliminating) the expression of a target gene in a cell or tissue of a subject.

[0817] In some aspects, the present disclosure provides the use of a conjugate of the present disclosure in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0818] In some aspects, the present disclosure provides the 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.

[0819] In some embodiments, the subject is a cell.

[0820] In some embodiments, the subject is a central nervous system, peripheral nervous system, adipose, muscle, cardiac, and / or pancreatic cell. In some embodiments, the subject is a pancreatic cell. In some embodiments, the subject is a pancreatic beta cell.

[0821] In some embodiments, the subject is a tissue.

[0822] In some embodiments, the subject is a central nervous system, peripheral nervous system, adipose, muscle, cardiac, and / or pancreatic tissue. In some embodiments, the subject is a pancreatic tissue.

[0823] In some embodiments, the subject is a human.

[0824] In some embodiments, the target gene is Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, HBV, HCV, RSV, PDGFβ 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 IIα gene, p73 gene, p21 (WAF1 / CIP1) gene, p27 (KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, tumor suppressor gene mutations, p53 tumor suppressor gene, LDHA or any combination thereof.

[0825] In some embodiments, the disease is characterized by an undesired expression of the target gene.

[0826] In some embodiments, administration results in a decrease or elimination of the expression of the target gene in the subject.

[0827] In some embodiments, the disease is a viral infection, such as HCV, HBV, HPV, HSV, or HIV infection.

[0828] In some embodiments, the disease is cancer.

[0829] In some embodiments, the cancer is biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary non-polyposis 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, Wilms tumor, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, solid pseudopapillary tumor, acinar cell carcinoma, prostate cancer, prostatic adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer or uterine sarcoma.

[0830] In some embodiments, the cancer is liver cancer, liver carcinoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma or hepatoblastoma.

[0831] In some embodiments, the disease is a proliferative disease, an inflammatory disease, an autoimmune disease, a neurological disease, an ophthalmic disease, a respiratory disease, a metabolic disease, a skin disease, an auditory disease, a liver disease, a kidney disease or an infectious disease. In some embodiments, the disease is a liver disease.

[0832] In some embodiments, the disease is a disease of the central nervous system, peripheral nervous system, adipose, muscle, heart and / or pancreas. In some embodiments, the disease is a disease of the pancreas.

[0833] Definitions

[0834] Unless otherwise stated, the following terms used in the specification and claims have the following meanings as set forth below.

[0835] Without wishing to be bound by this statement, it should be understood that although various options for variables are described herein, the present disclosure is intended to cover operable embodiments having combinations of these options. The present disclosure can be interpreted to exclude inoperable embodiments caused by certain combinations of these options.

[0836] As used herein, "alkyl", "C 1 , C 2 , C 3 , C 4 , C 5 or C 6 alkyl" or "C 1 -C 6 alkyl" is intended to include C 1 , C 2 , C 3 , C 4 , C 5 or C 6 linear (straight-chain) saturated aliphatic hydrocarbon groups and C 3 , C 4 , C 5 or C 6 branched saturated aliphatic hydrocarbon groups. For example, C 1 -C 6 alkyl is intended to include C 1 , C 2 , C 3 , C 4 , C 5 or C 6 alkyl groups. Examples of alkyl include moieties having from one to six carbon atoms, such as but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, or n-hexyl. In some embodiments, the straight-chain or branched alkyl has six or fewer carbon atoms (e.g., C 1 -C 6 for straight-chain and C 3 -C 6 for branched-chain), and in another embodiment, the straight-chain or branched alkyl has four or fewer carbon atoms.

[0837] As used herein, the term "optionally substituted alkyl" refers to an unsubstituted alkyl or an alkyl in which one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone are replaced by a specified substituent. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphono, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonylamino, nitro, trifluoromethyl, cyano, azide, heterocyclic group, alkylaryl, or an aromatic or heteroaromatic moiety.

[0838] Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl or heteroaryl) include both unsubstituted moieties and moieties having one or more specified substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl-piperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

[0839] As used herein, the term "substituted" means that any one or more hydrogen atoms on a specified atom are replaced by a selection from a specified group, provided that the normal valence of the specified atom is not exceeded and the substitution results in a stable compound. When the substituent is oxo or keto (i.e., =O), then two hydrogen atoms on the atom are replaced. There is no keto substituent on an aromatic moiety. 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 intended to refer to compounds that are sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulated as an effective therapeutic agent.

[0840] When a bond to a substituent is shown crossing a bond connecting two atoms in a ring, then such a substituent can be bonded to any atom in the ring. When a substituent is listed without indicating through which atom it is bonded to the remainder of the compound of a given formula, then such a substituent can be bonded to any atom in such a formula. Combinations of substituents and / or variables are permitted, but only if such combinations result in a stable compound.

[0841] When any variable (e.g., R) occurs more than once in any moiety or formula of a compound, the definition of that variable (e.g., R) at each occurrence is independent of its definition at each other occurrence. Thus, for example, if a group is shown to be substituted with 0 - 2 R moieties, then the group may optionally be substituted with up to two R moieties, and the R at each occurrence is selected independently of the definition of R. Additionally, combinations of substituents and / or variables are permitted, but only if such combinations result in stable compounds.

[0842] As used herein, the term “hydroxy” or “hydroxyl” includes groups having -OH or -O-.

[0843] As used herein, the term “halo” or “halogen” refers to fluorine, chlorine, bromine, and iodine.

[0844] The term “haloalkyl” or “haloalkoxy” refers to an alkyl or alkoxy group substituted with one or more halogen atoms.

[0845] As used herein, the term “optionally substituted haloalkyl” refers to an unsubstituted haloalkyl or a substituted haloalkyl in which one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms are replaced with a specified substituent. Such substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphono, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfonylamino, nitro, trifluoromethyl, cyano, azide, heterocyclic group, alkylaryl, or an aromatic or heteroaromatic moiety.

[0846] As used herein, the term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently attached to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, butoxy, and pentyloxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy group may be substituted with groups such as: alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxy, phosphate, phosphonic acid, phosphinic acid, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkanesulfinyl, sulfonic acid, sulfamoyl, sulfonylamino, nitro, trifluoromethyl, cyano, azide, heterocyclic group, 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.

[0847] As used herein, the term "overhang" refers to a segment of unpaired nucleotides at the end (e.g., the 3'-end or 5'-end) of an oligonucleotide chain. An overhang at the 3'-end of an oligonucleotide chain is a "3' overhang", and an overhang at the 5'-end of an oligonucleotide chain is a "5' overhang".

[0848] As used herein, the expressions "one or more of A, B, or C", "one or more of A, B, and C", "one or more of A, B, or 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", etc. are used interchangeably and all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more of A, one or more of B, one or more of C, or any combination thereof, unless otherwise indicated.

[0849] 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 a variety of disclosed compounds, scaffolds, and conjugates according to the protocols herein and the protocols shown in the examples.

[0850] It should be understood that throughout the description, when a composition is described as having, including, or containing a particular component, it is contemplated that the composition also consists essentially of or consists of the component. Similarly, when a method or process is described as having, including, or containing a particular process step, the process also consists essentially of or consists of the process step. In addition, it should be understood that as long as the present invention remains operable, the order of steps or the order of performing certain operations is not important. In addition, two or more steps or operations can be performed simultaneously.

[0851] It should be understood that the synthetic methods of the present disclosure can tolerate a variety of functional groups, and thus a variety of substituted starting materials can be used. The methods generally provide the desired final compound at or near the end of the overall method, although in some cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt.

[0852] It should be understood that the compounds, scaffolds, and conjugates of the present disclosure can be prepared in various ways by utilizing standard synthetic methods and procedures known to those skilled in the art or standard synthetic methods and procedures that will be apparent to those skilled in the art based on the teachings herein, using commercially available starting materials, compounds known in the literature, or intermediates readily prepared therefrom. Standard synthetic methods and procedures for the preparation of organic molecules and for the transformation and manipulation of functional groups can be obtained from relevant scientific literature or standard textbooks in the art. While not limited to any one or several sources, classic textbooks such as Smith, M.B., March, J., March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, John Wiley & Sons: New York, 2001; Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3rd 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), which are incorporated herein by reference, are useful and recognized reference textbooks on organic synthesis known to those skilled in the art.

[0853] One of ordinary skill in the art will note that during the reaction sequences and synthetic schemes described herein, the order of certain steps can be altered, such as the introduction and removal of protecting groups. One of ordinary skill in the art will recognize that certain groups may need to be protected from reaction conditions via the use of protecting groups. Lists of protecting groups and how to introduce and remove these groups can be found in Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons: New York, 1999.

[0854] It should be understood that unless otherwise stated, any description of a method of treatment or prevention includes the use of compounds, scaffolds, and conjugates to provide such treatment or prevention as described herein. It should also be understood that unless otherwise stated, any description of a method of treatment or prevention includes the use of compounds, scaffolds, and conjugates to prepare a medicament for treating or preventing such a condition. Treatment or prevention includes treating or preventing in a human or non-human animal, including rodents and other disease models.

[0855] It should be understood that unless otherwise stated, any description of a method of treatment includes the use of compounds, scaffolds, and conjugates to provide such treatment as described herein. It should also be understood that unless otherwise stated, any description of a method of treatment includes the use of compounds, scaffolds, and conjugates to prepare a medicament for treating such a condition. Treatment includes treating in a human or non-human animal, including rodents and other disease models.

[0856] As used herein, the term "subject" is interchangeable with the term "subject in need thereof", both referring to a subject having a disease or having an increased 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. A subject in need thereof can be a subject that has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be a subject suffering from a disease or disorder disclosed herein. Alternatively, a subject in need thereof can be a subject having an increased risk of developing such a disease or disorder relative to the general population (i.e., a subject predisposed to developing such a disorder relative to the general population). A subject in need thereof can be suffering from a refractory or drug-resistant disease or disorder disclosed herein (i.e., a disease or disorder that does not respond or has not responded to treatment as disclosed herein). The subject may be drug-resistant at the start of treatment or may become drug-resistant during treatment. In some embodiments, a subject in need thereof has received all known effective therapies for a disease or disorder disclosed herein, but all have failed. In some embodiments, a subject in need thereof has received at least one previous therapy.

[0857] As used herein, the term "treating" or "treat" describes 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 complications of the disease, condition, or disorder or to eliminate the disease, condition, or disorder. The term "treatment" can also include treatment of an in vitro cell or animal model. It should be understood that reference to "treating" or "treatment" includes alleviation of the established symptoms of a condition. Thus, "treating" or "treatment" of a state, disorder, or condition includes: (1) delaying the onset of clinical symptoms of the state, disorder, or condition in a human who is likely to develop or is predisposed to the state, disorder, or condition but has not yet experienced or exhibited clinical or subclinical symptoms of the state, disorder, or condition; (2) inhibiting the state, disorder, or condition, i.e., preventing, reducing, or delaying the development of the disease or its recurrence (in the case of maintenance therapy) or at least one of its clinical or subclinical symptoms; or (3) alleviating or abating the disease, i.e., causing the regression of the state, disorder, or condition or the regression of at least one of its clinical or subclinical symptoms.

[0858] It should be understood that the compounds, scaffolds, and conjugates of the present disclosure or pharmaceutically acceptable salts, polymorphs, or solvates thereof are also capable of or may also be used for preventing related diseases, conditions, or disorders or for identifying suitable candidates for such purposes.

[0859] As used herein, the terms "preventing," "prevent," or "protecting against" describe reducing or eliminating the onset of symptoms or complications of such a disease, condition, or disorder.

[0860] It should be understood that the present disclosure also provides pharmaceutical compositions comprising any of the compounds, scaffolds, or conjugates described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0861] As used herein, the term "pharmaceutical composition" refers to a formulation in a form suitable for administration to a subject that contains a compound, scaffold, or conjugate of the present disclosure. In some embodiments, the pharmaceutical composition is in a bulk dosage form or in a unit dosage form. A unit dosage form is any of a variety of forms, including, for example, capsules, IV bags, tablets, a single pump on an aerosol inhaler, or vials. The amount of the active ingredient (e.g., a formulation of the disclosed compound or its salt, hydrate, solvate, or isomer) in a unit dose of the composition is an effective amount and varies depending on the particular treatment involved. Those skilled in the art will understand that it is sometimes necessary to make routine variations in the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, and similar routes. Dosage forms for topical or transdermal administration of the compounds of the present 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 preservatives, buffers, or propellants as required.

[0862] As used herein, the term "pharmaceutically acceptable" refers to those compounds, scaffolds, conjugates, anions, cations, materials, compositions, carriers, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0863] As used herein, the term "pharmaceutically acceptable excipient" means an excipient useful in the preparation of a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable and includes excipients acceptable for veterinary use and human pharmaceutical use. As used in this specification and the claims, "pharmaceutically acceptable excipient" includes one and more than one such excipient.

[0864] It should be understood that the pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, oral (e.g., ingestion) administration, inhalation administration, transdermal (topical) administration, and transmucosal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH may be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0865] It should be understood that the compounds or pharmaceutical compositions of the present disclosure may be administered to a subject by many well-known methods currently used for chemotherapy treatment. For example, the compounds of the present disclosure may be injected into the bloodstream or body cavity, or administered orally or through skin application with a patch. The selected dose should be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects. The disease condition (e.g., the diseases or disorders disclosed herein) and the state of health of the patient should preferably be closely monitored during the treatment and for a reasonable period of time after the treatment.

[0866] As used herein, the term "therapeutically effective amount" refers to the amount of an agent that is used to treat, alleviate, or prevent a determined disease or condition, or exhibits a detectable therapeutic or inhibitory effect. The effect may be detected by any assay known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and 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 given situation can be determined by routine experimentation within the skill and judgment of a clinician.

[0867] As used herein, the term "therapeutically effective amount" refers to the amount of an agent that is used to treat or alleviate a determined disease or condition, or exhibits a detectable therapeutic or inhibitory effect. The effect may be detected by any assay known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and 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 given situation can be determined by routine experimentation within the skill and judgment of a clinician.

[0868] It should be understood that for any compound, a therapeutically effective amount can be initially estimated, for example, in cell culture assays of neoplastic cells or in animal models (usually rats, mice, rabbits, dogs, or pigs). Animal models can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine the useful dosage and route of administration for human use. Therapeutic / preventive efficacy and toxicity can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, and it can be expressed as the ratio LD 50 / ED 50 . Pharmaceutical compositions presenting a large therapeutic index are preferred. The dosage can vary within this range depending on the dosage form employed, the sensitivity of the patient, and the route of administration.

[0869] Adjust the dosage and administration to provide an adequate level of the active agent or maintain the desired effect. Factors that can be considered include the severity of the disease state, the general health of the subject, the age, weight, and sex of the subject, diet, the time and frequency of administration, drug combinations, response sensitivity, and tolerance / response to the therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, weekly, or biweekly, depending on the half-life and clearance rate of the particular formulation.

[0870] The pharmaceutical compositions containing the active compounds of the present disclosure can be manufactured in a generally known manner, for example, by means of conventional mixing processes, dissolving processes, granulating processes, dragee-making processes, levigating processes, emulsifying processes, encapsulating processes, entrapping processes, or lyophilization processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and / or auxiliaries, which facilitate processing the active compounds into a pharmaceutically usable article. Of course, the appropriate formulation depends on the chosen route of administration.

[0871] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. For example, the proper fluidity can be maintained by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersion and by using surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyols such as mannitol and sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0872] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent (optionally with a combination of one or more of the ingredients enumerated above), followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which comprises a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0873] Oral compositions generally contain an inert diluent or a pharmaceutically acceptable edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier to be used as a mouthwash, wherein the compounds in the fluid carrier are orally administered and swished and spat out or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, and the like can contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, acacia, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, orange flavoring.

[0874] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant such as a gas like carbon dioxide, or a nebulizer.

[0875] For nasal administration, the compounds are delivered in solution or solid formulation. In some embodiments, the compounds are delivered as a solution as a mist, drops, or swab. In some embodiments, the compounds are delivered as a powder. In some embodiments, the compounds are included in a kit that also includes a nasal applicator.

[0876] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, permeants suitable for the barrier to be permeated are used in the formulation. Such permeants 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 a nasal spray or a suppository. For transdermal administration, as is generally known in the art, the active compounds are formulated as an ointment, an ointment, a gel, or a cream.

[0877] The active compounds can be prepared with pharmaceutically acceptable carriers which will protect the compound from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions, including liposomes targeted to infected cells with monoclonal antibodies to viral antigens, can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0878] It is especially advantageous to formulate oral or parenteral compositions in dosage unit forms which are easy to administer and provide for a uniform dosage. As used herein, a dosage unit form is a physically discrete unit suitable as a unit dose for the subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are dictated by and directly depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.

[0879] In therapeutic applications, the dosage of the pharmaceutical compositions used in accordance with the present disclosure will vary in accordance with the dosage, the age, weight and clinical condition of the recipient patient, as well as the experience and judgment of the clinician or practitioner administering the therapy, and other factors which influence the selected dosage. Generally, the dosage should be sufficient to effect a slowing down of the symptoms of the disease or disorder disclosed herein and preferably to effect a regression of the symptoms of the disease or disorder disclosed herein, and more preferably to effect a complete regression of the disease or disorder. The dosage can range from about 0.01 mg / kg per day to about 5000 mg / kg per day. The effective amount of the agent is the amount which provides an objectively recognizable improvement as noted by a clinician or other qualified observer. Improvement in survival and growth indicates regression. As used herein, the term "dosage effective manner" means the amount of the active compound which produces the desired biological effect in a subject or cell.

[0880] It is to be understood that the pharmaceutical compositions can be included in a container, package, or dispenser together with instructions for administration.

[0881] It is to be understood that for the compounds, scaffolds, or conjugates of the present disclosure which are capable of further forming salts, all such forms are also contemplated within the scope of the claimed disclosure.

[0882] As used herein, the term "pharmaceutically acceptable salt" refers to derivatives of the compounds of the present disclosure, wherein the parent compound is modified by preparing its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali metal salts or organic salts of acidic residues such as carboxylic acids, and similar salts. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, salts derived from inorganic and organic acids selected from 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonic acid, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycollyarsanilic acid, hexylresorcinolic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, basic 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, arginine, etc.

[0883] In some embodiments, the pharmaceutically acceptable salt is a sodium salt, potassium salt, calcium salt, magnesium salt, diethylamine salt, choline salt, meglumine salt, benzathine salt, tromethamine salt, ammonium salt, arginine salt or lysine salt.

[0884] Other examples of pharmaceutically acceptable salts include salts of caproic 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 similar acids. The present disclosure also encompasses salts formed when an acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, alkaline earth metal ion or aluminum ion; or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. In salt form, it should be understood that the ratio of the compound to the cation or anion in the salt can be 1:1, or any ratio other than 1:1, such as 3:1, 2:1, 1:2 or 1:3.

[0885] It should be understood that all references to pharmaceutically acceptable salts include solvate addition forms (solvates) or crystal forms (polymorphs) of the same salt as defined herein.

[0886] The compound or a pharmaceutically acceptable salt thereof is administered orally, nasally, transdermally, by the pulmonary, inhalational, buccal, sublingual, intraperitoneal, subcutaneous, intramuscular, intravenous, rectal, intrapleural, intrathecal, and extraintestinal routes. In some embodiments, the compound is administered orally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0887] The dosage regimen utilizing the compound is selected according to a variety of factors including the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or its salt employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counteract, or arrest the progress of the condition. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to counteract or arrest the progress of the condition.

[0888] Techniques for formulating and administering the disclosed compounds of the present disclosure can be found in Remington: the Science and Practice of Pharmacy, 19th Edition, Mack Publishing Co., Easton, PA (1995). In an embodiment, the compounds and pharmaceutically acceptable salts thereof described herein are combined with a pharmaceutically acceptable carrier or diluent for use in a pharmaceutical product. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous organic solutions. The compound will be present in such a pharmaceutical composition in an amount sufficient to provide the desired dosage within the ranges described herein.

[0889] Unless otherwise indicated, all percentages and ratios used herein are by weight. Other features and advantages of the present disclosure are apparent from the various examples. The examples provided illustrate the different components and methods that can be used to practice the present disclosure. These examples do not limit the claimed disclosure. Based on the present disclosure, those skilled in the art can identify and employ other components and methods that can be used to practice the present disclosure.

[0890] In the synthetic schemes described herein, for simplicity, the compounds may be drawn in a specific configuration. Such a specific configuration should not be construed as limiting the present disclosure to one or the other isomer, tautomer, regioisomer, or stereoisomer, nor does it exclude a mixture of isomers, tautomers, regioisomers, or stereoisomers; however, it should be understood that a particular isomer, tautomer, regioisomer, or stereoisomer may have a higher level of activity than another isomer, tautomer, regioisomer, or stereoisomer.

[0891] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. The citation of publications and patent documents is not intended as an admission that any of them is relevant prior art, nor does it constitute any admission as to the content or date thereof. The present invention has now been described in terms of written description, and those skilled in the art will recognize that the present invention can be practiced in a variety of embodiments, and the above description and the following examples are for illustrative purposes and not limitations on the appended claims.

[0892] Exemplary embodiment

[0893] Exemplary embodiment No. 1. A nucleotide-based enhancer, which is a compound comprising from 2 to 30 nucleotides or a pharmaceutically acceptable salt thereof, wherein each nucleotide independently has formula (I) or formula (II):

[0894]

[0895] Wherein:

[0896] Each * independently represents an attachment to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancer, or represents H when the nucleotide is at the 5'-end of the nucleotide-based enhancer;

[0897] Each ** independently represents an attachment to the 5'-position of another nucleotide of the nucleotide-based enhancer, or represents H when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancer;

[0898] B is H, C 1 -C 6 alkyl or a nucleobase moiety;

[0899] V is -O-, -NR V -, or -C(R V ) 2 -;

[0900] Each R V independently is H or C 1 -C 6 alkyl;

[0901] X is H, halogen or -OR X ;

[0902] R X is H, C 1 -C 12 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C10 aryl), wherein said C 1 -C 6 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl) is optionally substituted by one or more R Xa substituents; or R X and R 4 together form C 1 -C 6 alkylene;

[0903] Each R Xa is independently halogen, C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl), wherein said C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl) is optionally substituted by one or more halogens;

[0904] 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 )-;

[0905] Each R Y is independently H or C 1 -C 6 alkyl optionally substituted by one or more halogens or cyano;

[0906] R 1 is H, halogen or C 1 -C 6 alkyl optionally substituted by one or more halogens;

[0907] R 2 is H, halogen or C 1 -C 6 alkyl optionally substituted by one or more halogens;

[0908] R 3is H, a halogen or C optionally substituted with one or more halogens 1 -C 6 alkyl;

[0909] R 4 is H, a halogen or C optionally substituted with one or more halogens 1 -C 6 alkyl; or R 4 and R X together form C 1 -C 6 alkylene; and

[0910] each R 5 is independently H, a halogen or C optionally substituted with one or more halogens 1 -C 6 alkyl.

[0911] Exemplary Embodiment No. 2. A conjugate or a pharmaceutically acceptable salt thereof, comprising:

[0912] (i) one or more nucleic acid agents;

[0913] (ii) one or more ligands; and

[0914] (iii) one or more nucleotide-based enhancing units, wherein each nucleotide-based enhancing unit independently comprises from 2 to 30 nucleotides, and wherein each nucleotide independently has:

[0915]

[0916] wherein:

[0917] the variables B, V, X, Y, R 1 , R 2 , R 3 , R 4 and R 5 are described herein;

[0918] each # independently represents an attachment to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, represents H or an attachment to the remainder of the conjugate; and

[0919] each ## independently represents an attachment to the 5'-position of another nucleotide of the nucleotide-based enhancing unit; or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, represents H or an attachment to the remainder of the conjugate.

[0920] Exemplary Embodiment No. 3. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein B is H.

[0921] Exemplary Embodiment No. 4. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein B is a nucleobase moiety.

[0922] Exemplary Embodiment No. 5. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0923] Exemplary Embodiment No. 6. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein the nucleobase moiety is a modified nucleobase.

[0924] Exemplary Embodiment No. 7. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein the nucleobase moiety is an artificial nucleobase.

[0925] Exemplary Embodiment No. 8. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein V is -O-.

[0926] Exemplary Embodiment No. 9. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein V is -NR V -.

[0927] Exemplary Embodiment No. 10. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein V is -NH-.

[0928] Exemplary Embodiment No. 11. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein V is -C(R V ) 2 -.

[0929] Exemplary Embodiment No. 12. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein V is -CH 2 -.

[0930] Exemplary Embodiment No. 13. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is H.

[0931] Exemplary Embodiment No. 14. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is a halogen.

[0932] Exemplary Embodiment No. 15. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is -OR X 。

[0933] Exemplary Embodiment No. 16. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is -OH.

[0934] Exemplary Embodiment No. 17. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is -O-(C 1 -C 6 alkyl).

[0935] Exemplary Embodiment No. 18. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is -O-(C 1 -C 6 alkyl)-O-(C 1 -C 6 alkyl).

[0936] Exemplary Embodiment No. 19. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein X is optionally substituted by one or more R Xa substituted -O-(C 1 -C 6 alkyl)-(C 6 -C 10 aryl).

[0937] Exemplary Embodiment No. 20. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R X is H.

[0938] Exemplary Embodiment No. 21. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R X is optionally substituted by one or more R Xa substituted C 1 -C 6 alkyl.

[0939] Exemplary Embodiment No. 22. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R X is optionally substituted by one or more R XaSubstituted-(C 1 -C 6 -alkyl)-(C 6 -C 10 -aryl).

[0940] Exemplary embodiment No. 23. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein R X is -(C 1 -C 6 -alkyl)-(C 6 -C 10 -aryl).

[0941] Exemplary embodiment No. 24. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein R X and R 4 together form C 1 -C 6 -alkylene.

[0942] Exemplary embodiment No. 25. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein Y is -P(R Y )-.

[0943] Exemplary embodiment No. 26. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein Y is -P(OR Y )-.

[0944] Exemplary embodiment No. 27. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein Y is -P(N(R Y )) 2 )-.

[0945] Exemplary embodiment No. 28. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein Y is -P(=O)(OR Y )-.

[0946] Exemplary embodiment No. 29. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein Y is -P(=O)(R Y )-.

[0947] Exemplary embodiment No. 30. A nucleotide-based enhancer or conjugate according to any one of the preceding exemplary embodiments, wherein Y is -P(=S)(OR Y )-.

[0948] Exemplary Embodiment No. 31. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein Y is -P(=S)(R Y )-.

[0949] Exemplary Embodiment No. 32. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein Y is -P(=O)(SR Y )-.

[0950] Exemplary Embodiment No. 33. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein Y is -P(=S)(SR Y )-.

[0951] Exemplary Embodiment No. 34. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein each R Y is H.

[0952] Exemplary Embodiment No. 35. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein at least one R Y is C 1 -C 6 alkyl optionally substituted with one or more halogens or cyano groups.

[0953] Exemplary Embodiment No. 36. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein each R Y is C 1 -C 6 alkyl optionally substituted with one or more halogens or cyano groups.

[0954] Exemplary Embodiment No. 37. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 1 is H.

[0955] Exemplary Embodiment No. 38. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 1 is a halogen.

[0956] Exemplary Embodiment No. 39. A nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 1 is C 1 -C 6 alkyl optionally substituted with one or more halogens.

[0957] Exemplary Embodiment No. 40. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 2 is H.

[0958] Exemplary Embodiment No. 41. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 2 is a halogen.

[0959] Exemplary Embodiment No. 42. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 2 is C 1 -C 6 alkyl optionally substituted with one or more halogens.

[0960] Exemplary Embodiment No. 43. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 3 is H.

[0961] Exemplary Embodiment No. 44. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 3 is a halogen.

[0962] Exemplary Embodiment No. 45. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 3 is C 1 -C 6 alkyl optionally substituted with one or more halogens.

[0963] Exemplary Embodiment No. 46. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 4 is H.

[0964] Exemplary Embodiment No. 47. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 4 is a halogen.

[0965] Exemplary Embodiment No. 48. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 4 is C 1 -C 6 alkyl optionally substituted with one or more halogens.

[0966] Exemplary Embodiment No. 49. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein R 4 and RX Together form C 1 -C 6 Alkylene group.

[0967] Exemplary embodiment No. 50. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein each R 5 is H.

[0968] Exemplary embodiment No. 51. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein at least one R 5 is a halogen.

[0969] Exemplary embodiment No. 52. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein at least one R 5 is C 1 -C 6 alkyl optionally substituted with one or more halogens.

[0970] Exemplary embodiment No. 53. The nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments, wherein each of R a , R b , R 1 , R 2 , R 3 , R 4 and R 5 is H.

[0971] Exemplary embodiment No. 54. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I’) or formula (II’):

[0972]

[0973] Exemplary embodiment No. 55. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I-A), formula (II-A), formula (III-A) or formula (IV-A):

[0974]

[0975] Exemplary embodiment No. 56. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I’-A), formula (II’-A), formula (III’-A) or formula (IV’-A):

[0976]

[0977] Exemplary embodiment No. 57. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, selected from the compounds described in Table L and pharmaceutically acceptable salts thereof.

[0978] Exemplary embodiment No. 58. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I-B) or formula (II-B):

[0979]

[0980] Exemplary embodiment No. 59. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I'-B) or formula (II'-B):

[0981]

[0982] Exemplary embodiment No. 60. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I-C), formula (II-C), formula (III-C), formula (IV-C):

[0983]

[0984] Exemplary embodiment No. 61. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer has formula (I'-C), formula (II'-C), formula (III'-C) or formula (IV'-C):

[0985]

[0986] Exemplary embodiment No. 62. The nucleotide-based enhancer according to any one of the foregoing exemplary embodiments, wherein at least one nucleotide of the nucleotide-based enhancer is selected from the nucleotides described in Table L.

[0987] Exemplary embodiment No. 63. An isotopic derivative of the nucleotide-based enhancer according to any one of the foregoing exemplary embodiments.

[0988] Exemplary embodiment No. 64. The conjugate according to any one of the foregoing exemplary embodiments, comprising double-stranded RNA.

[0989] Exemplary Embodiment No. 65. The conjugate according to any one of the foregoing exemplary embodiments, selected from Figure 1 the conjugates described in

[0990] Exemplary Embodiment No. 66. The conjugate according to any one of the foregoing exemplary embodiments, selected from the structures described in Table C.

[0991] Exemplary Embodiment No. 67. The conjugate according to any one of the foregoing exemplary embodiments, wherein the nucleotide-based enhancing unit corresponds to a nucleotide-based enhancer, and the * in the nucleotide is attached to the ligand, linker unit, and / or nucleic acid at the 5'-end.

[0992] Exemplary Embodiment No. 68. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises a carbohydrate moiety.

[0993] Exemplary Embodiment No. 69. The conjugate according to any one of the foregoing exemplary embodiments, wherein the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide, or tetrasaccharide.

[0994] Exemplary Embodiment No. 70. The conjugate according to any one of the foregoing exemplary embodiments, wherein the carbohydrate moiety comprises galactose or a derivative thereof.

[0995] Exemplary Embodiment No. 71. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises

[0996] Exemplary Embodiment No. 72. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand

[0997]

[0998] Exemplary Embodiment No. 73. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises

[0999] Exemplary Embodiment No. 74. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises a moiety capable of binding to the glucagon-like peptide-1 receptor (GLP-1 receptor).

[1000] Exemplary Embodiment No. 75. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises glucagon-like peptide-1 (GLP-1) or a derivative thereof.

[1001] Exemplary Embodiment No. 76. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises glucagon or a derivative thereof.

[1002] Exemplary Embodiment No. 77. The conjugate according to any one of the foregoing exemplary embodiments, wherein the ligand comprises:

[1003] one, two or three sugars (e.g., N-acetyl-D-galactosamine);

[1004] one, two or three lipid moieties;

[1005] one, two or three peptide moieties;

[1006] one, two or three antibody moieties;

[1007] one, two or three oligonucleotides; or

[1008] any combination thereof.

[1009] Exemplary Embodiment No. 78. A pharmaceutical composition comprising a nucleotide-based enhancer or conjugate according to any one of the foregoing exemplary embodiments.

[1010] Exemplary Embodiment No. 79. A method of modulating the expression of a target gene in a subject, comprising administering to the subject a conjugate according to any one of the foregoing exemplary embodiments.

[1011] Exemplary Embodiment No. 80. A method of delivering a nucleic acid agent to a subject, comprising administering to the subject a conjugate according to any one of the foregoing exemplary embodiments.

[1012] Exemplary Embodiment No. 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 according to any one of the foregoing exemplary embodiments.

[1013] Exemplary Embodiment No. 82. The conjugate according to any one of the foregoing exemplary embodiments, for modulating the expression of a target gene in a subject.

[1014] Exemplary Embodiment No. 83. The conjugate according to any one of the foregoing exemplary embodiments, for delivering a nucleic acid agent to a subject.

[1015] Exemplary Embodiment No. 84. The conjugate according to any one of the foregoing exemplary embodiments, for treating or preventing a disease in a subject in need thereof.

[1016] Use of the conjugate according to any of the foregoing exemplary embodiments in the manufacture of a medicament for modulating the expression of a target gene in a subject.

[1017] Exemplary embodiment No. 86. Use of the conjugate according to any of the foregoing exemplary embodiments in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[1018] Exemplary embodiment No. 87. Use of the conjugate according to any of the foregoing exemplary embodiments in the manufacture of a medicament for treating or preventing a disease in a subject in need thereof.

[1019] Exemplary embodiment No. 88. The method, conjugate or use according to any of the foregoing exemplary embodiments, wherein the subject is human. Examples

[1020] Example 1. Synthesis of duplexes 1 - 5.

[1021] Scheme 1. Synthesis of conjugate duplex 1.

[1022]

[1023] Duplex 1 was synthesized according to Scheme 1. The sense strand 1a and the antisense strand 1 were prepared by solid-phase synthesis according to the manufacturer's protocol.

[1024] The sense strand 1b was generated by post-synthetic conjugation. Add the sense strand 1a (10.00 mg, 0.71 μmol) and H 2 O (0.50 mL) to Eppendorf tube 1 and mix vigorously to obtain a clear solution. Add DMF (0.50 mL), N-β-maleimidopropionyloxysuccinimide ester (0.38 mg, 1.42 μmol) and DIPEA (1.24 μL, 7.10 μmol) to Eppendorf tube 2 and mix vigorously. Then add the DMF solution in Eppendorf tube 2 to Eppendorf tube 1, and stir the resulting mixture at room temperature. After the reaction is indicated to be complete by LC-MS analysis, quench it with H 2 O (10 mL). Dialyze the reaction mixture against water (3 × 15 mL) using an Ultra-15 centrifuge with an MW cut-off of 3K. Freeze-dry the solution to provide the crude sense strand 1b (8.23 mg, 82% yield), which was used directly in the next step without further purification.

[1025] Sense strand 1 was prepared via post-synthesis conjugation. Sense strand 1b (8.23 mg, 0.58 μmol) and H 2 O (0.20 mL) 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. The DMA solution in Eppendorf tube 2 was then added to Eppendorf tube 1, and the resulting mixture was stirred at room temperature. After the reaction was completed as indicated by LC-MS analysis, it was washed with H 2 O (2 mL) quenched. The reaction mixture was purified by reverse phase HPLC (C18 column, at I and H 2 The fractions were pooled and purified using a 3K cutoff The solution was then lyophilized to provide sense strand 1 as a white solid (6.42 mg, 58% yield).

[1026] Duplex 1 is produced by annealing of sense strand 1 and antisense strand 1. Sense strand 1 solution (10 mg / mL) and antisense strand 1 solution (10 mg / mL) are prepared according to the OD amount of sense strand 1 solution (10 mg / mL) and antisense strand 1 solution (10 mg / mL). Based on equimolar amounts, the sense strand and antisense strand are mixed and annealed at 95° C. for 5 min, and cooled to room temperature to provide a conjugate duplex with a purity of> 90%. The solution of the duplex is lyophilized to provide the desired duplex 1 (96% yield).

[1027] Duplexes 2-5 were prepared in comparable yield and purity using a similar procedure as described for the preparation of duplex 1 (see Figure 2 ).

[1028] Example 2. In vivo KD activity of duplex 1-duplex 5.

[1029] CD-1 female mice were subcutaneously administered 30 mg / kg of duplexes 1 - 5. The control group was dosed with phosphate buffered saline (PBS). Animals were sacrificed 7 days after treatment. Specific tissues of interest were collected and stored in 10% for 24 hours. The tissues were then transferred to 75% ethanol and embedded in paraffin blocks. 4-μm tissue sections were made from the paraffin blocks. The slides were dewaxed twice in 100% xylene for 5 min and twice in 100% ethanol for 5 min. The RNASCOPE assay for the target gene was performed according to the RNASCOPE kit manual (RNASCOPE 2.5HD assay - Red, catalog number: 322350, Advanced Cell Diagnostics). The slides after RNASCOPE were imaged with a Keyence microscope, and the images were quantified with Indica Halo software. All samples were normalized to the PBS-treated control samples and plotted using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). For detailed results, see Figures 3 - 5 .

[1030] equivalents

[1031] The details of one or more embodiments of the present disclosure are set forth in the foregoing specification. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the present disclosure will be apparent from the specification and claims. In this specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. 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 pertains, unless otherwise defined. All patents and publications cited in this specification are incorporated by reference.

[1032] The foregoing description is presented for purposes of illustration only and is not intended to limit the present disclosure to the exact form disclosed, but is presented by the appended claims.

Claims

1. A conjugate or a pharmaceutically acceptable salt thereof, comprising: (i) one or more nucleic acid agents; (ii) one or more ligands; and (iii) one or more nucleotide-based enhancing units, wherein each nucleotide-based enhancing unit independently comprises from 2 to 30 nucleotides, and wherein each nucleotide independently has: Wherein: The nucleic acid agent includes an oligonucleotide; The ligand is a moiety that is capable of mediating its entry into or promoting its delivery to a target site when covalently attached to the nucleic acid agent; The one or more nucleic acid agents, the one or more ligands, and the one or more nucleotide-based enhancing units are covalently linked to each other directly or via a linker unit; B is H, C 1 -C 6 an alkyl or nucleobase moiety; V is -O-, -NR V -, or -C(R V ) 2 -; Each R V is independently H or C 1 -C 6 alkyl; X is H, a halogen or -OR X ; R X is H, C 1 -C 12 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl), wherein the C 1 -C 6 alkyl or -(C 1 -C 6 alkyl)-(C 6 -C 10 aryl) is optionally substituted with one or more R Xa ; or R X and R 4 together form C 1 -C 6 alkylene; Each R Xa is independently a halogen, C 1 -C 6 alkyl or -O-(C 1 -C 6 alkyl), wherein the C 1 -C 6 alkyl or -O-(C 1 -C 6 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 )-; Each R Y is independently H or C optionally substituted by one or more halogens or cyano 1 -C 6 alkyl; R 1 is H, halogen or C optionally substituted by one or more halogens 1 -C 6 alkyl; R 2 is H, halogen or C optionally substituted by one or more halogens 1 -C 6 alkyl; R 3 is H, a halogen or C 1 -C 6 alkyl optionally substituted by one or more halogens; R 4 is H, a halogen or a C 1 -C 6 alkyl optionally substituted by one or more halogens; or R 4 and R X together form a C 1 -C 6 alkylene; Each R 5 is independently H, halogen or C optionally substituted by one or more halogens 1 -C 6 -alkyl; Each # independently represents an attachment to the 2'-position or 3'-position of another nucleotide of the nucleotide-based enhancing unit, or when the nucleotide is at the 5'-end of the nucleotide-based enhancing unit, represents H or an attachment to the remainder of the conjugate; and Each ## independently represents an attachment to the 5'-position of another nucleotide of the nucleotide-based enhancing unit, or when the nucleotide is at the 2'-end or 3'-end of the nucleotide-based enhancing unit, represents H or an attachment to the remainder of the conjugate.

2. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit comprises from 3 to 20 nucleotides.

3. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit comprises from 6 to 15 nucleotides.

4. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit comprises 9 nucleotides.

5. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit is attached to the nucleic acid agent directly or via a linker unit.

6. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit is attached to the sense strand of the nucleic acid agent directly or via a linker unit.

7. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit is attached to the 3'-end of the nucleic acid agent directly or via a linker unit.

8. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit does not bind to any part of the antisense strand of the nucleic acid agent.

9. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit is attached to the ligand directly or via a linker unit.

10. The conjugate according to any one of the preceding claims, wherein at least one nucleotide-based enhancing unit: (i) is attached directly to both the nucleic acid agent and the ligand; (ii) is attached directly to the nucleic acid agent and attached to the ligand via a linker unit; (iii) is attached directly to the ligand and attached to the nucleic acid agent via a linker unit; or (iv) is attached to both the nucleic acid agent and the ligand via a linker unit.

11. The conjugate according to any one of the preceding claims, wherein the nucleic acid agent is double-stranded RNA.

12. The conjugate according to any one of the preceding claims, wherein the conjugate has the structure described in FIG.

1.

13. The conjugate according to any one of the preceding claims, wherein the conjugate has the structure described in Table D.

14. The conjugate according to any one of the preceding claims, wherein the conjugate has the structure described in Table C.

15. The conjugate according to any one of the preceding claims, wherein the ligand comprises a carbohydrate moiety.

16. The conjugate according to any one of the preceding claims, wherein the carbohydrate moiety comprises a monosaccharide, disaccharide, trisaccharide or tetrasaccharide.

17. The conjugate according to any one of the preceding claims, wherein the carbohydrate moiety comprises galactose or a derivative thereof.

18. The conjugate according to any one of the preceding claims, wherein the ligand comprises a moiety capable of binding to the glucagon-like peptide-1 receptor (GLP-1 receptor).

19. The conjugate according to any one of the preceding claims, wherein the ligand comprises glucagon-like peptide-1 (GLP-1) or a derivative thereof.

20. The conjugate according to any one of the preceding claims, wherein the ligand comprises glucagon or a derivative thereof.

21. An isotopic derivative of the conjugate according to any one of the preceding claims.

22. A pharmaceutical composition comprising the conjugate according to any one of the preceding claims.

23. A method of modulating the expression of a target gene in a subject, comprising administering to the subject the conjugate according to any one of claims 1-21.

24. A method of delivering a nucleic acid agent to a subject, comprising administering to the subject the conjugate according to any one of claims 1-21.

25. A method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate according to any one of claims 1-21.

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