Una amidites and uses thereof

Abstract

To provide novel linkers for better stabilizing and / or lower toxicity, optimization of binding moiety for increased target specificity and / or target delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effect.SOLUTION: Disclosed herein are a composition and a pharmaceutical formulation that comprise a binding moiety conjugated to a modified polynucleic acid molecule and a polymer. Also described herein is a method for treating a disease which utilize a composition or a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 858,289, filed June 6, 2019, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference. The ASCII copy created on June 5, 2020 is named 45532-735_601_SL.txt and is 3,172,165 bytes in size. [Background technology]

[0003] Gene suppression by RNA-induced gene silencing provides multiple levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA downregulation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) confers long-lasting effects over multiple cell divisions. Thus, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease treatment. Summary of the Invention

[0004] In an embodiment, disclosed herein is a compound of formula (II):

[0005] [ka] During the ceremony, Ring G is

[0006] [ka] and X is O, NH, or C(=O); R 1 is hydrogen, deuterium, halogen, substituted or unsubstituted C1 -C 10 Alkyl, substituted or unsubstituted C 2 -C 10 Alkenyl, substituted or unsubstituted C 2 -C 10 Alkynyl, substituted or unsubstituted C 1 -C 10 Fluoroalkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted -O-aryl, -OR 6 , -C(=O)R 6 , -C(=O)OR 6 , -C(=O)NR 7 R 7 , -OCH 2 C(=O)R 6 , -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 and R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 is alkyl, Or two R's 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forming a heterocycloalkyl; R 3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R 6 , -OR 6 , or -NR 7 R 7 and R 4 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R 5is -C(=O)R 6 and R 6 and R 7 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 1 -C 6 Fluoroalkyl, substituted or unsubstituted C 3 -C 6 It is cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0007] In some instances, R 1 is halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 1 -C 10 Fluoroalkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted -O-aryl, -OH, -OR 6 , -C(=O)R 6 , -C(=O)OR 6 , -C(=O)NR 7 R 7 , -OCH 2 C(=O)R 6 , or -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 In some cases, R 1 is halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted -O-aryl, -OCH 2 C(=O)R 6 , or -OCH 2C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 In some cases, R 1 is a halogen or a substituted or unsubstituted C 1 -C 20 It is heteroalkyl.

[0008] In some instances, R 1 teeth,

[0009] [ka] where n is 0, 1, 2, 3, 4, or 5; R 8 is hydrogen or substituted or unsubstituted C 1 -C 3 In some embodiments, R 8 is a substituted or unsubstituted C 1 -C 3 In some embodiments, R 8 is methyl or ethyl. In some embodiments, n is 1 or 2.

[0010] In some instances, R 1 HA-OCH 2 C(=O)NR 7 R 7 and R 7 each independently represents hydrogen or a substituted or unsubstituted C 1 -C 3 It is an alkyl.

[0011] In some examples, ring G is

[0012] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzoyl. In some examples, ring G is

[0013] [ka] and R 5 is -C(=O)CH(CH 3 ) 2 In some examples, ring G is

[0014] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzoyl, and R 5 is -C(=O)R 6 It is.

[0015] In some embodiments, R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 In some embodiments, R 2 each independently represents a substituted or unsubstituted C 1 -C 10 In some embodiments, R 2 In some embodiments, the two R's, together with the nitrogen atom to which they are attached, are substituted or unsubstituted C 3 -C 10 In some embodiments, X is O and R 3 is 4,4'-dimethoxytrityl (DMT).

[0016] In certain embodiments, the compound of Formula (IIa) is described herein:

[0017] [ka] During the ceremony, Ring G is

[0018] [ka] and X is O, NH, or C(=O); n is 0, 1, 2, 3, or 4; R 8 is a substituted or unsubstituted C 1 -C 6 is alkyl, R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 is alkyl, Or two R's 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forming a cycloalkyl; R 3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R 6 , -OR 6 , or -NR 7 R 7 and R 4 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R 5 is -C(=O)R 6 and R 6 and R 7 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 1 -C 6 Fluoroalkyl, substituted or unsubstituted C 3 -C 6 It is cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0019] In some examples, ring G is

[0020] [ka] and R 4 are each independently hydrogen, acetylisobutyl, or substituted or unsubstituted benzoyl. In some embodiments, G is

[0021] [ka] In some examples, ring G is

[0022] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzyl, and R 5 is -C(=O)R 6 In some examples, ring G is

[0023] [ka] and R 5 is -C(=O)CH(CH 3 ) 2 It is.

[0024] In some instances, R 6 is a substituted or unsubstituted C 1 -C 6 Alternatively and / or additionally, R 8 is methyl or ethyl. Alternatively and / or additionally, n is 1 or 2. In some embodiments, n is 1. In some embodiments, R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 In some embodiments, R 2 each independently represents a substituted or unsubstituted C 1 -C 10 In some embodiments, R 2In some embodiments, the two R's, together with the nitrogen atom to which they are attached, are substituted or unsubstituted C 3 -C 10 In some instances, R 3 is 4,4'-dimethoxytrityl (DMT).

[0025] In some instances, the compound is

[0026] [ka]

[0027] [ka] is selected from.

[0028] In some embodiments, the compounds are suitable for the synthesis of oligonucleotides.

[0029] In certain embodiments, disclosed herein are oligonucleotides comprising a compound of Formula (II) or Formula (IIa).

[0030] In certain embodiments, compositions and pharmaceutical formulations are disclosed herein that include a polynucleic acid molecule and a binding moiety, optionally conjugated to a polymer. Thus, disclosed herein is an oligonucleotide conjugate of formula (I):AB, where A is a binding moiety and B is an oligonucleotide comprising a compound of formula (II-1), (II-2), or (II-3), or a compound derived from formula (II-1), (II-2), or (II-3);

[0031] [ka] Ring G is

[0032] [ka] X is O, NH, or C(=O), and L 1 and L 2 are each independently absent or a linking moiety selected from a phosphodiester bond, an amide bond, and an ester bond; R 1 is hydrogen, deuterium, halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 2 -C 10 Alkenyl, substituted or unsubstituted C 2 -C 10 Alkynyl, substituted or unsubstituted C 1 -C 10 Fluoroalkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted -O-aryl, -OR 6 , -C(=O)R 6 , -C(=O)OR 6 , -C(=O)NR 7 R 7 , -OCH 2 C(=O)R 6 , -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 and R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 alkyl or two R 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Form a heterocycloalkyl, R 3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R 6 , -OR 6 , or -NR 7 R 7 and R 4each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R 5 -C(=O)OR 6 and R 6 and R 7 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 1 -C 6 Fluoroalkyl, substituted or unsubstituted C 3 -C 6 In some embodiments, the compound derived from formula (e.g., the derivative compound from formula II-3) includes modified forms of the formula with substituents.For example, in one embodiment, the compound derived from formula II-3 has a phosphorothioate group replacing the phosphodiester group, phosphotriester group, or phosphoramidite group.

[0033] In some embodiments, the oligonucleotide is an RNA oligonucleotide.In some embodiments, the oligonucleotide comprises at least one modification, or at least one 2'-modified nucleotide selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide, or alternatively selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA). Alternatively and / or in addition, the oligonucleotide comprises at least one modified internucleotide linkage optionally selected from a phosphorothioate linkage, a phosphorodithioate linkage, a methylphosphonate linkage, a phosphotriester linkage, or an amide linkage.

[0034] In some embodiments, the derivative compound of formula (II-3) is located at the 5' end of the oligonucleotide. Alternatively and / or in addition, the compound of formula (II-2) is located at the 3' end of the oligonucleotide. Alternatively and / or in addition, the derivative compound of formula (II-3) is located at the 5' end of the oligonucleotide and the binding moiety is conjugated to the 3' end of the oligonucleotide. Alternatively and / or in addition, the compound of formula (II-1) is located at an internal position within the oligonucleotide. Alternatively and / or in addition, the compound of formula (II-2) is located at the 5' end of the oligonucleotide and the binding moiety is conjugated to the 3' end of the oligonucleotide. Alternatively and / or in addition, the compound of formula (II-2) is located at the 3' end of the oligonucleotide and the binding moiety is conjugated to the 5' end of the oligonucleotide. Alternatively and / or in addition, the compound of formula (II-1) is located at the internal position of the oligonucleotide and the binding moiety is conjugated to the 5' end or the 3' end of the oligonucleotide. In some embodiments, formula (I) further comprises A to form ABC (formula IA), where A and C are not attached to B at the same end. In some embodiments, C is a polymer, which can include polyethylene glycol.

[0035] In some examples, the binding moiety comprises an antibody or binding fragment thereof. In some embodiments, the binding moiety comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , single chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb), or camelid antibody or binding fragment thereof. In some embodiments, the binding moiety comprises a peptide or a small molecule.

[0036] In some embodiments, the oligonucleotide comprises from about 8 to about 50 nucleotides, or from about 10 to about 30 nucleotides.

[0037] In some examples, B of formula I comprises an RNA oligonucleotide, has a length of about 10 to about 30 nucleotides, comprises at least one 2' modified nucleotide, and comprises at least one modified internucleotide linkage. In some embodiments, B hybridizes to at least 8 adjacent bases of a target gene sequence. In some embodiments, B mediates RNA interference. In some embodiments, B is a sense strand. In some embodiments, B hybridizes to a second oligonucleotide to form a double-stranded oligonucleic acid molecule. In some embodiments, the second oligonucleotide is an antisense strand. In some embodiments, the second oligonucleotide is an RNA oligonucleotide. In some embodiments, the second oligonucleotide comprises at least one modification. In some embodiments, the second oligonucleotide comprises a compound of formula (II) or a compound of formula (IIa). In some embodiments, the second oligonucleotide comprises at least one 2' modified nucleotide. In some embodiments, the second oligonucleotide comprises at least two 2' modified nucleotides selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotides, or alternatively and / or additionally selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA).

[0038] In some embodiments, the second oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the second oligonucleotide comprises at least one or at least three modified internucleotide linkages selected from phosphorothioate linkages or phosphorodithioate linkages.

[0039] In some embodiments, the oligonucleotide is a sense strand, is an RNA oligonucleotide, is conjugated to a binding moiety, a polymer, or a combination thereof, is about 10 to about 30 nucleotides, contains at least one 2' modified nucleotide, and contains at least one modified internucleotide linkage.

[0040] In some embodiments, methods of treating a disease or condition (e.g., cancer) are also described herein that utilize compositions and pharmaceutical formulations that include polynucleic acid molecules and binding moieties optionally conjugated to a polymer. Thus, disclosed herein is a method of treating a subject with a disease or condition characterized by insufficient expression of a protein, comprising administering to the subject an oligonucleotide conjugate as described herein to regulate expression of a gene encoding the protein, thereby treating the disease or condition characterized by insufficient expression of the protein. Disclosed herein is a method of treating a subject with a disease or condition characterized by overexpression of a protein, comprising administering to the subject an oligonucleotide conjugate as described herein to regulate expression of a gene encoding the protein, thereby treating the disease or condition characterized by overexpression of the protein. In some embodiments, the disease or condition is cancer, a neuromuscular disease, a muscular dystrophy, muscle atrophy, muscle wasting, a genetic disease, a genetic disease, or a cardiovascular disease.

[0041] In certain embodiments, disclosed herein is a method of inhibiting expression of a target gene in a primary cell of a patient, comprising administering to the primary cell a molecule as described above. In some embodiments, the method is an in vivo method. In some embodiments, the patient is a human.

[0042] In certain embodiments, disclosed herein are immuno-oncology therapies comprising vapor molecules for the treatment of a disease or disorder in a patient.

[0043] In certain embodiments, disclosed herein are kits that contain the above-described molecules. [Brief description of the drawings]

[0044] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1A] 1 shows a cartoon representation of a polynucleotide duplex comprising a compound of formula (II) attached to the 5' end of a guide (antisense) strand and further comprising a binding moiety A, which can be attached to the 5' end of a passenger (sense) strand directly or via a linker to the polynucleotide. [Figure 1B] 1 shows a cartoon representation of a polynucleotide duplex comprising one or more compounds of formula (II) attached to the 5' end of the guide (antisense) strand and inserted into the guide (antisense) strand, and further comprising a binding moiety A which can be attached to the 5' end of the passenger (sense) strand directly or via a linker to the polynucleotide. [Diagram 2] 1 is a cartoon representation of an exemplary compound of formula (II) in an oligonucleotide. [Diagram 3] 1 shows a graph of MSTN knockdown in SJCRH30 cells in vitro using four UNA siRNAs. [Figure 4]13 shows a graph of MSTN knockdown in gastrocnemius tissue in vitro using α-CD71-UNA siRNA conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Nucleic acid (e.g., RNAi) therapy is a targeted therapy that boasts high selectivity and high specificity. However, in some cases, nucleic acid therapy is also hindered by poor intracellular uptake, limited blood stability, and non-specific immune stimulation. To address these issues, various modifications of nucleic acid compositions are being explored, such as novel linkers for better stabilization and / or lower toxicity, optimization of binding moieties for increased target specificity and / or targeted delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0046] In some embodiments, the arrangement or order of the various components that make up the nucleic acid composition further effects cellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation. For example, when the nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide), the order or arrangement of the binding moiety, polymer, and / or polynucleic acid molecule (or polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule) further effects cellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation.

[0047] In some embodiments, the present disclosure provides oligonucleotide conjugates whose nucleic acid moiety configuration provides intracellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation.In some examples, the oligonucleotide conjugates comprise a binding moiety conjugated to a polynucleic acid molecule and a polymer.In some embodiments, the oligonucleotide conjugates comprise a compound of formula (II) or formula (IIa).

[0048] [ka]

[0049] In some embodiments, oligonucleotide conjugates comprising binding moieties conjugated to polynucleic acid molecules and polymers arranged as described herein enhance intracellular uptake, stability, and / or efficacy. In some examples, oligonucleotide conjugates comprising binding moieties conjugated to polynucleic acid molecules and polymers arranged as described herein reduce toxicity and / or non-specific immune stimulation. In some examples, the oligonucleotide conjugate comprises a compound of formula (II) or formula (IIa).

[0050] [ka]

[0051] In additional embodiments, kits are described herein that include one or more of the molecules or compositions described herein.

[0052] Therapeutic Molecular Platforms In some embodiments, the oligonucleotide conjugates described herein (e.g., therapeutic oligonucleotide conjugates) comprise a binding moiety conjugated to a polynucleic acid molecule and a polymer that comprises one or more modified nucleotides. In some embodiments, the oligonucleotide conjugate comprises a compound of formula (I) or formula (IA). A.B. Formula (I) (In the formula, A is a linking moiety, and B is an oligonucleotide comprising a compound of formula (II) or (IIa) A.B.C. Formula (IA) (In the formula, A is a linking moiety, and B is an oligonucleotide comprising a compound of formula (II) or (IIa), C is optionally a polymer.

[0053] In some embodiments, the oligonucleotide comprises a compound of formula (II):

[0054] [ka] During the ceremony, Ring G is

[0055] [ka] and X is O, NH, or C(=O); R 1 is hydrogen, deuterium, halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 2 -C 10 Alkenyl, substituted or unsubstituted C 2 -C 10 Alkynyl, substituted or unsubstituted C 1 -C 10 Fluoroalkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted -O-aryl, -OR 6 , -C(=O)R 6 , -C(=O)OR 6 , -C(=O)NR 7 R 7 , -OCH 2 C(=O)R 6 , -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 and R 2each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 is alkyl, Or two R's 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forming a heterocycloalkyl; R 3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R 6 , -OR 6 , or -NR 7 R 7 and R 4 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R 5 is -C(=O)R 6 and R 6 and R 7 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 1 -C 6 Fluoroalkyl, substituted or unsubstituted C 3 -C 6 It is cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0056] In some embodiments of Formula (II), R 1 is halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 1 -C 10 Fluoroalkyl, substituted or unsubstituted C 1 -C 20Heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted -O-aryl, -OH, -OR 6 , -C(=O)R 6 , -C(=O)OR 6 , -C(=O)NR 7 R 7 , -OCH 2 C(=O)R 6 , or -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 It is.

[0057] In some embodiments of Formula (II), R 1 is halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted -O-aryl, -OCH 2 C(=O)R 6 , or -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 In some embodiments of formula (II), R 1 is a halogen or a substituted or unsubstituted C 1 -C 20 In some embodiments of Formula (II), R 1 is fluoro, chloro, or bromo. In some embodiments of formula (II), R 1 is fluoro.

[0058] In some embodiments of Formula (II), R 1 teeth,

[0059] [ka] where n is 0, 1, 2, 3, 4, or 5, and R 8 is hydrogen or substituted or unsubstituted C 1 -C 3 In some embodiments, n is alkyl. In some embodiments, n is 1, 2, or 3. In some embodiments, n is 1 or 2. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 0.

[0060] In some embodiments of Formula (II), R 1 teeth,

[0061] [ka] and R 8 is a substituted or unsubstituted C 1 -C 3 In some embodiments, R 8 is methyl or ethyl. In some embodiments, R 8 is methyl. In some embodiments, R 8 is ethyl. In some embodiments, R 8 is H.

[0062] In some embodiments of Formula (II), ring G is

[0063] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzoyl. In some embodiments of Formula (II), Ring B is

[0064] [ka] In some embodiments of Formula (II), ring G is:

[0065] [ka] In some embodiments of Formula (II), ring G is:

[0066] [ka] It is.

[0067] In some embodiments of Formula (II), ring G is

[0068] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzoyl, and R 5 is -C(=O)R 6 In some embodiments of formula (II), ring G is

[0069] [ka] and R 5 is -C(=O)R 6 In some embodiments of formula (II), ring G is

[0070] [ka] and R 5 is -C(=O)CH(CH 3 ) 2 In some embodiments of formula (II), ring G is

[0071] [ka] and R 4 Each is independently hydrogen or substituted or unsubstituted benzoyl.

[0072] In some embodiments of Formula (II), R 2each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 In some embodiments of formula (II), R 2 each independently represents a substituted or unsubstituted C 1 -C 10 In some embodiments of formula (II), R 2 are each isopropyl.

[0073] In some embodiments of Formula (II), two R 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forms a heterocycloalkyl.

[0074] In some embodiments of Formula (II), X is O and R 3 is 4,4'-dimethoxytrityl (DMT).

[0075] In some embodiments, the oligonucleotide comprises a compound of Formula (IIa):

[0076] [ka] During the ceremony, Ring G is

[0077] [ka] and X is O, NH, or C(=O); n is 0, 1, 2, 3, or 4; R 8 is a substituted or unsubstituted C 1 -C 6 is alkyl, R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 is alkyl, Or two R's2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forming a cycloalkyl; R 3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R 6 , -OR 6 , or -NR 7 R 7 and R 4 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R 5 is -C(=O)R 6 and R 6 and R 7 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 Alkyl, substituted or unsubstituted C 1 -C 6 Fluoroalkyl, substituted or unsubstituted C 3 -C 6 It is cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0078] In some embodiments of Formula (IIa), ring G is

[0079] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzyl. In some embodiments of Formula (IIa), ring G is

[0080] [ka] In some embodiments of formula (II), ring G is

[0081] [ka] In some embodiments of Formula (IIa), ring G is

[0082] [ka] It is.

[0083] In some embodiments of Formula (IIa), ring G is

[0084] [ka] and R 4 are each independently hydrogen or substituted or unsubstituted benzyl, and R 5 is -C(=O)R 6 In some embodiments of Formula (IIa), ring G is

[0085] [ka] and R 5 is -C(=O)R 6 In some embodiments of Formula (IIa), ring G is

[0086] [ka] and R 5 is -C(=O)CH(CH 3 ) 2 In some embodiments of Formula (IIa), ring G is

[0087] [ka] and R 4 Each is independently hydrogen or substituted or unsubstituted benzoyl.

[0088] In some embodiments of Formula (IIa), n is 0, 1, 2, 3, 4, or 5. In some embodiments of Formula (IIa), n is 0, 1, 2, 3, or 4. In some embodiments of Formula (IIa), n is 1 or 2. In some embodiments of Formula (IIa), n is 0. In some embodiments of Formula (IIa), n is 1. In some embodiments of Formula (IIa), n is 2.

[0089] In some embodiments of Formula (IIa), R 8 is hydrogen or substituted or unsubstituted C 1 -C 3 In some embodiments of Formula (IIa), R 8 is a substituted or unsubstituted C 1 -C 3 In some embodiments of Formula (IIa), R 8 is methyl or ethyl. In some embodiments of Formula (IIa), R 8 is methyl.

[0090] In some embodiments of Formula (IIa), R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 In some embodiments of Formula (IIa), R 2 each independently represents a substituted or unsubstituted C 1 -C 10 In some embodiments of Formula (IIa), R 2 are each isopropyl.

[0091] In some embodiments of Formula (IIa), two R 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forms a heterocycloalkyl.

[0092] In some embodiments of Formula (IIa), R 3is 4,4'-dimethoxytrityl (DMT).

[0093] In some embodiments of the compound of Formula (IIa), the compound is selected from the group consisting of:

[0094] [ka]

[0095] In some embodiments, the oligonucleotide comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, or more compounds of formula (II) (e.g., formula IIa). In some cases, the oligonucleotide comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or more compounds of formula (II) (e.g., formula IIa). In some embodiments, the compounds of formula (II) (e.g., formula IIa) are present in tandem within the oligonucleotide. In other embodiments, the compounds of Formula (II) (eg, Formula IIa) are interspersed within an oligonucleotide, where the nucleotides are modified with one or more additional modifications described below.

[0096] In some embodiments, the oligonucleotide comprises at least one of about 5% to about 100% modifications, about 10% to about 100% modifications, about 20% to about 100% modifications, about 30% to about 100% modifications, about 40% to about 100% modifications, about 50% to about 100% modifications, about 60% to about 100% modifications, about 70% to about 100% modifications, about 80% to about 100% modifications, and about 90% to about 100% modifications, where the modifications are compounds of formula (II) (e.g., formula (IIa)). For example, if an oligonucleotide has 20 nucleosides, an oligonucleotide having about 60% modifications comprises about 12 nucleosides substituted with 12 compounds of formula (II) (e.g., formula IIa).

[0097] In some embodiments, the oligonucleotide conjugate is a molecule as exemplified:

[0098] [ka]

[0099] In some embodiments, the oligonucleotide conjugate is a molecule as exemplified:

[0100] [ka]

[0101] The above

[0102] [ka] are for illustrative purposes only and include humanized antibodies or binding fragments thereof, chimeric antibodies or binding fragments thereof, anti-human antibodies, anti-murine antibodies (e.g., anti-mouse antibodies, anti-rat antibodies, etc.), monoclonal antibodies or binding fragments thereof, monovalent Fab', bivalent Fab 2 , single chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb) or camelid antibody or binding fragment thereof.

[0103] Additional Modifications In some examples, the additional modifications include synthetic or artificial nucleotide analogs or bases that contain modifications at one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or combinations thereof.

[0104] In some embodiments, modifications at the 2' hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0105] In some embodiments, the nucleotide analogs include modified bases, such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-(2 -amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine) , 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queousine, archaeosine, naphthyl, and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine, 5-oxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. 5'-modified nucleotides further include those nucleotides that are modified on the sugar moiety and 5'-modified nucleotides having a sugar that is not ribosyl or an analog thereof, for example, the sugar moiety optionally is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles.The term nucleotide further includes what are known in the art as universal bases, including, but not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0106] In some embodiments, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some instances, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving cellular uptake properties due to its zwitterionic properties.

[0107] In some instances, the 5'-modified nucleotide is further modified at the 2' hydroxyl group in a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene linked bicyclic ribonucleotide monomer.

[0108] In some embodiments, the additional modifications further include morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or 1',5'-anhydrohexitol nucleic acid (HNA). Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics the natural nucleic acid structure but deviates from the normal sugar and phosphonate structure. In some instances, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some instances, the ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some instances, the backbone changes remove all positive and negative charges, allowing the morpholino neutral molecule to cross cell membranes without the aid of a cellular delivery agent as used with charged oligonucleotides. Non-limiting examples of 5'-modified morpholino oligonucleotides are illustrated below.

[0109] [ka]

[0110] In some embodiments, the 5'-modified morpholino or PMO described above is a PMO that contains positive or cationic charge. In some examples, the PMO is PMOplus (Sarepta). PMOplus refers to phosphorodiamidate morpholino oligomers that contain any number of (1-piperazino)phosphinylideneoxy, (1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy bonds (such as those described in PCT Publication No. WO2008 / 036127). In some examples, the PMO is a PMO described in U.S. Patent No. 7,943,762.

[0111] In some embodiments, the morpholino or PMO is PMO-X (Sarepta). In some cases, PMO-X refers to a phosphorodiamidate morpholino oligomer that includes at least one bond or at least one of the disclosed terminal modifications, such as those disclosed in PCT Publication No. WO2011 / 150408 and U.S. Patent Application Publication No. 2012 / 0065169.

[0112] In some embodiments, the morpholino or PMO is a PMO as described in Table 5 of U.S. Patent Application Publication No. 2014 / 0296321.

[0113] In some embodiments, peptide nucleic acids (PNAs) contain no sugar rings or phosphonate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating the backbone charge.

[0114] [ka]

[0115] In some embodiments, one or more of the above modifications occur at the internucleotide bond. In some examples, the modified internucleotide bond may include, but is not limited to, phosphorothioate; phosphorodithioate; methyl phosphonate; 5'-alkylene phosphonate; 5'-methyl phosphonate; 3'-alkylene phosphonate; boron trifluoride; 3'-5' or 2'-5' boranophosphate and selenophosphate; phosphotriester; thionoalkyl phosphotriester; hydrogen phosphonate bond; alkyl phosphonate; alkyl phosphonothioate; aryl phosphonothioate; phosphoroselenoate; phosphorodiselenoate; phosphinate; phosphoramidate; 3'-alkyl phosphoramidate; aminoalkyl phosphoramidate; thionophosphoramidate; phosphoropiper ... radidate; phosphoroanilothioate; phosphoroanilidate; ketone; sulfone; sulfonamide; carbonate; carbamate; methylenehydrazo; methylenedimethylhydrazo; formacetal; thioformacetal; oxime; methyleneimino; methylenemethylimino; thioamidate; bond with riboacetyl group; aminoethylglycine; silyl or siloxane bond; alkyl or cycloalkyl bond, e.g., saturated or unsaturated and / or substituted and / or containing heteroatoms, with or without heteroatoms of 1 to 10 carbons; bond with morpholino structure, amide, or polyamide in which base is directly or indirectly attached to the aza nitrogen of the backbone; and combinations thereof.

[0116] In some examples, the modification is a methyl or thiol modification, such as a methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left), phosphorodithioate (middle), and methylphosphonate nucleotides (right) are illustrated below.

[0117] [ka]

[0118] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, phosphoramidites exemplified as follows:

[0119] [ka]

[0120] In some examples, the modified internucleotide linkage is a phosphorodiamidate linkage. Non-limiting examples of phosphorodiamidate linkages with morpholino systems are shown below.

[0121] [ka]

[0122] In some examples, the modified internucleotide linkage is a methylphosphonate linkage. Non-limiting examples of methylphosphonate linkages are shown below.

[0123] [ka]

[0124] In some instances, the modified internucleotide bond is an amide bond. Non-limiting examples of amide bonds are shown below.

[0125] [ka]

[0126] In some embodiments, the one or more modifications include a modified phosphate backbone, where the modification produces a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation to produce an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some instances, alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing 1-6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3.3-dimethylbutyl, and 2-ethylbutyl groups. In some instances, the modified phosphate is a phosphate group described in U.S. Pat. No. 9,481,905.

[0127] In some embodiments, the further modified phosphate backbone comprises a methylphosphonate, an ethylphosphonate, a methylthiophosphonate, or a methoxyphosphonate. In some embodiments, the modified phosphate is a methylphosphonate. In some embodiments, the modified phosphate is an ethylphosphonate. In some embodiments, the modified phosphate is a methylthiophosphonate. In some embodiments, the modified phosphate is a methoxyphosphonate.

[0128] In some embodiments, the one or more modifications further optionally include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or the nucleotide analog at the 3' or 5' end. For example, the 3' end optionally includes a 3' cationic group, or inverts the nucleoside at the 3' end with a 3'-3' bond. In another alternative, the 3' end is optionally conjugated with an aminoalkyl group, e.g., a 3'C5-aminoalkyl dT. In an additional alternative, the 3' end is optionally conjugated with an abasic site, e.g., an apurinic or apyrimidinic site.

[0129] In some embodiments, the oligonucleotides comprising the compound of formula (II) (e.g., formula IIa) further comprise one or more of the artificial nucleotide analogs described herein. In some embodiments, the oligonucleotides comprising the compound of formula (II) (e.g., formula IIa) further comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more, additional modifications, such as, but not limited to, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-aminopropyl), 2'-aminopropyl, ... 2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof. In some examples, an oligonucleotide comprising a compound of Formula (II) (e.g., Formula IIa) can further comprise 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyloxyethyl ...P), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAOE), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2 In some embodiments, the nucleotide sequence includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogs selected from 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5' phosphoramidites, or combinations thereof.In some examples, oligonucleotides comprising a compound of Formula (II) (e.g., Formula IIa) contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methyl modified nucleotides. In some examples, oligonucleotides comprising a compound of Formula (II) (e.g., Formula IIa) contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, an oligonucleotide comprising a compound of Formula (II) (e.g., Formula IIa) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiol phosphonate nucleotides.

[0130] In some examples, about 5 to about 100% of the oligonucleotides comprising a compound of formula (II) (e.g., formula IIa) comprise an artificial nucleotide analogue as described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules comprise an artificial nucleotide analogue as described herein. In some embodiments, the artificial nucleotide analog comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof.

[0131] In some embodiments, the oligonucleotide (or B) described herein comprises RNA or DNA. In some cases, the oligonucleotide comprises RNA. In some examples, the RNA comprises small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some examples, the RNA comprises shRNA. In some examples, the RNA comprises miRNA. In some examples, the RNA comprises dsRNA. In some examples, the RNA comprises tRNA. In some examples, the RNA comprises rRNA. In some examples, the RNA comprises hnRNA. In some examples, the RNA comprises siRNA. In some cases, the oligonucleotide comprises the sense strand (or passenger strand) of an siRNA. In other cases, the oligonucleotide comprises the antisense (or guide strand) of an siRNA.

[0132] In some embodiments, the oligonucleotide is about 10 to about 50 nucleotides in length. In some examples, the oligonucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length.

[0133] In some embodiments, the oligonucleotide is about 50 nucleotides long. In some examples, the oligonucleotide is about 45 nucleotides long. In some examples, the oligonucleotide is about 40 nucleotides long. In some examples, the oligonucleotide is about 35 nucleotides long. In some examples, the oligonucleotide is about 30 nucleotides long. In some examples, the oligonucleotide is about 25 nucleotides long. In some examples, the oligonucleotide is about 20 nucleotides long. In some examples, the oligonucleotide is about 19 nucleotides long. In some examples, the oligonucleotide is about 18 nucleotides long. In some examples, the oligonucleotide is about 17 nucleotides long. In some examples, the oligonucleotide is about 16 nucleotides long. In some examples, the oligonucleotide is about 15 nucleotides long. In some examples, the oligonucleotide is about 14 nucleotides long. In some examples, the oligonucleotide is about 13 nucleotides long. In some examples, the oligonucleotide is about 12 nucleotides long. In some examples, the oligonucleotide is about 11 nucleotides long. In some examples, the oligonucleotide is about 10 nucleotides long. In some examples, the oligonucleotide is about 10 to about 50 nucleotides long. In some examples, the oligonucleotide is about 10 to about 45 nucleotides long. In some examples, the oligonucleotide is about 10 to about 40 nucleotides long. In some examples, the oligonucleotide is about 10 to about 35 nucleotides in length. In some examples, the oligonucleotide is about 10 to about 30 nucleotides in length. In some examples, the oligonucleotide is about 10 to about 25 nucleotides in length. In some examples, the oligonucleotide is about 10 to about 20 nucleotides in length. In some examples, the oligonucleotide is about 15 to about 25 nucleotides in length. In some examples, the oligonucleotide is about 19 to about 23 nucleotides in length. In some examples, the oligonucleotide is about 15 to about 30 nucleotides in length. In some examples, the oligonucleotide is about 12 to about 30 nucleotides in length.

[0134] In some embodiments, the oligonucleotide further hybridizes to a second oligonucleotide to form a duplex. In some examples, the oligonucleotide is a sense strand or a passenger strand. In some examples, the oligonucleotide is an antisense strand or a guide strand.

[0135] In some embodiments, the second oligonucleotide is about 10 to about 50 nucleotides in length. In some embodiments, the second oligonucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length.

[0136] In some examples, the second oligonucleotide is about 50 nucleotides in length. In some examples, the second oligonucleotide is about 45 nucleotides in length. In some examples, the second oligonucleotide is about 40 nucleotides in length. In some examples, the second oligonucleotide is about 35 nucleotides in length. In some examples, the second oligonucleotide is about 30 nucleotides in length. In some examples, the second oligonucleotide is about 25 nucleotides in length. In some examples, the second oligonucleotide is about 20 nucleotides in length. In some examples, the second oligonucleotide is about 19 nucleotides in length. In some examples, the second oligonucleotide is about 18 nucleotides in length. In some examples, the second oligonucleotide is about 17 nucleotides in length. In some examples, the second oligonucleotide is about 16 nucleotides in length. In some examples, the second oligonucleotide is about 15 nucleotides in length. In some examples, the second oligonucleotide is about 14 nucleotides in length. In some examples, the second oligonucleotide is about 13 nucleotides in length. In some examples, the second oligonucleotide is about 12 nucleotides in length. In some examples, the second oligonucleotide is about 11 nucleotides in length. In some examples, the second oligonucleotide is about 10 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 50 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 45 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 40 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 35 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 30 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 25 nucleotides in length. In some examples, the second oligonucleotide is about 10 to about 20 nucleotides in length. In some examples, the first oligonucleotide is about 15 to about 25 nucleotides in length. In some examples, the first oligonucleotide is about 15 to about 25 nucleotides in length.In some examples, the first oligonucleotide is about 15 to about 30 nucleotides in length. In some examples, the second oligonucleotide is about 12 to about 30 nucleotides in length.

[0137] In some embodiments, the polynucleic acid molecule comprises a first oligonucleotide and a second oligonucleotide. In some examples, the polynucleic acid molecule further comprises a blunt end, an overhang, or a combination thereof. In some examples, the blunt end is a 5' blunt end, a 3' blunt end, or both. In some cases, the overhang is a 5' overhang, a 3' overhang, or both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base paired nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base paired nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base paired nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4 non-base paired nucleotides. In some cases, the overhang comprises 1 non-base paired nucleotide. In some cases, the overhang comprises 2 non-base paired nucleotides. In some cases, the overhang comprises 3 non-base paired nucleotides. In some cases, the overhang comprises 4 non-base paired nucleotides.

[0138] In some embodiments, the sequence of the polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to the target sequences described herein. Exemplary target sequences include, but are not limited to, the sequence of the DMD gene or any of its mRNAs, the sequence of the DMPK gene or any of its mRNAs, the sequence of a cancer gene or any of its mRNAs, any gene associated with a genetic disease or genetic disorder (e.g., GYS1), any gene associated with muscle atrophy, muscular dystrophy, or muscle wasting (e.g., DMD, DMPK, DUX4) and any of its mRNAs. In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to the target sequence described herein. In some examples, the sequence of the polynucleic acid molecule is 100% complementary to the target sequence described herein.

[0139] In some embodiments, the sequence of the polynucleic acid molecule has 5 or less mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has 4 or less mismatches to the target sequences described herein. In some examples, the sequence of the polynucleic acid molecule has 3 or less mismatches to the target sequences described herein. In some cases, the sequence of the polynucleic acid molecule has 2 or less mismatches to the target sequences described herein. In some cases, the sequence of the polynucleic acid molecule has 1 or less mismatch to the target sequences described herein.

[0140] In some embodiments, the specificity of a polynucleic acid molecule hybridizing to a target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some examples, the hybridization is under high stringency hybridization conditions.

[0141] In some embodiments, the polynucleic acid molecule hybridizes to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 8 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 9 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 10 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 11 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 12 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 13 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 14 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 15 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 16 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 17 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 18 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 19 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 20 contiguous bases of the target sequence described herein.

[0142] In some embodiments, the polynucleic acid molecule has reduced off-target effects. In some instances, "off-target" or "off-target effect" refers to any instance where a polynucleic acid polymer directed to a given target causes unintended effects by directly or indirectly interacting with another mRNA sequence, DNA sequence, or cellular protein or other moiety. In some instances, "off-target effect" occurs when there is simultaneous degradation of other transcripts due to partial homology or complementarity between other transcripts and the sense and / or antisense strands of the polynucleic acid molecule.

[0143] In some cases, one or more of the artificial nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNases, or exonucleases, e.g., 5'-3' exonuclease and 3'-5' exonuclease, as compared to naturally occurring polynucleic acid molecules. In some examples, the compounds of Formula II (e.g., Formula IIa), 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are capable of inhibiting RNase activity. Resistant to nucleases such as ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some examples, 2'-O-methyl modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the 2'-O-aminopropyl modified polynucleic acid molecule is nuclease resistant (eg, RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, 2'-deoxy modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-deoxy-2'-fluoro modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, LNA-modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, ENA-modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, HNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). Morpholinos are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, PNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, methylphosphonate nucleotide-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some examples, the thiophosphonate nucleotide modified polynucleic acid molecule is nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the polynucleic acid molecule comprising a 2'-fluoro N3-P5'-phosphoramidite is nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some examples, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0144] In some embodiments, one or more of the artificial nucleotide analogs comprising the compounds of formula II (e.g., formula IIa) described herein have increased binding affinity to their mRNA targets compared to the equivalent naturally occurring polynucleic acid molecules. One or more of the compounds of formula II (e.g., formula IIa), 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or artificial nucleotide analogs including 2'-fluoro N3-P5' phosphoramidites, can have increased binding affinity to their mRNA compared to a comparable naturally occurring polynucleic acid molecule. In some examples, a 2'-O-methyl modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a 2'-O-aminopropyl modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a 2'-deoxy modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a 2'-deoxy-2'-fluoro modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule.In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, LNA modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, an ENA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a PNA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, an HNA modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a morpholino modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a methylphosphonate nucleotide modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some examples, a thiol phosphonate nucleotide modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent native polynucleic acid molecule. In some instances, a polynucleic acid molecule containing a 2'-fluoro N3-P5'-phosphoramidite has increased binding affinity for its mRNA target as compared to a comparable native polynucleic acid molecule.In some cases, the increased affinity is demonstrated by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0145] In some embodiments, the artificial nucleotide analogs comprising the compounds of formula II (e.g., formula IIa) described herein are chirally pure (or stereochemically pure) polynucleic acid molecules or polynucleic acid molecules comprising a single enantiomer. In some examples, the polynucleic acid molecules comprise L-nucleotides. In some examples, the polynucleic acid molecules comprise D-nucleotides. In some examples, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some cases, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some examples, the polynucleic acid molecule is a polynucleic acid molecule described in U.S. Patent Application Publication Nos. 2014 / 194610 and 2015 / 211006; and PCT International Publication No. WO2015107425.

[0146] In some embodiments, the polynucleic acid molecules described herein are further modified to include aptamer conjugate moieties. In some examples, the aptamer conjugate moieties are DNA aptamer conjugate moieties. In some examples, the aptamer-conjugate moieties are Alphamer (Centauri Therapeutics), which include an aptamer moiety that recognizes a specific cell surface target and a moiety that displays a specific epitope for binding to circulating antibodies. In some examples, the polynucleic acid molecules described herein are further modified to include aptamer conjugate moieties as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0147] In further embodiments, the polynucleic acid molecule described herein is modified to increase its stability.In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA), and the polynucleic acid molecule is modified to increase its stability.In some examples, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecules are modified at the 2' hydroxyl position, such as with a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, or with a locked or bridged ribose structure (e.g., LNA or ENA). In some cases, the polynucleic acid molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule further comprises morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability.In some examples, the polynucleic acid molecule is a chirally pure (or stereopure) polynucleic acid molecule.In some examples, the chirally pure (or stereopure) polynucleic acid molecule is modified to increase its stability.The appropriate modification of RNA to increase the stability of delivery will be clear to those skilled in the art.

[0148] In some embodiments, the polynucleic acid molecules described herein have RNAi activity that regulates the expression of the RNA encoded by the gene. In some examples, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of a gene, where one strand of the double-stranded siRNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of the RNA encoded by the gene or a portion thereof, and the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence that is substantially similar to the nucleotide sequence of the RNA encoded by the gene or a portion thereof. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of a gene, where each strand of the siRNA molecule comprises about 15-25, 18-24, or 19-about 23 nucleotides, and each strand comprises at least about 14, 17, or 19 nucleotides that are complementary to the nucleotides of the other strand. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate expression of a gene, wherein each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to nucleotides in the other strand.

[0149] In some embodiments, the polynucleic acid molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions, using procedures known in the art.For example, polynucleic acid molecules are chemically synthesized using naturally occurring nucleotides, or using various modified nucleotides designed to increase the biological stability of the molecule, or to increase the physical stability of the duplex formed between the polynucleic acid molecule and the target nucleic acid.Exemplary methods include those described in U.S. Patent Nos. 5,142,047, 5,185,444, 5,889,136, 6,008,400, and 6,111,086, PCT International Publication No. WO2009099942, or European Patent Publication No. 1579015.Additional exemplary methods include Griffey et al., “2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al. “Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering”. Tetrahedron Letters 38 (50): 8735 1997; Koizumi, M. “ENA oligonucleotides as therapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149 (2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits-antisense technologies: new chemical possibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, the polynucleic acid molecule is produced biologically using an expression vector into which the polynucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted polynucleic acid molecule will be in an antisense orientation relative to the desired target polynucleic acid molecule).

[0150] One embodiment provides an oligonucleotide conjugate of formula (I) or formula (IA). A.B. Formula (I) (In the formula, A is a binding moiety, B is an oligonucleotide comprising a compound of formula (II) or (IIa) A.B.C. Formula (IA) (In the formula, A is a linking moiety, and B is an oligonucleotide comprising a compound of formula (II) or (IIa), C is optionally a polymer.

[0151] One embodiment provides an oligonucleotide conjugate of formula (I) or formula (IA): A.B. Formula (I) A.B.C. Formula (IA) During the ceremony, A is a binding moiety, C is optionally a polymer, and B is an oligonucleotide comprising a compound of formula (II-1), (II-2), or (II-3),

[0152] [ka] During the ceremony, Ring G is

[0153] [ka] and X is O, NH, or C(=O); L 1 and L 2 are each independently absent or a linking moiety selected from a phosphodiester bond, an amide bond, and an ester bond; R 1 is hydrogen, deuterium, halogen, substituted or unsubstituted C 1 -C 10 Alkyl, substituted or unsubstituted C 2 -C 10 Alkenyl, substituted or unsubstituted C 2 -C10 Alkynyl, substituted or unsubstituted C 1 -C 10 Fluoroalkyl, substituted or unsubstituted C 1 -C 20 Heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted -O-aryl, -OR 6 , -C(=O)R 6 , -C(=O)OR 6 , -C(=O)NR 7 R 7 , -OCH 2 C(=O)R 6 , -OCH 2 C(=O)OR 6 , or -OCH 2 C(=O)NR 7 R 7 and R 2 each independently represents hydrogen, deuterium, or a substituted or unsubstituted C 1 -C 10 is alkyl, Or two R's 2 are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C 3 -C 10 Forming a heterocycloalkyl; R 3 is hydrogen, 4,4'-dimethoxytrityl (DMT), -R 6 , -OR 6 , or -NR 7 R 7 and R 4 each independently represents hydrogen, a substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R 5 -C(=O)OR 6 and R 6 and R 7 each independently represents hydrogen, a substituted or unsubstituted C 1-C 6 Alkyl, substituted or unsubstituted C 1 -C 6 Fluoroalkyl, substituted or unsubstituted C 3 -C 6 It is cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0154] In some embodiments of the oligonucleotide conjugate of formula (I), B is a compound having the structure of formula (II-1). In some embodiments of the oligonucleotide conjugate of formula (I), B is a compound having the structure of formula (II-2). In some embodiments of the oligonucleotide conjugate of formula (I), B is a compound having the structure of formula (II-3).

[0155] One embodiment provides an oligonucleotide conjugate of formula (Xa): AX-B'-YC Formula (Xa) During the ceremony, A is a binding moiety, B' is an oligonucleotide comprising a compound of formula (II) or (IIa), C is optionally a polymer; X is a bond or a first linker, and Y is a bond or a second linker; The polynucleotide further comprises one or more additional non-naturally occurring nucleotides; and A and C do not bind to B at the same end.

[0156] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein the oligonucleotide further comprises at least one modified internucleotide linkage, or at least one inverted abasic moiety.

[0157] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein the compound of formula II is located at the 5' end of the oligonucleotide.

[0158] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein the compound of formula II is located at an internucleotide linkage of the oligonucleotide.

[0159] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein the compound of formula II is further modified at the 2' position.

[0160] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein the one or more additional non-natural nucleosides comprise a 2'-modification selected from a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide.

[0161] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein the compound of formula II is selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

[0162] Another embodiment provides an oligonucleotide comprising a compound of formula (I), or an oligonucleotide conjugate of formula (Xa), wherein at least one inverted abasic moiety is at least one terminus.

[0163] Another embodiment provides an oligonucleotide comprising a compound of formula (I) or an oligonucleotide conjugate of formula (Xa), wherein the oligonucleotide is single-stranded.Another embodiment provides an oligonucleotide of formula (Xa), wherein the oligonucleotide is double-stranded.

[0164] Another embodiment provides an oligonucleotide comprising a compound of formula (I) or an oligonucleotide conjugate of formula (Xa), wherein the oligonucleotide is 2 to about 100 residues long. Another embodiment provides an oligonucleotide comprising a compound of formula (I) or an oligonucleotide conjugate of formula (Xa), wherein the oligonucleotide is 2 to about 90 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 80 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 70 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 60 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 50 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 40 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 20 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 2 to about 10 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 8 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (Xa), wherein the oligonucleotide is 10 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 14 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (Xa), wherein the oligonucleotide is 18 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 22 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is 26 to about 30 residues long.

[0165] One embodiment provides compounds suitable for the synthesis of oligonucleotides selected from the group consisting of:

[0166] [ka]

[0167] Conjugation Chemistry In some embodiments, the polynucleic acid molecule is conjugated to a binding moiety. In some examples, the binding moiety includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and all analogs or derivatives of these classes of substances. Additional examples of binding moieties further include steroids, such as cholesterol, phospholipids, diacylglycerol and triacylglycerol, fatty acids, carbohydrates (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some examples, the binding moiety is an antibody or a binding fragment thereof. In some examples, the polynucleic acid molecule is further conjugated to a polymer, and optionally to an endosomolytic moiety.

[0168] In some embodiments, the polynucleic acid molecule is conjugated to the binding moiety by a chemical ligation process. In some examples, the polynucleic acid molecule is conjugated to the binding moiety by native ligation. In some examples, the conjugation can be carried out using methods such as those described in Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation:Expanded scope by using straightforward methodology.," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleic acid molecule is conjugated to the binding moiety site-specifically or non-specifically via native ligation chemistry.

[0169] In some instances, polynucleic acid molecules are conjugated to binding moieties in a site-directed manner utilizing "traceless" coupling technology (PhiloChem). In some instances, the "traceless" coupling technology utilizes N-terminal 1,2-aminothiol groups of binding moieties that are conjugated to polynucleic acid molecules that contain aldehyde groups. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13): 5887-5892 (2012)).

[0170] In some examples, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an unnatural amino acid introduced into the binding moiety. In some examples, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some examples, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derivative conjugate moiety to form an oxime bond. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40): 16101-16106 (2012)).

[0171] In some examples, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method that utilizes an enzyme-catalyzed process. In some examples, the site-directed method utilizes SMARTag™ technology (Redwood). In some examples, SMARTag™ technology involves the generation of formylglycine (FGly) residues from cysteine ​​by formylglycine generating enzyme (FGE) through an oxidation process in the presence of an aldehyde tag, and the subsequent conjugation of FGly to an alkylhydrazine-functionalized polynucleic acid molecule through hydrazino-Pictet-Spengler (HIPS) ligation. (Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51 (2013)).

[0172] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some examples, the polynucleic acid molecule is conjugated to the binding moiety using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some examples, mTG is produced from Streptomyces mobarensis (see Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013)).

[0173] In some examples, the polynucleic acid molecules are conjugated to the binding moiety by the methods described in PCT Publication No. WO2014 / 140317, which utilize sequence-specific transpeptidases.

[0174] In some examples, the polynucleic acid molecules are conjugated to binding moieties by methods such as those described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0175] joining part In some embodiments, binding moiety A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a human antibody or binding fragment thereof, an anti-murine antibody (e.g., an anti-mouse antibody, an anti-rat antibody, etc.), an anti-human antibody, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , F(ab)' 3Fragment, single chain variable fragment (scFv), bis-scFv, (scFv) 2 , diabodies, minibodies, nanobodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv), single domain antibodies (sdAbs), Ig NARs, camelid antibodies or binding fragments thereof, bispecific antibodies or binding fragments thereof, or chemically modified derivatives thereof.

[0176] In some examples, A is an antibody or binding fragment thereof. In some examples, A is a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , F(ab)' 3 Fragment, single chain variable fragment (scFv), bis-scFv, (scFv) 2 , a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some examples, A is a humanized antibody or binding fragment thereof. In some examples, A is a murine antibody or binding fragment thereof. In some examples, A is a chimeric antibody or binding fragment thereof. In some examples, A is a monoclonal antibody or binding fragment thereof. In some examples, A is a full size antibody. In some examples, A is a monovalent Fab'. In some examples, A is a bivalent Fab 2 In some examples, A is a single chain variable fragment (scFv).

[0177] In some embodiments, binding moiety A is a bispecific antibody or a binding fragment thereof. In some examples, the bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, the bispecific antibody is a trifunctional antibody. In some examples, the trifunctional antibody is a full-length monoclonal antibody that contains binding sites for two different antigens. Exemplary trifunctional antibodies include catumaxomab (targeting EpCAM and CD3; Fresenius Biotech / Trion Pharma), ertumaxomab (targeting HER2 / neu / CD3; Fresenius Biotech / Trion Pharm), lymphomun FBTA05 (targeting CD20 / CD3; Fresenius Biotech / Trion Pharm), and erythromycin (targeting HER2 / neu / CD3; Fresenius Biotech / Trion Pharm). Pharm), RG7221 (RO5520985; targets Angiopoietin 2 / VEGF; Roche), RG7597 (targets Her1 / Her3; Genentech / Roche), MM141 (targets IGF1R / Her3; Merrimack), ABT122 (targets TNFα / IL17; Abbvie), ABT981 (targets IL1α / IL1β; Abbott), LY3164530 (targets Her1 / cMET; Eli Lilly), and TRBS07 (Ektomab; targets GD2 / CD3; Trion Research Gmbh). Further exemplary trifunctional antibodies include the mAb from F-star Biotechnology Ltd. 2In some examples, A is a bispecific trifunctional antibody. In some embodiments, A is selected from the group consisting of catumaxomab (targets EpCAM and CD3; Fresenius Biotech / Trion Pharma), ertumaxomab (targets HER2 / neu / CD3; Fresenius Biotech / Trion Pharm), lymphomun FBTA05 (targets CD20 / CD3; Fresenius Biotech / Trion Pharm), RG7221 (RO5520985; angiopoietin 2 / VEGF; Roche), RG7597 (targeting Her1 / Her3; Genentech / Roche), MM141 (targeting IGF1R / Her3; Merrimack), ABT122 (targeting TNFα / IL17; Abbvie), ABT981 (targeting IL1α / IL1β; Abbott), LY3164530 (targeting Her1 / cMET; Eli Lilly), TRBS07 (Ektomab; targeting GD2 / CD3; Trion Research Gmbh), and mAbs from F-star Biotechnology Ltd. 2 A bispecific trifunctional antibody selected from the group consisting of

[0178] In some cases, the bispecific antibody is a bispecific miniantibody. In some examples, the bispecific miniantibody is a bivalent Fab 2 , F(ab)' 3 Fragment, bis-scFv, (scFv) 2, diabodies, minibodies, triabodies, tetrabodies, or bispecific T cell engagers (BiTEs). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single chain variable fragments (scFvs), where the two scFvs target epitopes of two different antigens. Exemplary bispecific miniantibodies include, but are not limited to, DART (Dual Affinity Retargeting Platform; MacroGenics), blinatumomab (MT103 or AMG103; targeting CD19 / CD3; Micromet), MT111 (targeting CEA / CD3; Micromet / Amegen), MT112 (BAY2010112; targeting PSMA / CD3; Micromet / Bayer), MT110 (AMG These include 110 (targeting EPCAM / CD3; Amgen / Micromet), MGD006 (targeting CD123 / CD3; MacroGenics), MGD007 (targeting GPA33 / CD3; MacroGenics), BI1034020 (targeting two different epitopes on β-amyloid; Ablynx), ALX0761 (targeting IL17A / IL17F; Ablynx), TF2 (targeting CEA / Heptene; Immunomedics), IL-17 / IL-34 biAb (BMS), AFM13 (targeting CD30 / CD16; Affimed), AFM11 (targeting CD19 / CD3; Affimed), and domain antibodies (dAbs from Domantis / GSK).

[0179] In some embodiments, binding moiety A is a bispecific miniantibody. In some examples, A is a bispecific Fab 2 In some examples, A is a bispecific F(ab)' 3 Optionally, A is a bispecific bis-scFv. Optionally, A is a bispecific (scFv) 2In some embodiments, A is a bispecific diabody. In some embodiments, A is a bispecific minibody. In some embodiments, A is a bispecific triabody. In other embodiments, A is a bispecific tetrabody. In other embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). In further embodiments, A is a bispecific T cell engager (BiTE). The bispecific mini-antibodies selected from: 110 (targeting EPCAM / CD3; Amgen / Micromet), MGD006 (targeting CD123 / CD3; MacroGenics), MGD007 (targeting GPA33 / CD3; MacroGenics), BI1034020 (targeting two different epitopes on β-amyloid; Ablynx), ALX0761 (targeting IL17A / IL17F; Ablynx), TF2 (targeting CEA / Heptene; Immunomedics), IL-17 / IL-34 biAb (BMS), AFM13 (targeting CD30 / CD16; Affimed), AFM11 (targeting CD19 / CD3; Affimed), and domain antibodies (dAbs from Domantis / GSK).

[0180] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody is F(ab)' 3 In some instances, A comprises a trispecific F(ab)' fragment or triabody. 3In some embodiments, A is a triabody. In some embodiments, A is a trispecific antibody as described in Dimas, et al., "Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells," Mol. Pharmaceutics, 12(9): 3490-3501 (2015).

[0181] In some embodiments, binding moiety A is an antibody or binding fragment thereof that recognizes a cell surface protein. In some examples, the cell surface protein is an antigen expressed by a cancer cell. Exemplary cancer antigens include, but are not limited to, alpha fetoprotein, ASLG659, B7-H3, BAFF-R, brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, S PAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB (β subunit of MHC class II molecule (Ia antigen)), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate-specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, p97, purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (lymphocyte antigen 64 (RP105), gp100, P21, six transmembrane epithelial antigen of prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72), transferrin receptor, TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), etc. In some examples, the binding moiety is an α-transferrin receptor antibody or binding fragment thereof.In some examples, the binding moiety is an alpha-human transferrin receptor antibody. In some examples, the binding moiety is an alpha-human transferrin receptor antibody, such as those described in PCT / US2019 / 068078, which is incorporated herein by reference.

[0182] In some instances, the cell surface proteins include cluster of differentiation (CD) cell surface markers. Exemplary CD cell surface markers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD 49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, C D61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD7 1, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), etc.

[0183] In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a cancer antigen. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a cancer antigen. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a cancer antigen. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a cancer antigen. AP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB (β subunit of MHC class II molecule (Ia antigen)), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, immunoglobulin superfamily receptor translocation-associated 2 (IRTA2), L6, Lewis Y, Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MART1, mesothelin, MCP1 (CCL2), MDP, macrophage migration inhibitory factor (MIF), MPF (SMR, MSLN), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate-specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, p97, purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (lymphocyte antigen 96 (RP105), gp100, P21, six transmembrane epithelial antigen of the prostate (STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), or a combination thereof, or a binding fragment thereof.

[0184] In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD 43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49 f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g., CD79a, CD79b), CD 90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), or a combination thereof, or a binding fragment thereof.

[0185] In some embodiments, the antibody or binding fragment thereof is selected from the group consisting of zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), avituzumab (Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®, MabCampath or Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), asculinbacumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech / Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics), cetuximab (Erbitux®, ImClone), sitatuzumab vogatox (VB6-845), cizutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab (MAb171A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek Inc.), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), framvotumab (ImClone Systems), frezolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), direntuximab (Rencarex®, Wilex AG), IMAB362 (claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImClone Systems Inc.), imgatuzumab (Genentech / Roche), intetumumab (Centocor), ipilimumab (Yervoy®, Bristol-Myers Squibb), Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), rumiliximab, mapatuzumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek Inc.), oregovomab (OvaRex® (AltaRex, Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotopex GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech / Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumumab (Schering-Plough), seribantomab (Sanofi / Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab-cell moleukin (EMD These include ublituximab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), and zatuximab.

[0186] In some embodiments, binding moiety A is selected from the group consisting of zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®, MabCampath or Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), asculinbacumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech / Roche), B43.13 (OvaRex, AltaRex Corporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics), cetuximab (Erbitux®, ImClone), sitatuzumab vogatox (VB6-845), cizutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab (MAb171A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek Inc.), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), framvotumab (ImClone Systems), frezolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), direntuximab (Rencarex®, Wilex AG), IMAB362 (claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImClone Systems Inc.), imgatuzumab (Genentech / Roche), intetumumab (Centocor), ipilimumab (Yervoy®, Bristol-Myers Squibb), Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), rumiliximab, mapatuzumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher;. Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratu-zumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek Inc.), oregovomab (OvaRex® (AltaRex, Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech / Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumumab (Schering-Plough), seribantomab (Sanofi / Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), or zatuximab. In some embodiments, binding moiety A is zalutumumab (HuMax-EFGr by Genmab).

[0187] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. In some examples, the plasma protein comprises albumin. In some examples, the binding moiety A is albumin. In some examples, the albumin is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some examples, the albumin is conjugated to the polynucleic acid molecule by native ligation chemistry. In some examples, the albumin is conjugated to the polynucleic acid molecule by lysine bond.

[0188] In some examples, the binding moiety A is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (saturated, unsaturated, substituted, or combinations thereof). In some examples, the steroid is cholesterol. In some examples, the binding moiety is cholesterol. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by native ligation chemistry. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by lysine conjugation.

[0189] In some examples, the binding moiety is a polymer, including but not limited to a polynucleic acid molecule aptamer, that binds to a specific surface marker on a cell. In this example, the binding moiety is a polynucleic acid that does not hybridize to a target gene or mRNA, but instead can selectively bind to the cell surface marker, similar to an antibody that binds to that specific epitope of the cell surface marker.

[0190] In some cases, the binding moiety A is a peptide. In some cases, the peptide comprises about 1 to about 3 kDa. In some cases, the peptide comprises about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some examples, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some examples, the peptide moiety is a bicyclic peptide (e.g., Bicycle from Bicycle Therapeutics). In further cases, the binding moiety is a small molecule. In some examples, the small molecule is an antibody-recruiting small molecule. In some cases, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, the target-binding end being capable of recognizing and interacting with a cell surface receptor. For example, in some examples, the target-binding end comprising a glutamic acid urea compound allows for interaction with PSMA, thereby enhancing antibody interaction with cells expressing PSMA (e.g., cancer cells). In some examples, the binding moiety is a small molecule described in Zhang et al., "A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules," J Am Chem Soc. 132(36): 12711-12716 (2010); or McEnaney, et al., "Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease," ACS Chem Biol. 7(7):1139-1151 (2012).

[0191] Polynucleic acid molecular target In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule (or polynucleotide) that hybridizes to a target region on an oncogene. In some examples, oncogenes are further classified into several categories: growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, and transcription factors. Exemplary growth factors include c-Sis. Exemplary receptor tyrosine kinases include epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and HER2 / neu. Exemplary cytoplasmic tyrosine kinases include the Src family tyrosine kinases, the Syk-ZAP-70 family of tyrosine kinases, the BTK family of tyrosine kinases, and the Abl gene in CML. Exemplary cytoplasmic serine / threonine kinases include Raf kinases and cyclin-dependent kinases. Exemplary regulatory GTPases include the Ras family of proteins, such as KRAS. Exemplary transcription factors include the MYC gene. In some examples, the oncogenes described herein include oncogenes selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, or transcription factors. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of an oncogene selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, or transcription factors.

[0192] In some embodiments, the oncogenes described herein are Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2,3,6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1 , LMO2, LYL1, MAS1, MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11 / CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51 B), including PAX5, PIM1, PRAD-1, RAF, RAR / PML, HRAS, KRAS, NRAS, REL / NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2. In some embodiments, the polynucleic acid molecule is selected from the group consisting of Abl, AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2,3,6, BRAF, c-MYC, EGFR, ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS 1, MDM2, MET, MLL (KMT2A), MOS, MYB, MYH11 / CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B), PAX5, PIM1, PRAD-1, RAF, RAR / PML, HRAS, KRAS, NRAS, REL / NRG, RET, ROS, SKI, SRC, TIAM1, or TSC2.

[0193] In some embodiments, the oncogenes described herein include KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related genes. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to the target region of KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related genes. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to the target region of KRAS. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to the target region of EGFR. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to the target region of AR. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to the target region of CNNTB1 (β-catenin). In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to the target region of CNNTB1 (β-catenin)-related genes. In some examples, the β-catenin related genes include PIK3CA, PIK3CB, and Myc. In some examples, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of HPRT1.

[0194] In some embodiments, the binding moiety A is conjugated to a polynucleic acid molecule (B) and optionally a polymer (C) as described herein according to formula (I). In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 142-255, 256-369, 481-591, 592-702, 3407-14222, 14222-14238, or 14239-14352. In some examples, the polynucleic acid molecule comprises a sequence selected from SEQ ID NOs: 142-255, 256-369, 481-591, 592-702, 3407-14222, 14222-14238, or 14239-14352. In some examples, polymer C optionally comprises a polyalkylene oxide (e.g., polyethylene glycol).

[0195] In some embodiments, binding moiety A is conjugated to a polynucleic acid molecule (B) and optionally a polymer (C). In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0196] In some embodiments, binding moiety A is conjugated non-specifically to polynucleic acid molecule (B). In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site specific manner via a lysine residue or a cysteine ​​residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site specific manner via a lysine residue. In some cases, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site specific manner via a cysteine ​​residue. In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0197] In some embodiments, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner via a lysine residue, a cysteine ​​residue, at the 5' end, at the 3' end, at an unnatural amino acid, or at an enzyme-modified or enzyme-catalyzed residue. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner via a lysine residue. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner via a cysteine ​​residue. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner at the 5' end. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner at the 3' end. In some examples, the binding moiety A is conjugated to the polynucleic acid molecule (B) in a site-specific manner via an unnatural amino acid. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) via an enzyme-modified or enzyme-catalyzed residue in a site-specific manner. In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0198] In some embodiments, one or more regions of binding moiety A (e.g., an antibody or binding fragment thereof) are conjugated to polynucleic acid molecule (B). In some examples, one or more regions of binding moiety A include N-terminal, C-terminal, within the constant region, to the hinge region, or to the Fc region of binding moiety A. In some examples, polynucleic acid molecule (B) is conjugated to the N-terminus of binding moiety A (e.g., the N-terminus of an antibody or binding fragment thereof). In some examples, polynucleic acid molecule (B) is conjugated to the C-terminus of binding moiety A (e.g., the N-terminus of an antibody or binding fragment thereof). In some examples, polynucleic acid molecule (B) is conjugated to the constant region of binding moiety A (e.g., the constant region of an antibody or binding fragment thereof). In some examples, polynucleic acid molecule (B) is conjugated to the hinge region of binding moiety A (e.g., the constant region of an antibody or binding fragment thereof). In some examples, polynucleic acid molecule (B) is conjugated to the Fc region of binding moiety A (e.g., the constant region of an antibody or binding fragment thereof).

[0199] In some embodiments, one or more polynucleic acid molecules (B) are conjugated to a binding moiety A. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 1 polynucleic acid molecule is conjugated to one binding moiety A. In some examples, about 2 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 3 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 4 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 5 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 6 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 7 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 8 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 9 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 10 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 11 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 12 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 13 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 14 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 15 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 16 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, one or more polynucleic acid molecules are the same. In other examples, one or more polynucleic acid molecules are different. In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0200] In some embodiments, the number of polynucleic acid molecules (B) conjugated to binding moiety A (e.g., an antibody or binding fragment thereof) forms a ratio. In some examples, the ratio is referred to as a DAR (drug-to-antibody) ratio, and the drug as referred to herein is the polynucleic acid molecule (B). In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 2 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 3 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 4 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 5 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 6 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 7 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 8 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 9 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 10 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 11 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 12 or greater.

[0201] In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A (e.g., an antibody or binding fragment thereof) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 2. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 3. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 4. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 5. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 6. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 7. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 8. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 9. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 10. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 11. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 12. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 13. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 14. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 15. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 16.

[0202] In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 1. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 2. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 4. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 6. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 8. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 12.

[0203] In some embodiments, the antibody or binding fragment thereof is further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, alone or in combination, and / or by recombination and / or other modifications known in the art (e.g., post-translational modifications such as glycosylation and phosphorylation and chemical modifications). In some examples, the modifications further include modifications to modulate interaction with Fc receptors. In some examples, one or more modifications include, for example, those described in International Publication No. WO97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence underlying the amino acid sequence of the antibody or binding fragment thereof are well known to those skilled in the art.

[0204] In some instances, an antibody binding fragment further includes derivatives thereof and comprises a polypeptide sequence comprising at least one CDR.

[0205] In some instances, the term "single-chain" as used herein means that the first and second domains of a bispecific single-chain construct are covalently linked, preferably in the form of a co-linear amino acid sequence that can be encoded by a single nucleic acid molecule.

[0206] In some examples, the bispecific single chain antibody construct relates to a construct comprising binding domains from two antibodies. In such an embodiment, the bispecific single chain antibody construct is a tandem bi-scFv or diabody. In some examples, the scFv comprises a VH and a VL domain connected by a linker peptide. In some examples, the linker is of sufficient length and sequence to allow each of the first and second domains to retain their differential binding specificity independently of each other.

[0207] In some embodiments, as used herein, binding with or interacting with defines the binding / interaction of at least two antigen interaction sites with each other. In some examples, the antigen interaction sites define a motif of a polypeptide that exhibits a capacity for specific interaction with a specific antigen or a specific group of antigens. In some cases, the binding / interaction is also understood to define specific recognition. In such cases, specific recognition refers to the ability of an antibody or a binding fragment thereof to specifically interact and / or bind to at least two amino acids of each of the target molecule. For example, specific recognition relates to the specificity of an antibody molecule or its ability to distinguish a specific region of a target molecule. In additional examples, the specific interaction of the antigen interaction site with a specific antigen results in the initiation of a signal, for example, by inducing a conformational change in the antigen, oligomerization of the antigen, etc. In further embodiments, the binding is exemplified by the specificity of the "key-lock-principle". Thus, in some instances, specific motifs in the amino acid sequence of the antigen interaction site and the antigen bind to each other as a result of their primary, secondary, or tertiary structure, as well as as a result of secondary modifications of said structure. In such cases, the specific interaction of the antigen interaction site with its specific antigen causes facile binding of the site to the antigen.

[0208] In some examples, specific interaction also refers to reduced cross-reactivity of an antibody or its binding fragment, or reduced off-target effects.For example, an antibody or its binding fragment that binds to a polypeptide / protein of interest, but does not or does not essentially bind to any other polypeptide, is considered to be specific for the polypeptide / protein of interest.Examples of specific interaction of an antigen interaction site with a specific antigen include the specificity of a ligand with its receptor, for example, the interaction of an antigenic determinant (epitope) with an antigen binding site of an antibody.

[0209] Production of antibodies or binding fragments thereof In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced using any method known in the art to aid in the synthesis of polypeptides (e.g., antibodies), inter alia, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0210] In some examples, antibodies or binding fragments thereof are recombinantly expressed and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesis of overlapping oligonucleotides containing portions of the antibody encoding sequence, annealing and ligation of those oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0211] Alternatively, nucleic acid molecules encoding antibodies are optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0212] In some examples, the antibody or binding thereof is optionally produced by immunizing an animal such as a rabbit to produce polyclonal antibodies, or, more preferably, by producing monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72), or by Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of the antibody are optionally obtained by screening a Fab expression library (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of FAb fragments that bind to a specific antigen, or by screening an antibody library (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0213] In some embodiments, techniques developed for the production of "chimeric antibodies" by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.

[0214] In some embodiments, techniques described for the production of single chain antibodies (US Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region by an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0215] In some embodiments, an expression vector containing the antibody nucleotide sequence or the antibody nucleotide sequence is introduced into a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0216] In some embodiments, a variety of host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent not only the vehicles in which the antibody coding sequences are produced and subsequently purified, but also cells which, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or binding fragments thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody or binding fragment coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus (CaMV) and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or binding fragment coding sequence; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0217] For long-term and high-yield production of recombinant proteins, stable expression is preferred. In some examples, cell lines that stably express the antibody are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the foreign DNA, engineered cells are grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow to form foci that are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or binding fragments thereof.

[0218] In some examples, a number of selection systems are used including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes utilized in tk cells, hgprt cells, or aprt cells, respectively. Similarly, antimetabolite resistance has been used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods generally known in the art of recombinant DNA technology which may be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0219] In some instances, antibody expression levels are increased by vector amplification (for review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is linked to the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0220] In some examples, any method for purification of antibodies known in the art is used, for example, by chromatography (e.g., ion exchange, affinity, especially affinity to a specific antigen followed by Protein A and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purification of proteins.

[0221] Polymer conjugate moiety In some embodiments, the polymer moiety C is further conjugated to a polynucleic acid molecule described herein, a binding moiety described herein, or a combination thereof. In some examples, the polymer moiety C is conjugated to a polynucleic acid molecule. In some cases, the polymer moiety C is conjugated to a binding moiety. In other cases, the polymer moiety C is conjugated to a polynucleic acid molecule binding moiety molecule. In further cases, the polymer moiety C is conjugated as discussed in the therapeutic molecular platform section.

[0222] In some examples, the polymer moiety C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or cross-linked networks of monomers in two or three dimensions. In some examples, the polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C includes, but is not limited to, alpha-dihydroxyl polyethylene glycol, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, a mixture refers to the use of various polymers within the same compound, as in the context of a block copolymer. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some examples, polymer portion C includes a polyalkylene oxide. In some examples, polymer portion C includes a PEG. In some examples, polymer portion C includes a polyethyleneimide (PEI) or a hydroxyethyl starch (HES).

[0223] In some examples, C is a PEG moiety. In some examples, the PEG moiety is conjugated to the 5' end of the polynucleic acid molecule, while the linking moiety is conjugated to the 3' end of the polynucleic acid molecule. In some examples, the PEG moiety is conjugated to the 3' end of the polynucleic acid molecule, while the linking moiety is conjugated to the 5' end of the polynucleic acid molecule. In some examples, the PEG moiety is conjugated to an internal site of the polynucleic acid molecule. In some examples, the PEG moiety, the linking moiety, or a combination thereof, is conjugated to an internal site of the polynucleic acid molecule. In some examples, the conjugation is direct conjugation. In some examples, the conjugation is via natural ligation.

[0224] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some examples, polydisperse materials contain a disperse distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some examples, monodisperse PEG contains molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight given represents the average molecular weight of the polyalkylene oxide, e.g., PEG molecules.

[0225] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 83 00, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0226] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, , 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 290 The C has a molecular weight of 0, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some examples, the molecular weight of C is about 200 Da. In some examples, the molecular weight of C is about 300 Da. In some examples, the molecular weight of C is about 400 Da. In some examples, the molecular weight of C is about 500 Da. In some examples, the molecular weight of C is about 600 Da. In some examples, the molecular weight of C is about 700 Da. In some examples, the molecular weight of C is about 800 Da. In some examples, the molecular weight of C is about 900 Da. In some examples, the molecular weight of C is about 1000 Da. In some examples, the molecular weight of C is about 1100 Da. In some examples, the molecular weight of C is about 1200 Da. In some examples, the molecular weight of C is about 1300 Da. In some examples, the molecular weight of C is about 1400 Da. In some examples, the molecular weight of C is about 1450 Da.In some examples, the molecular weight of C is about 1500 Da. In some examples, the molecular weight of C is about 1600 Da. In some examples, the molecular weight of C is about 1700 Da. In some examples, the molecular weight of C is about 1800 Da. In some examples, the molecular weight of C is about 1900 Da. In some examples, the molecular weight of C is about 2000 Da. In some examples, the molecular weight of C is about 2100 Da. In some examples, the molecular weight of C is about 2200 Da. In some examples, the molecular weight of C is about 2300 Da. In some examples, the molecular weight of C is about 2400 Da. In some examples, the molecular weight of C is about 2500 Da. In some examples, the molecular weight of C is about 2600 Da. In some examples, the molecular weight of C is about 2700 Da. In some examples, the molecular weight of C is about 2800 Da. In some examples, the molecular weight of C is about 2900 Da. In some examples, the molecular weight of C is about 3000 Da. In some examples, the molecular weight of C is about 3250 Da. In some examples, the molecular weight of C is about 3350 Da. In some examples, the molecular weight of C is about 3500 Da. In some examples, the molecular weight of C is about 3750 Da. In some examples, the molecular weight of C is about 4000 Da. In some examples, the molecular weight of C is about 4250 Da. In some examples, the molecular weight of C is about 4500 Da. In some examples, the molecular weight of C is about 4600 Da. In some examples, the molecular weight of C is about 4750 Da. In some examples, the molecular weight of C is about 5000 Da. In some examples, the molecular weight of C is about 5500 Da. In some examples, the molecular weight of C is about 6000 Da. In some examples, the molecular weight of C is about 6500 Da. In some examples, the molecular weight of C is about 7000 Da. In some examples, the molecular weight of C is about 7500 Da. In some examples, the molecular weight of C is about 8000 Da. In some examples, the molecular weight of C is about 10,000 Da. In some examples, the molecular weight of C is about 12,000 Da. In some examples, the molecular weight of C is about 20,000 Da. In some examples, the molecular weight of C is about 35,000 Da. In some examples, the molecular weight of C is about 40,000 Da. In some examples, the molecular weight of C is about 50,000 Da.In some examples, the molecular weight of C is about 60,000 Da. In some examples, the molecular weight of C is about 100,000 Da.

[0227] In some embodiments, the polyalkylene oxide (e.g., PEG) is a dispersed PEG, which is a polymeric PEG that contains more than one repeating ethylene oxide unit. In some examples, the dispersed PEG (dPEG) contains 2-60, 2-50, or 2-48 repeating ethylene oxide units. In some examples, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some examples, the dPEG contains about 2 or more repeating ethylene oxide units. In some examples, the dPEG contains about 3 or more repeating ethylene oxide units. In some examples, the dPEG contains about 4 or more repeating ethylene oxide units. In some examples, the dPEG contains about 5 or more repeating ethylene oxide units. In some examples, the dPEG contains about 6 or more repeating ethylene oxide units. In some examples, dPEG contains about 7 or more repeating ethylene oxide units. In some examples, dPEG contains about 8 or more repeating ethylene oxide units. In some examples, dPEG contains about 9 or more repeating ethylene oxide units. In some examples, dPEG contains about 10 or more repeating ethylene oxide units. In some examples, dPEG contains about 11 or more repeating ethylene oxide units. In some examples, dPEG contains about 12 or more repeating ethylene oxide units. In some examples, dPEG contains about 13 or more repeating ethylene oxide units. In some examples, dPEG contains about 14 or more repeating ethylene oxide units. In some examples, dPEG contains about 15 or more repeating ethylene oxide units. In some examples, dPEG contains about 16 or more repeating ethylene oxide units. In some examples, dPEG contains about 17 or more repeating ethylene oxide units. In some examples, dPEG contains about 18 or more repeating ethylene oxide units. In some examples, dPEG contains about 19 or more repeating ethylene oxide units. In some examples, dPEG contains about 20 or more repeating ethylene oxide units. In some examples, dPEG contains about 22 or more repeating ethylene oxide units.In some examples, dPEG comprises about 24 or more repeating ethylene oxide units. In some examples, dPEG comprises about 26 or more repeating ethylene oxide units. In some examples, dPEG comprises about 28 or more repeating ethylene oxide units. In some examples, dPEG comprises about 30 or more repeating ethylene oxide units. In some examples, dPEG comprises about 35 or more repeating ethylene oxide units. In some examples, dPEG comprises about 40 or more repeating ethylene oxide units. In some examples, dPEG comprises about 42 or more repeating ethylene oxide units. In some examples, dPEG comprises about 48 or more repeating ethylene oxide units. In some examples, dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized stepwise from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials as a single molecular weight compound. In some cases, the dPEG has a specific molecular weight rather than an average molecular weight. In some cases, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0228] In some embodiments, the polymer portion C comprises a cationic mucic acid-based polymer (cMAP). In some examples, the cMPA comprises one or more subunits of at least one repeating subunit, the subunit structure being represented as formula (III):

[0229] [ka] wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4-6, or 5, and n is independently at each occurrence 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0230] In some examples, the cMAP is further conjugated to a PEG moiety to produce a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some examples, the PEG moiety ranges from about 500 Da to about 50,000 Da. In some examples, the PEG moiety ranges from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

[0231] In some examples, the polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the polymer moiety C is a cMAP-PEG copolymer. In other cases, the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In further cases, the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0232] Endosomolytic or cell membrane permeabilizing moiety In some embodiments, the formula (Xa):AX 1 -B'-X 2The molecule of -C further comprises an additional conjugate moiety. In some instances, the additional conjugate moiety is an endosomolytic moiety and / or a cell membrane penetrating moiety. In some instances, the endosomolytic moiety is a compound capable of releasing from any of the cell compartments known in the art, such as a cell compartment releasing component, for example, an endosome, a lysosome, an endoplasmic reticulum (ER), a Golgi apparatus, a microtubule, a peroxisome, or other endoplasmic reticulum with a cell. In some instances, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some instances, the endosomolytic moiety comprises an endosomolytic polypeptide. In other instances, the endosomolytic moiety comprises an endosomolytic polymer. In some instances, the cell membrane penetrating moiety comprises a cell membrane penetrating peptide (CPP). In other instances, the cell membrane penetrating moiety comprises a cell membrane penetrating lipid. In other instances, the cell membrane penetrating moiety comprises a cell membrane penetrating small molecule.

[0233] Endosomolytic and cell membrane-permeable polypeptides In some embodiments, the formula (Xa):AX 1 -BX 2The -C molecule is further conjugated to an endosomolytic polypeptide. In some cases, the endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the endosomolytic polypeptide is an amphipathic polypeptide. In additional cases, the endosomolytic polypeptide is a peptidomimetic. In some examples, the endosomolytic polypeptide comprises INF, melittin, mucin, or their respective derivatives. In some examples, the endosomolytic polypeptide comprises INF or a derivative thereof. In other cases, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further cases, the endosomolytic polypeptide comprises mucin or a derivative thereof. In some examples, the endosomolytic polypeptide is Pep-1 (originating from the simian virus 40 large antigen and the NLS of HIV reverse transcriptase), Pvec (originating from VE-cadherin), VT5 (originating from a synthetic peptide), C105Y (originating from 1-antitrypsin), transportan (originating from galanin and mastoparan), TP10 (originating from galanin and mastoparan), MPG (originating from the fusion sequence of HIV gp41 and SV40 originating from the hydrophobic domain from the NLS of T antigen), GH625 (originating from the glycoprotein gH of HSVI type IV), CADY (PPTG1 peptide), GALA (synthetic peptide), INF (influenza HA2 fusion peptide), HA2E5-TAT (influenza HA2 subunit of influenza virus strain X31 fusion peptide), HA2-penetratin (influenza HA2 subunit of influenza virus strain X31 fusion peptide), HA-K4 (influenza HA2 subunit of influenza virus strain X31 fusion peptide), HA2E4 (influenza HA2 subunit of influenza virus strain X31 fusion peptide), H5WYG (HA2 analog), GALA-INF3-(PEG)6-NH (INF3 fusion peptide), or CM18-TAT11 (cecropin-A-melittin) 2-12 (CM 18 ) fusion peptide).

[0234] In some cases, the endosomolytic moiety is selected from the group consisting of Bcl-2 and / or Bcl-x L In some examples, the endosomolytic moiety comprises a Bak BH3 polypeptide described in Albarran, et al., "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive & Functional Polymers 71:261-265 (2011).

[0235] In some examples, the endosomolytic moiety comprises a polypeptide (e.g., a cell membrane-permeable polypeptide) described in PCT Publication WO2013 / 166155 or WO2015 / 069587.

[0236] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the endosomolytic moiety is represented by formula (Xa):AX 1 -B'-X 2The -C molecule is further conjugated to an endosomolytic lipid (eg, a fusogenic lipid). Exemplary fusogenic lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC). In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in PCT Publication WO09 / 126,933.

[0237] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, the endosomolytic moiety is represented by formula (Xa):AX 1 -B'-X 2The -C molecule is further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquines (carnoquines), amopyrroquine, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, the quinoline endosomolytic moiety may include, but is not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline; 4-(4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline. Phosphorus;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino-)quinoline;7-Hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;Hydroxychloroquine phosphate;7-Chloro-4-(4-ethyl-(2-hydroxyethyl)-1-amino-1-butylamino)quinoline (desmethylhydroxychloroquine);7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline 7-Hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-Fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-Hydroxy-4-(1-carboxy 7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline Amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline;1-Acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-Butyryl-1,2,3,4-tetrahydroquinoline;3-Chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde; 1,1'-pentamethylenequinolinium iodide; 8-quinolinol sulfate, and its amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs. In some examples, the endosomolytic moiety is a quinolinol derivative or analog as described by Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Pat. No. 5,736,557. Cell-penetrating polypeptides (CPPs) In some embodiments, the cell membrane penetrating polypeptide comprises a positively charged short peptide having 5-30 amino acids. In some embodiments, the cell membrane penetrating polypeptide comprises an amino acid sequence rich in arginine or lysine. In some embodiments, the cell membrane penetrating polypeptide comprises any polypeptide or combination thereof, including Antennapedia Penetratin (43-58), HIV-1 TAT protein (48-60), pVEC cadherin (615-632), transportangalanin / mastoparan, MPG HIV-gp41 / SV40 T antigen, Pep-1 HIV reverse transcriptase / SV40 T antigen, polyarginine, MAP, R6W3, NLS, 8-lysine, ARF (1-22), and azurin-p28.

[0238] Linker In some embodiments, the linkers described herein are cleavable or non-cleavable linkers. In some examples, the linker is a cleavable linker. In some examples, the linker is an acid cleavable linker. In some examples, the linker is a non-cleavable linker. In some examples, the linker is a C 1 -C 6 Alkyl groups (e.g., C 5 , C 4 , C 3 , C 2 , or C 1 In some examples, the linker includes a homobifunctional cross-linker, a heterobifunctional cross-linker, etc. In some examples, the linker is a traceless linker (or a zero-length linker). In some examples, the linker is a non-polymeric linker. In some cases, the linker is a non-peptide linker or a linker that does not include an amino acid residue.

[0239] In some examples, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyl dithio) thio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0240] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterofunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-crosslinking linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl Succinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysuccinimide ester (sulfo-MBs), N-succinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succini ... Succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl) butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl) butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy) succinimide esters (GMBs), N-(γ-maleimidobutyryloxy) sulfosuccinimide esters (sulfo-GM Bs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA),Carbonyl-reactive and sulfhydryl-reactive cross-linkers, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M, 2 C 2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(ρ- Azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate Sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo -sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as ρ-azidophenylglyoxal (APG).

[0241] In some examples, the linker comprises a reactive functional group. In some cases, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present in the linking moiety. Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0242] In some embodiments, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further includes caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), as described above.

[0243] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination via a retro-Michael reaction. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0244] In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some examples, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some examples, the peptide moiety is a non-cleavable peptide moiety. In some examples, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224), or Gly-Phe-Leu-Gly (SEQ ID NO: 14225). In some examples, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 14223), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 14224), or Gly-Phe-Leu-Gly (SEQ ID NO: 14225). In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

[0245] In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some examples, the benzoic acid group or a derivative thereof comprises para-aminobenzoic acid (PABA). In some examples, the benzoic acid group or a derivative thereof comprises gamma-aminobutyric acid (GABA).

[0246] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises a mc-val-cit-PABA group.

[0247] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some examples, the linker comprises a linker described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0248] In some embodiments, the linker is a dendritic linker. In some examples, the dendritic linker comprises a branched multifunctional linker moiety. In some examples, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some examples, the dendritic linker comprises a PAMAM dendrimer.

[0249] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or a linker group) on the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D after cleavage. Exemplary traceless linkers include, but are not limited to, a germanium linker, a selenium linker, a sulfur linker, a selenium linker, a nitrogen linker, a phosphorus linker, a boron linker, a chromium linker, or a phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497 (2013). In some examples, the linker is a traceless linker as described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem. Rev. 102:2607-2024 (2002). In some examples, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0250] In some examples, the linker includes a functional group that exerts steric hinderance at the attachment site between the linker and the conjugate moiety (e.g., A, B, C, or D described herein). In some examples, the steric hinderance is around a disulfide bond. Exemplary linkers that exhibit steric hinderance include heterobifunctional linkers, such as the heterobifunctional linkers described above. In some cases, linkers that exhibit steric hinderance include SMCC and SPDB.

[0251] In some examples, the linker is an acid-cleavable linker. In some examples, the acid-cleavable linker comprises a hydrazone bond that is susceptible to hydrolytic cleavage. In some examples, the acid-cleavable linker comprises a thiomaleamic acid linker. In some examples, the acid-cleavable linker is a thiomaleamic acid linker as described in "Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation," Chem. Commun. 49:8187-8189 (2013)".

[0252] In some examples, the linker may be any of the compounds described in U.S. Patent Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 2013 / 030 9256; 2015 / 037360; or 2014 / 0294851; or in PCT Publication Nos. WO2015057699; WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

[0253] In some embodiments, X, Y, and L are independently a single bond or a linker. In some examples, X, Y, and L are independently a single bond. In some cases, X, Y, and L are independently a linker.

[0254] In some examples, X is a single bond or a linker. In some examples, X is a single bond. In some examples, X is a linker. In some examples, the linker is C 1 -C 6 In some cases, X is, for example, C 5 , C 4 , C 3 , C 2 , or C1 C such as alkyl group 1 -C 6 It is an alkyl group. In some cases, C 1 -C 6 The alkyl group is unsubstituted C 1 -C 6 It is an alkyl group. When used in the context of a linker, especially in the context of X, alkyl means a saturated straight or branched chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X is a non-polymeric linker. In some examples, X includes a homobifunctional linker or a heterobifunctional linker as described above. In some cases, X includes a heterobifunctional linker. In some cases, X includes sMCC. In other examples, X includes C 1 -C 6 In another embodiment, X is a heterobifunctional linker optionally conjugated to an alkyl group. 1 -C 6 In some additional examples, X does not comprise a homobifunctional linker or a heterobifunctional linker as described above.

[0255] In some examples, Y is a single bond or a linker. In some examples, Y is a single bond. In other cases, Y is a linker. In some embodiments, Y is C 1 -C 6is an alkyl group. In some examples, Y is a homobifunctional linker or a heterobifunctional linker as described above. In some examples, Y is a homobifunctional linker as described above. In some examples, Y is a heterobifunctional linker as described above. In some examples, Y comprises a maleimide group, such as maleimidocaproyl (mc), or a self-stabilizing maleimide group as described above. In some examples, Y comprises a peptide moiety, such as Val-Cit. In some examples, Y comprises a benzoic acid group, such as PABA. In further examples, Y comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In additional examples, Y comprises a mc group. In additional examples, Y comprises a mc-val-cit group. In additional examples, Y comprises a val-cit-PABA group. In additional examples, Y comprises a mc-val-cit-PABA group.

[0256] In some examples, L is a single bond or a linker. In some cases, L is a single bond. In some cases, L is a linker. In some embodiments, L is C 1 -C 6 is an alkyl group. In some examples, L is a homobifunctional linker or a heterobifunctional linker as described above. In some examples, L is a homobifunctional linker as described above. In some examples, L is a heterobifunctional linker as described above. In some examples, L comprises a maleimide group, such as maleimidocaproyl (mc), or a self-stabilizing maleimide group as described above. In some examples, L comprises a peptide moiety, such as Val-Cit. In some examples, L comprises a benzoic acid group, such as PABA. In further examples, L comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In further examples, L comprises a mc group. In further examples, L comprises a mc-val-cit group. In further examples, L comprises a val-cit-PABA group. In further examples, L comprises a mc-val-cit-PABA group.

[0257] How to use In some embodiments, the compositions or pharmaceutical preparations described herein, including the binding moieties conjugated to the polynucleic acid molecules and polymers, are used to treat a disease or disorder. In some examples, the disease or disorder is muscular dystrophy, muscular atrophy, and / or muscular wasting. Muscular dystrophy refers to the loss of muscle mass and / or the progressive weakening and degeneration of muscle. In some cases, the loss of muscle mass and / or the progressive weakening and degeneration of muscle is caused by a high rate of protein degradation, a low rate of protein synthesis, or a combination of both. In some cases, the high rate of muscle protein degradation is caused by muscle protein catabolism (i.e., the breakdown of muscle proteins to use amino acids as substrates for gluconeogenesis). In some examples, the disease or disorder is cancer. In some embodiments, the compositions or pharmaceutical preparations described herein are used as immunotherapy for the treatment of a disease or disorder. In some examples, the immunotherapy is cancer immunotherapy.

[0258] cancer In some embodiments, the compositions or pharmaceutical formulations described herein are used to treat cancer. In some examples, the cancer is a solid tumor. In some examples, the cancer is a hematological malignancy. In some examples, the cancer is a recurrent or refractory cancer, or a metastatic cancer. In some examples, the solid tumor is a recurrent or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematological malignancy is a recurrent or refractory hematological malignancy, or a metastatic hematological malignancy.

[0259] In some embodiments, the cancer is a solid tumor. In some examples, the compositions or pharmaceutical preparations described herein, which include oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat solid tumors. In some examples, the compositions or pharmaceutical preparations described herein, which include oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat anal cancer, appendix cancer, bile duct cancer (i.e., intrahepatic cholangiocarcinoma), bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, cancer of unknown primary (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some examples, the solid tumor is a recurrent or refractory solid tumor, or a metastatic solid tumor.

[0260] In some examples, the cancer is a hematological malignancy. In some examples, the compositions or pharmaceutical preparations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat hematological malignancies. In some examples, the compositions or pharmaceutical preparations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat leukemia, lymphoma, myeloma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma. In some instances, hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and idiopathic ... In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy, or a metastatic hematologic malignancy.

[0261] In some examples, the cancer is KRAS-related, EGFR-related, AR-related, HPRT1-related, or β-catenin-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat KRAS-related, EGFR-related, AR-related, HPRT1-related, or β-catenin-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat KRAS-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat EGFR-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to binding moieties, are used to treat AR-related cancer. In some examples, the compositions or pharmaceutical formulations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to a binding moiety, are used to treat HPRT1-associated cancers. In some examples, the compositions or pharmaceutical formulations described herein, including oligonucleotides, polymers, or combinations thereof, optionally conjugated to a binding moiety, are used to treat β-catenin-associated cancers. In some examples, the cancer is a solid tumor. In some examples, the cancer is a hematological malignancy. In some examples, the solid tumor is a recurrent or refractory solid tumor, or a metastatic solid tumor. In some cases, the hematological malignancy is a recurrent or refractory hematological malignancy, or a metastatic hematological malignancy. In some examples, the cancer comprises bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, acute myeloid leukemia, CLL, DLBCL, or multiple myeloma. In some examples, the β-catenin associated cancer further includes a PIK3C associated cancer and / or a MYC associated cancer.

[0262] immunotherapy In some embodiments, the compositions or pharmaceutical formulations described herein are used as immunotherapy for the treatment of diseases or disorders. In some examples, the immunotherapy is cancer immunotherapy. In some examples, cancer immunotherapy is classified as active, passive, or combined (active and passive) methods. In active cancer immunotherapy, for example, tumor-associated antigens (TAA) are presented to the immune system, triggering an attack on cancer cells presenting these TAAs. In some examples, active cancer immunotherapy includes tumor targeting and / or immune targeting agents (e.g., checkpoint inhibitors such as monoclonal antibodies), and / or vaccines, for example, in situ vaccination and / or cell-based or non-cell-based (e.g., dendritic cell-based, tumor cell-based, antigen, anti-idiotype, DNA, or vector-based) vaccines. In some examples, cell-based vaccines are vaccines generated using activated immune cells obtained from the patient's own immune system and then activated by the patient's own cancer. In some examples, active cancer immunotherapy is further subdivided into non-specific active immunotherapy and specific active immunotherapy. In some instances, non-specific active immunotherapy utilizes cytokines and / or other cell signaling components to induce a general immune system response. In some cases, specific active immunotherapy utilizes specific TAAs to induce an immune response.

[0263] In some embodiments, the compositions or pharmaceutical preparations described herein are used as active cancer immunotherapy for the treatment of a disease or disorder (e.g., cancer). In some embodiments, the compositions or pharmaceutical preparations described herein include a tumor targeting agent. In some examples, the tumor targeting agent is encompassed by the binding moiety A. In other examples, the tumor targeting agent is an additional agent used in combination with the molecule of formula (I). In some examples, the tumor targeting agent is a tumor-directed polypeptide (e.g., a tumor-directed antibody). In some examples, the tumor targeting agent is a tumor-directed antibody that exerts its anti-tumor activity by mechanisms such as direct killing (e.g., signal transduction-induced apoptosis), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In further examples, the tumor targeting agent induces an adaptive immune response involving the induction of anti-tumor T cells.

[0264] In some embodiments, binding moiety A is a tumor-targeting polypeptide (e.g., a tumor-targeting antibody). In some examples, binding moiety A is a tumor-targeting antibody that exerts its anti-tumor activity by mechanisms such as direct killing (e.g., signaling-induced apoptosis), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In further examples, binding moiety A induces an adaptive immune response involving the induction of anti-tumor T cells.

[0265] In some embodiments, the compositions or pharmaceutical formulations described herein include an immune targeting agent. In some examples, the immune targeting agent is encompassed by binding moiety A. In other examples, the immune targeting agent is an additional agent used in combination with the molecule of formula (I). In some examples, the immune targeting agent includes a cytokine, a checkpoint inhibitor, or a combination thereof.

[0266] In some embodiments, the immune targeting agent is a checkpoint inhibitor. In some cases, the immune checkpoint molecule is a molecule displayed on the cell surface of CD4 T cells and / or CD8 T cells. Exemplary immune checkpoint molecules include, but are not limited to, programmed death-ligand 1 (PD-L1 (also known as B7-H1, CD274)), programmed death 1 (PD-1), CTLA-4, B7H1, B7H4, OX-40, CD137, CD40, 2B4, IDO1, IDO2, VISTA, CD27, CD28, PD-L2 (B7-DC, CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM, BTLA, KIR, GAL9, TIM3, A2aR, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), ICOS (inducible T cell costimulatory molecule), HAVCR2, CD276, VTCN1, CD70, and CD160.

[0267] In some examples, immune checkpoint inhibitors refer to any molecule that modulates or inhibits the activity of an immune checkpoint molecule. In some examples, immune checkpoint inhibitors include antibodies, antibody derivatives (e.g., FAb fragments, scFv, minibodies, diabodies), antisense oligonucleotides, siRNA, aptamers, or peptides. In some embodiments, Immune checkpoint inhibitors are inhibitors of programmed death-ligand 1 (PD-L (also known as B7-H1, CD274)), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof.

[0268] In some embodiments, exemplary checkpoint inhibitors include:

[0269] PD-L1 inhibitors, such as MPDL3280A (RG7446) from Genentech, anti-mouse PD-L1 antibody clone 10F.9G2 (Cat# BE0101) from BioXcell, anti-PD-L1 monoclonal antibodies MDX-1105 (BMS-936559) and BMS-935559, MSB0010718C, mouse anti-PD-L1 clone 29E.2A3, and MEDI4736 from AstraZeneca;

[0270] PD-L2 inhibitors, e.g., GlaxoSmithKline's AMP-224 (Amplimmune), and rHIgM12B7;

[0271] PD-1 inhibitors, such as anti-mouse PD-1 antibody clone J43 (Cat# BE0033-2) from BioXcell, anti-mouse PD-1 antibody clone RMP1-14 (Cat# BE0146) from BioXcell, mouse anti-PD-1 antibody clone EH12, MK-3475 anti-mouse PD-1 antibody from Merck (Keytruda, pembrolizumab, lambrolizumab), anti-PD-1 antibody from AnaptysBio known as ANB011, antibody MDX-1 106 (ONO-4538), human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106) from Bristol-Myers Squibb, AMP-514 and AMP-224 from AstraZeneca, and pidilizumab (CT-011) from CureTech Ltd;

[0272] CTLA-4 inhibitors, such as Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101), the anti-CTLA4 antibody, clone 9H10 from Millipore, Pfizer's tremelimumab (CP-675,206, ticilimumab), and Abcam's anti-CTLA4 antibody clone BNI3;

[0273] LAG3 inhibitors, such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016, and LAG-3 chimeric antibody A9H12;

[0274] B7-H3 inhibitors, e.g., MGA271;

[0275] KIR inhibitors, e.g., Lirilumab (IPH2101);

[0276] CD137 (41BB) inhibitors, such as urelumab (BMS-663513 Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

[0277] PS inhibitors, such as Bavituximab; as well as inhibitors, such as antibodies or fragments thereof (e.g., monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies), RNAi molecules, or small molecules against TIM3, CD52, CD30, CD20, CD33, CD27, OX40 (CD134), GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[0278] In some embodiments, binding moiety A comprising an immune checkpoint inhibitor is used to treat a disease or disorder (e.g., cancer). In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. 3. Binding moiety A comprising an inhibitor of GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof, is used to treat a disease or disorder (e.g., cancer).

[0279] In some embodiments, the molecule of formula (I) in combination with an immune checkpoint inhibitor is used to treat a disease or disorder (e.g., cancer). In some examples, the immune checkpoint inhibitor is an inhibitor of programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD1 37, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof. In some cases, the molecule of formula (I) is used in combination with ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, MK-3475, or BMS-936559 for the treatment of a disease or disorder (e.g., cancer).

[0280] In some embodiments, the immune targeting agent is a cytokine. In some cases, cytokines are further subgrouped into chemokines, interferons, interleukins, and tumor necrosis factors. In some embodiments, chemokines act as chemoattractants that guide cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include the CC subfamily: CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (alternatively, CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL 27, and CCL28; CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; XC subfamily: XCL1 and XCL2; and chemokines from the CX3C subfamily; CX3CL1.

[0281] Interferons (IFNs) include type I interferons (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), type II interferons (e.g., IFN-γ), and type III interferons. In some embodiments, IFN-α is further classified into about 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0282] Interleukins are expressed by white blood cells or leukocytes and promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Exemplary interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36.

[0283] Tumor necrosis factors (TNFs) are a group of cytokines that regulate apoptosis. In some instances, there are about 19 members within the TNF family, including, but not limited to, TNFα, lymphotoxin-α (LT-α), lymphotoxin-β (LT-β), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-related apoptosis-inducing ligand (TRAIL).

[0284] In some embodiments, a molecule of formula (I) in combination with a cytokine is used to treat a disease or disorder (e.g., cancer). In some cases, a molecule of formula (I) in combination with a chemokine is used to treat a disease or disorder (e.g., cancer). In some cases, a molecule of formula (I) in combination with an interferon is used to treat a disease or disorder (e.g., cancer). In some cases, a molecule of formula (I) in combination with an interleukin is used to treat a disease or disorder (e.g., cancer). In some cases, a molecule of formula (I) in combination with a tumor necrosis factor is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of formula (I) in combination with IL-1β, IL-2, IL-7, IL-8, IL15, MCP-1 (CCL2), MIP-1α, RANTES, MCP-3, MIP5, CCL19, CCL21, CXCL2, CXCL9, CXCL10, or CXCL11 is used to treat a disease or disorder (e.g., cancer).

[0285] In some embodiments, the compositions or pharmaceutical formulations described herein include a vaccine. In some examples, the vaccine is an in situ vaccination. In some examples, the vaccine is a cell-based vaccine. In some examples, the vaccine is a non-cell-based vaccine. In some examples, the molecule of formula (I) in combination with a dendritic cell-based vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, the molecule of formula (I) in combination with a tumor cell-based vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, the molecule of formula (I) in combination with an antigen vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, the molecule of formula (I) in combination with an anti-idiotype vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, the molecule of formula (I) in combination with a DNA vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, the molecule of formula (I) in combination with a vector-based vaccine is used to treat a disease or disorder (e.g., cancer).

[0286] In some embodiments, the compositions or pharmaceutical preparations described herein are used as passive cancer immunotherapy for the treatment of a disease or disorder (e.g., cancer). In some examples, passive methods utilize adaptive immune system components, such as exogenously generated T cells, natural killer (NK) T cells, and / or chimeric antigen receptor (CAR) T cells, to attack cancer cells.

[0287] In some embodiments, the molecule of formula (I) combined with a T cell-based therapeutic agent is used to treat a disease or disorder (e.g., cancer). In some cases, the T cell-based therapeutic agent is an activating T cell agent that recognizes one or more of the CD cell surface markers described above. In some examples, the T cell-based therapeutic agent includes an activating T cell agent that recognizes one or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357. In some examples, a molecule of formula (I) in combination with an activating T cell agent that recognizes one or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274, CD278, CD279, or CD357 is used to treat a disease or disorder (e.g., cancer).

[0288] In some embodiments, the molecules of formula (I) in combination with natural killer (NK) T cell-based therapeutics are used to treat diseases or disorders (e.g., cancer). In some examples, the NK-based therapeutics are activating NK agents that recognize one or more of the CD cell surface markers described above. In some cases, the NK-based therapeutics are activating NK agents that recognize one or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a / b, CD158c, CD158e / f / k, CD158h / j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319. In some examples, a molecule of formula (I) in combination with an activating NK agent that recognizes one or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a / b, CD158c, CD158e / f / k, CD158h / j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319 is used to treat a disease or disorder (e.g., cancer).

[0289] In some embodiments, molecules of Formula (I) in combination with CAR-T cell-based therapeutics are used to treat a disease or disorder (e.g., cancer).

[0290] In some embodiments, the molecule of formula (I) is combined with an additional agent that destabilizes endosomal membrane (or interferes with endosomal-lysosomal membrane trafficking) to treat a disease or disorder (e.g., cancer). In some embodiments, the additional agent comprises a mitotic inhibitor. Exemplary mitotic inhibitors include, but are not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine; cabazitaxel; colchicine; eribulin; estramustine; etoposide; ixabepilone; podophyllotoxin; teniposide; or griseofulvin. In some examples, the additional agent comprises paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, cabazitaxel, colchicine, eribulin, estramustine, etoposide, ixabepilone, podophyllotoxin, teniposide, or griseofulvin. In some examples, the additional agent comprises taxol. In some examples, the additional agent comprises paclitaxel. In some examples, the additional agent comprises etoposide. In other examples, the additional agent comprises vitamin K3.

[0291] In some embodiments, the compositions or pharmaceutical formulations described herein are used as combination methods (including both active and passive methods) in the treatment of a disease or disorder (e.g., cancer).

[0292] Muscle dystrophy, muscle atrophy, muscle wasting In one embodiment, muscular dystrophy refers to a significant decrease in muscle strength. A significant decrease in muscle strength refers to a decrease in the strength of diseased, damaged or unused muscle tissue of a subject, compared to the same muscle tissue of a control subject. In an embodiment, a significant decrease in muscle strength refers to a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the same muscle tissue of a control subject. In another embodiment, a significant decrease in muscle strength refers to a decrease in the strength of unused muscle tissue, compared to the muscle strength of the same muscle tissue of the same subject before a period of disuse. In an embodiment, a significant decrease in muscle strength refers to a decrease in strength of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the muscle strength of the same muscle tissue of the same subject before a period of disuse.

[0293] In another embodiment, muscular dystrophy refers to a significant reduction in muscle mass. A significant reduction in muscle mass refers to a reduction in muscle volume in diseased, damaged, or disused muscle tissue of a subject, compared to the same muscle tissue of a control subject. In an embodiment, a significant reduction in muscle volume refers to a reduction in muscle volume in disused muscle tissue, compared to the same muscle tissue of a control subject, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more. In another embodiment, a significant reduction in muscle mass refers to a reduction in muscle volume in disused muscle tissue, compared to the muscle volume in the same muscle tissue of the same subject before a period of disuse. In an embodiment, a significant reduction in muscle tissue refers to a reduction in muscle volume in the same muscle tissue of a disuse subject, compared to the muscle volume in the same muscle tissue of the same subject before a period of disuse. Muscle volume is optionally measured by assessing muscle cross-sectional area, such as by magnetic resonance imaging (eg, by muscle volume / cross-sectional area (CSA) MRI method).

[0294] Myotonic dystrophy is a multisystemic neuromuscular disease that includes two major types, namely myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by a dominantly inherited "CTG" repeat expansion in the gene DM protein kinase (DMPK), which when transcribed into mRNA, forms a hairpin that binds with high affinity to the Muscleblind-like (MBNL) family of proteins. MBNL proteins are involved in the control of post-transcriptional splicing and polyadenylatin sites, and loss of MBNL protein function causes downstream accumulation of nuclear foci and increased mis-splicing events, subsequently resulting in myotonia and other clinical symptoms.

[0295] In some embodiments, methods of treating muscular dystrophy, muscle atrophy, and / or muscle wasting in a subject are described herein, comprising providing a polynucleic acid molecule or a polynucleic acid molecule conjugate described herein, and administering a therapeutically effective amount of the polynucleic acid molecule or polynucleic acid molecule conjugate to the subject to treat muscular dystrophy, muscle atrophy, and / or muscle wasting. In some embodiments, the polynucleic acid molecule or polynucleic acid molecule conjugate targets a gene transcript, which is mutated or upregulated such that downregulation, deletion, exon skipping of the transcript is desired to treat the disease. In some embodiments, the polynucleic acid molecule or polynucleic acid molecule conjugate targets DMPK mRNA, DMD mRNA, or GYS1 mRNA.

[0296] Pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal routes of administration. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet other examples, the pharmaceutical compositions described herein are formulated for intranasal administration.

[0297] In some embodiments, pharmaceutical compositions include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and combined immediate and controlled release formulations.

[0298] In some examples, the pharmaceutical formulation comprises a multiparticulate formulation. In some examples, the pharmaceutical formulation comprises a nanoparticle formulation. In some examples, the nanoparticle comprises cMAP, cyclodextrin, or lipid. In some cases, the nanoparticle comprises a solid lipid nanoparticle, a polymeric nanoparticle, a self-emulsifying nanoparticle, a liposome, a microemulsion, or a micellar solution. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those with covalently bound metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, the nanoparticles are metal nanoparticles, such as nanoparticles of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys or oxides thereof.

[0299] In some instances, the nanoparticles include a core or a core and a shell, such as core-shell nanoparticles.

[0300] In some examples, the nanoparticles are further coated with molecules (e.g., one or more of the polynucleic acid molecules or binding moieties described herein) for attachment of functional elements. In some examples, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyldextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (alternatively, chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histones, protamine, ovalbumin, dextrin, or cyclodextrin. In some examples, the nanoparticles comprise graphene-coated nanoparticles.

[0301] In some cases, the nanoparticles have at least one dimension less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

[0302] In some examples, the nanoparticle formulation comprises a paramagnetic nanoparticle, a superparamagnetic nanoparticle, a metal nanoparticle, a fullerene-like material, an inorganic nanotube, a dendrimer (such as one with a covalently bound metal chelate), a nanofiber, a nanohorn, a nanoonion, a nanorod, a nanorope, or a quantum dot. In some examples, the polynucleic acid molecule or binding moiety described herein is directly or indirectly conjugated to the nanoparticle. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticle.

[0303] In some embodiments, the pharmaceutical formulation comprises a carrier or carrier material selected based on compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, humectants, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0304] In some examples, the pharmaceutical preparation further comprises a pH adjusting or buffering agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amount necessary to maintain the pH of the composition in an acceptable range.

[0305] In some examples, pharmaceutical preparations contain one or more salts in an amount necessary to make the osmolality of the composition acceptable.Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0306] In some instances, pharmaceutical formulations further include a diluent that is used to stabilize the compound, because the diluent can provide a more stable environment. Salts dissolved in buffers (which can also control or maintain pH) are utilized as diluents in the art, including but not limited to phosphate buffered saline solutions. In some instances, the diluent increases the volume of the composition to facilitate compression or create sufficient volume for homogeneous blending for capsule filling. Such compounds may include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; calcium hydrogen phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0307] In some cases, pharmaceutical formulations contain disintegrants or disintegrants that promote the breakup or disintegration of substances. The term "disintegrate" includes both the disintegration and dispersion of the dosage form when it comes into contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches, such as corn starch or potato starch, pregelatinized starches, such as National 1551 or Amijel®, or sodium starch glycolate, such as Promogel® or Explotab®, cellulose, such as wood products, methyl crystalline cellulose, such as Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Min. Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or crosslinked cellulose, such as crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®, crosslinked carboxymethylcellulose, or crosslinked sodium croscarmellose, crosslinked starch, such as sodium starch glycolate, crosslinked polymers, such as crospovidone, crosslinked polyvinylpyrrolidone, alginates, such as alginic acid, or salts of alginic acid, such as sodium alginate, clays, such as Veegum® HV (magnesium aluminum silicate), gums, such as agar, gaur, carob, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, sponge, surfactants, resins, such as cation exchange resins, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, and the like.

[0308] In some examples, the pharmaceutical formulation comprises a filler such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0309] Lubricants and glidants are optionally included in the pharmaceutical formulations described herein to prevent, reduce, or inhibit adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons, such as mineral oil or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol, such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, Syloid™, colloidal-silica, such as Cab-O-Sil®, starch, such as corn starch, silicone oil, surfactants, and the like.

[0310] Plasticizers include compounds that are used to soften the microencapsulation material or film coating, making them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers may also act as dispersants or wetting agents.

[0311] Solubilizing agents include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide.

[0312] Stabilizing agents include any mixture of antioxidants, buffers, acids, preservatives, and the like.

[0313] Suspending agents include, for example, polyvinylpyrrolidone such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose acetate stearate, poly sorbate 80, hydroxyethylcellulose, sodium alginate, gums such as gum tragacanth and gum arabic, guar gum, xanthans including xanthan gum, sugars, cellulosic compounds such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[0314] Surfactants include compounds such as sodium lauryl sulfate, docusate sodium, or Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, such as Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40. Often surfactants are included to enhance physical stability or for other purposes.

[0315] Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosan, and combinations thereof.

[0316] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, docusate sodium, sodium oleate, sodium lauryl sulfate, docusate sodium, triacetin, Tween 80, Vitamin E TPGS, ammonium salts, and the like.

[0317] Treatment regimen In some embodiments, the pharmaceutical composition described herein is administered for therapeutic use. In some embodiments, the pharmaceutical composition is administered once a day, twice a day, three times a day, or more. The pharmaceutical composition is administered every day, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0318] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at an interval (e.g., a first administration of a first pharmaceutical composition is on day 1, followed by at least 1, 2, 3, 4, 5 or more days before administration of at least a second pharmaceutical composition).

[0319] In some embodiments, two or more different pharmaceutical compositions are administered simultaneously. In some examples, two or more different pharmaceutical compositions are administered simultaneously. In some cases, two or more different pharmaceutical compositions are administered simultaneously in sequence with no interval between administrations. In other cases, two or more different pharmaceutical compositions are administered simultaneously in sequence with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days or more between administrations.

[0320] If the patient's condition improves, at the physician's discretion, administration of the composition may continue or the dose of the composition being administered may be temporarily reduced or discontinued for a specified period of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday may vary between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can range from 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0321] Once the patient's condition has improved, a maintenance dose is administered if necessary, after which the dosage or frequency of administration, or both, are optionally reduced, depending on the symptoms, to a level at which the improved disease, disorder, or condition is maintained.

[0322] In some embodiments, the amount of a given agent that corresponds to such an amount will depend on factors such as the particular compound, the severity of the disease, the nature (e.g., body weight) of the subject or host in need of treatment, but will nevertheless be routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dose is conveniently presented as a single dose, or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example, two, three, four or more sub-doses per day.

[0323] Because of the large number of variables associated with any particular treatment regimen, the foregoing ranges are merely suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on a number of variables, including, but not limited to, the activity of the compound being used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the physician.

[0324] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form used and the route of administration utilized.

[0325] Kits / Products In an embodiment, the present specification discloses kits and products that can be used with one or more compositions and methods described herein.Such kits include a carrier, packaging, or container that is partitioned to accommodate one or more containers, such as vials, tubes, and each container includes one of the separate elements for using the methods described herein.Suitable containers include, for example, bottles, vials, syringes, and test tubes.In one embodiment, containers are formed from various materials, such as glass or plastic.

[0326] The products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, jars, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0327] For example, the container contains a molecule of formula (Xa): AX-B'-YC, as disclosed herein. Such kits optionally include an identifying statement or label, or instructions for its use in the methods described herein.

[0328] The kit typically includes a label listing the contents and / or instructions for use, and a package insert containing the instructions. A set of instructions is also typically included.

[0329] In one embodiment, a label is on or associated with a container. In one embodiment, a label is on a container if letters, numbers, or other indicia forming the label are affixed to, molded into, or engraved into the container itself. A label is associated with a container if the label is present in a receptacle or carrier that holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates how to use the contents, e.g., in the manner described herein.

[0330] In some embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms comprising the compounds provided herein. For example, the pack comprises metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accompanied by a notice attached to the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the drug form for human or animal administration. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved package insert. In one embodiment, compositions comprising the compounds provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for the treatment of an indicated disease.

[0331] Specific Terms Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used within the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0332] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

[0333] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0334] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms are limited to situations characterized by the supervision (e.g., full-time or intermittent) of a health care worker (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker).

[0335] chemical definition The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0336] Where substituents are specified by their conventional chemical formula written from left to right, the substituents equally encompass the chemically identical substituents that result from writing the structure from right to left, e.g., -CH 2 O-OCH 2 - Equal to.

[0337] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, straight-chain (i.e., unbranched) or branched carbon chains (or carbons), or combinations thereof, which may be fully saturated, monounsaturated, and polyunsaturated, and which can include mono-, di-, and polyvalent radicals having the specified number of carbon atoms (i.e., C 1 -C 10means 1 to 10 carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, e.g., homologs and isomers of n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers. Alkoxy is an alkyl that is attached to the remainder of the molecule through an oxygen linker (-O-).

[0338] The term "alkylene," by itself or as part of another substituent, unless otherwise stated, includes, but is not limited to, -CH 2 CH 2 CH 2 CH 2 "Alkyl" refers to a divalent radical derived from an alkyl, as exemplified by -. Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A "lower alkyl" or "lower alkylene" is generally a shorter chain alkyl or alkylene group having eight or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means a divalent radical derived from an alkene, unless otherwise stated.

[0339] The term "heteroalkyl", by itself or in combination with another term, means, unless otherwise specified, a stable straight or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom (e.g., N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is a non-cyclized chain. Examples include, but are not limited to, -CH 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-CH 3 , -CH 2 -S-CH 2 -CH 3 , -CH 2 -CH 2 , -S(O)-CH 3 , -CH 2 -CH 2 -S(O) 2 -CH 3 , -CH=CH-O-CH 3 , -Si(CH 3 ) 3 , -CH 2 -CH=N-OCH 3 , -CH=CH-N(CH 3 )-CH 3 , -O-CH 3 , -O-CH 2 -CH 3 Up to two or three heteroatoms include, for example, -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3The heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include two, optionally different, heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include three, optionally different, heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include four, optionally different, heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include five, optionally different, heteroatoms (e.g., O, N, S, Si, or P). The heteroalkyl moiety may include up to eight, optionally different, heteroatoms (e.g., O, N, S, Si, or P).

[0340] Similarly, the term "heteroalkylene," by itself or as part of another substituent, unless otherwise stated, includes but is not limited to -CH 2 -CH 2 -S-CH 2 -CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 - refers to a divalent radical derived from heteroalkyl, as exemplified by -. In the case of heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Additionally, in the case of alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, a group of the formula -C(O) 2 R'- is -C(O) 2 R'- and -R'C(O) 2 As mentioned above, heteroalkyl groups as used herein include -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SO 2It is understood that when "heteroalkyl" is described after a recitation of a specific heteroalkyl group, such as -NR'R'', the terms "heteroalkyl" and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl group is recited to add clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding a specific heteroalkyl group, such as -NR'R".

[0341] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, refer to cyclic versions of "alkyl" and "heteroalkyl", respectively, unless otherwise specified. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. "Cycloalkylene" and "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. "Cycloalkyl" is also meant to refer to bicyclic and polycyclic hydrocarbon rings, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.

[0342] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, "halo(C 1 -C 4 The term "alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0343] The term "acyl," unless otherwise specified, means -C(O)R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0344] The term "aryl", unless otherwise specified, refers to a polyunsaturated, aromatic, hydrocarbon substituent, which may be one ring or multiple rings (preferably 1-3 rings) fused together (i.e., fused-ring aryl) or covalently linked. Fused-ring aryl refers to multiple rings fused together, where at least one of the fused rings is an aryl group. The term "heteroaryl" refers to an aryl group (or ring) containing at least one heteroatom, such as N, O, or S, where the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Thus, the term "heteroaryl" includes fused-ring heteroaryl groups (i.e., multiple rings fused together, where at least one of the fused rings is an aromatic heterocycle). 5,6-fused-ring heteroarylene refers to two rings fused together, where one ring has 5 members and the other ring has 6 members, where at least one ring is a heteroaryl ring. Similarly, 6,6-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other ring has 6 members, and where at least one ring is a heteroaryl ring. And, 6,5-fused ring heteroarylene refers to two rings fused together, where one ring has 6 members and the other ring has 5 members, and where at least one ring is a heteroaryl ring. Heteroaryl groups can be attached to the remainder of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazolylbenzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, aryl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms "arylene" and "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent can be -O- attached to a ring heteroatom nitrogen.

[0345] A spiro ring is two or more rings in which adjacent rings are connected through one atom. The individual rings in a spiro ring may be the same or different. The individual rings in a spiro ring may be substituted or unsubstituted and may have different substituents than the other individual rings in a set of spiro rings. The possible substituents for the individual rings in a spiro ring are the possible substituents for the same ring when not part of a spiro ring (e.g., the substituents for a cycloalkyl ring or a heterocycloalkyl ring). A spiro ring may be a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heterocycloalkylene, and the individual rings in a spiro ring group may be any of those in the list immediately above, including all rings of one type (e.g., all rings are substituted heterocycloalkylene, and each ring may be the same or different substituted heterocycloalkylene). When referring to a spiro ring system, a heterocyclic spiro ring means a spiro ring in which at least one ring is a heterocycle and each ring is different. When referring to a spiro ring system, a substituted spiro ring means one in which at least one ring is substituted and each substituent can optionally be different.

[0346] The following symbol "

[0347] [ka] " indicates the point of attachment of the chemical moiety to the remainder of the molecule or chemical formula.

[0348] The term "oxo" as used herein means an oxygen that is double bonded to a carbon atom.

[0349] The term "alkylarylene" as an arylene moiety is covalently linked to an alkylene moiety (also referred to herein as an alkylene linker). In certain embodiments, the alkylarylene group has the formula:

[0350] [ka] The alkylarylene portion is halogen, oxo, -N 3 , -CF 3 , -CCl 3 , -CBr 3 , -CI 3 , -CN, -CHO, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 2 CH 3 -SO 3 H, -OSO 3 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC(O)NHNH 2 , substituted or unsubstituted C 1 -C 5 The alkyl or substituted or unsubstituted 2-5 membered heteroalkyl can be substituted (e.g., by a substituent) on the alkylene portion or on the arylene linker (e.g., at carbons 2, 3, 4, or 6). In certain embodiments, the alkylarylene is unsubstituted.

[0351] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cycloalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0352] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO 2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O) 2 R', -NR-C(NR'R''R''')=NR''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O) 2 R', -S(O) 2 NR'R'', -NRSO 2 R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO 2 , -NR'SO 2 R may be one or more of a variety of groups selected from R'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', where m' is the total number of carbon atoms in such radical. Each of R, R', R'', R''', and R'''' preferably independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When the compounds described herein contain more than one R group, for example, each of the R groups is independently selected as are the R', R'', R''', and R'''' groups, respectively, when more than one of these groups is present. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the description of substituents above, one of skill in the art will understand that the term "alkyl" includes haloalkyl (e.g., -CF 3 and -CH 2 CF 3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH 3It is understood that the term is intended to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as aryl, aryloxy ...

[0353] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and include, for example, -OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO 2 R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O) 2 R', -NR-C(NR'R''R''')=NR''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O) 2 R', -S(O) 2 NR'R'', -NRSO 2 R', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO 2 , -R', -N 3 , -CH(Ph) 2 , Fluoro(C 1 -C 4 ) alkoxy, and fluoro (C 1 -C 4 ) alkyl, -NR'SO 2 and R', R'', R''', and R'''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When the compounds described herein include more than one R group, for example, each of the R groups is independently selected as are each of the R', R'', R''', and R'''' groups when more than one of these groups is present.

[0354] Substituents for a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be represented as a substituent on the ring rather than on a specific atom of the ring (commonly referred to as a floating substituent). In such cases, the substituent may be attached (in accordance with the rules of chemical valency) to any of the ring atoms, and, in the case of a fused or spiro ring, a substituent represented as relating to a member of the fused or spiro ring (a floating substituent on one ring) may also be a substituent on any of the fused or spiro rings (floating substituents on multiple rings). When a substituent is attached to a ring rather than to a specific atom (floating substituent), and the substituent subscript is an integer greater than 1, the multiple substituents may be on the same atom, the same ring, different atoms, different fused rings, different spiro rings, and each substituent may optionally be different. If the point of attachment of the ring to the rest of the molecule is not limited to one atom (a floating substituent), the point of attachment may be any atom of the ring, and in the case of a fused or spiro ring, any atom of either the fused or spiro ring, subject to the rules of chemical valency. If a ring, fused or spiro ring contains one or more ring heteroatoms, and the ring, fused or spiro ring is shown with one or more floating substituents (including but not limited to the point of attachment to the rest of the molecule), said floating substituents may be attached to the heteroatoms. If a ring heteroatom is shown in a structure or formula with floating substituents as being attached to one or more hydrogens (e.g., a nitrogen atom with two bonds to a ring atom and a third bond to a hydrogen), it is understood that when a heteroatom is attached to a floating substituent, the substituent replaces the hydrogen, subject to the rules of chemical valency.

[0355] Two or more substituents can be optionally bonded to form an aryl group, a heteroaryl group, a cycloalkyl group, or a heterocycloalkyl group. Such so-called ring-forming substituents are typically, but not necessarily, found to be bonded to a cyclic base structure. In one embodiment, the ring-forming substituents are bonded to adjacent members of the base structure. For example, two ring-forming substituents bonded to adjacent members of the cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are bonded to one member of the base structure. For example, two ring-forming substituents bonded to one member of the cyclic base structure create a spiro ring structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0356] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring are optionally represented by the formula -TC(O)-(CRR') q -U-, where T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -A-(CH 2 ) r A and B may be independently substituted with a substituent of -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2 NR'-, or a single bond, where r is an integer from 1 to 4. One of the single bonds in the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be represented by the formula -(CRR') s -X'-(C''R''R'') d where s and d are independently an integer of 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O) 2 - or -S(O) 2The substituents R, R', R'', and R''' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0357] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0358] "Substituent," as used herein, means a group selected from the following moieties: (A) Oxo, halogen, -CF 3 , -CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 3 H, -SO 4 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC=(O)NHNH 2 , -NHC=(O)NH 2 , -NHSO 2 H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF 3 , -OCHF 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (i) oxo, halogen, -CF 3 , -CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 3 H, -SO4 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC=(O)NHNH 2 , -NHC=(O)NH 2 , -NHSO 2 H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF 3 , -OCHF 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: (a) Oxo, halogen, -CF 3 , -CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 3 H, -SO 4 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC=(O)NHNH 2 , -NHC=(O)NH 2 , -NHSO 2 H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF 3 , -OCHF 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from the following: oxo, halogen, -CF 3 , -CN, -OH, -NH 2 , -COOH, -CONH 2 , -NO 2 , -SH, -SO 3 H, -SO4 H, -SO 2 NH 2 , -NHNH 2 , -ONH 2 , -NHC=(O)NHNH 2 , -NHC=(O)NH 2 , -NHSO 2 H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF 3 , -OCHF 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0359] A "size-limited substituent" or "size-limited substituent group" as used herein means a group selected from all of the substituents described above for "substituent", where each substituted or unsubstituted alkyl is, respectively, a substituted or unsubstituted C 1 -C 20 each of the substituted or unsubstituted heteroalkyl groups is a substituted or unsubstituted 2- to 20-membered heteroalkyl group; each of the substituted or unsubstituted cycloalkyl groups is a substituted or unsubstituted C 3 -C 8 Each of the substituted or unsubstituted heterocycloalkyl groups is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl group, and each of the substituted or unsubstituted aryl groups is a substituted or unsubstituted C 6 -C 10 Aryl and the substituted or unsubstituted heteroaryl are each substituted or unsubstituted 5-10 membered heteroaryl.

[0360] "Lower substituent" or "lower substituent group" as used herein means a group selected from all of the substituents listed above for "substituent", where substituted or unsubstituted alkyl is, respectively, substituted or unsubstituted C 1 -C8 each of the substituted or unsubstituted heteroalkyl groups is a substituted or unsubstituted 2- to 8-membered heteroalkyl group; each of the substituted or unsubstituted cycloalkyl groups is a substituted or unsubstituted C 3 -C 7 Each of the substituted or unsubstituted heterocycloalkyl groups is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group, and each of the substituted or unsubstituted aryl groups is a substituted or unsubstituted C 6 -C 10 Aryl and the substituted or unsubstituted heteroaryl are each substituted or unsubstituted 5- to 9-membered heteroaryl.

[0361] In some particular embodiments, each of the substituents described in the compounds herein is substituted with at least one substituent.More specifically, in some particular embodiments, the substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are each substituted with at least one substituent.In other particular embodiments, at least one or all of such groups are substituted with at least one size-limited substituent.In other particular embodiments, at least one or all of such groups are substituted with at least one lower substituent.

[0362] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 20 each of the substituted or unsubstituted heteroalkyl groups is a substituted or unsubstituted 2- to 20-membered heteroalkyl group; each of the substituted or unsubstituted cycloalkyl groups is a substituted or unsubstituted C 3 -C 8Each of the substituted or unsubstituted heterocycloalkyl groups is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl group, and each of the substituted or unsubstituted aryl groups is a substituted or unsubstituted C 6 -C 10 In some embodiments of the compounds herein, each of the substituted or unsubstituted alkylenes is a substituted or unsubstituted C 1 -C 20 Each of the substituted or unsubstituted heteroalkylenes is a substituted or unsubstituted 2- to 20-membered heteroalkylene, and each of the substituted or unsubstituted cycloalkylenes is a substituted or unsubstituted C 3 -C 8 Each of the substituted or unsubstituted heterocycloalkylenes is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene, and each of the substituted or unsubstituted arylenes is a substituted or unsubstituted C 6 -C 10 The arylene and / or substituted or unsubstituted heteroarylene are each substituted or unsubstituted 5-10 membered heteroarylene.

[0363] In some particular embodiments, each of the substituted or unsubstituted alkyl groups is a substituted or unsubstituted C 1 -C 8 each of the substituted or unsubstituted heteroalkyl groups is a substituted or unsubstituted 2- to 8-membered heteroalkyl group; each of the substituted or unsubstituted cycloalkyl groups is a substituted or unsubstituted C 3 -C 7 Each of the substituted or unsubstituted heterocycloalkyl groups is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl group, and each of the substituted or unsubstituted aryl groups is a substituted or unsubstituted C 6 -C 10In some particular embodiments, each of the substituted or unsubstituted alkylenes is a substituted or unsubstituted C 1 -C 8 Each of the substituted or unsubstituted heteroalkylenes is a substituted or unsubstituted 2- to 8-membered heteroalkylene, and each of the substituted or unsubstituted cycloalkylenes is a substituted or unsubstituted C 3 -C 7 Each of the substituted or unsubstituted heterocycloalkylenes is a substituted or unsubstituted 3- to 7-membered heterocycloalkylene, and each of the substituted or unsubstituted arylenes is a substituted or unsubstituted C 6 -C 10 arylene, and / or each of the substituted or unsubstituted heteroarylenes is a substituted or unsubstituted 5- to 9-membered heteroarylene. In some particular embodiments, the compound is a species described in the Examples section, Figures, or Tables below.

[0364] Certain compounds of the present invention have asymmetric carbon atoms (optical or chiral centers) or double bonds, which may be defined with respect to absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- for amino acids; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms, and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those known in the art to be too unstable to synthesize and / or separate. The present invention is intended to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents or separated using conventional techniques. Where the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0365] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and therefore the same molecular weight, but differ with regard to the structural arrangement or configuration of the atoms.

[0366] The term "tautomer," as used herein, refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another.

[0367] It will be apparent to one skilled in the art that certain compounds of this invention exist in tautomeric forms, and all such tautomeric forms of the compounds are within the scope of the invention.

[0368] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure, i.e., the R and S configurations of each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and astereomeric mixtures of the present compounds are within the scope of the invention.

[0369] Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of hydrogen with deuterium or tritium, or 13 C or 14 Compounds having the present structures except for the replacement of a carbon with a C-rich carbon are within the scope of the invention.

[0370] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may contain isotopes of, for example, tritium ( 3 H), Iodine-125( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0371] Please note that throughout this application, options are written in Markush groups, e.g., each amino acid position contains more than one possible amino acid. It is specifically contemplated that each member of a Markush group should be considered separately, thereby including alternative embodiments, and that the Markush group is not to be read as a single unit.

[0372] "Analog" or "analogous" is used according to its plain ordinary meaning within chemistry and biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called "reference" compound) but differs in composition, such as, for example, the replacement of one atom with an atom of a different element, or the presence of a particular functional group, or the replacement of one functional group with another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Thus, an analog is a compound that is similar or equivalent to the reference compound in function and appearance, but not in structure or origin.

[0373] The terms "a" or "an" as used herein means one or more. Additionally, the phrase "substituted with a[n]" as used herein means that the particular group may be substituted with one or more of any or all of the specified substituents. For example, a group such as an alkyl group or a heteroaryl group may be substituted with an unsubstituted C 1 -C 20 When substituted with alkyl, or unsubstituted 2-20 membered heteroalkyl, the group may further comprise one or more unsubstituted C 1 -C 20 The alkyl may include one or more unsubstituted 2-20 membered heteroalkyl groups.

[0374] Additionally, when a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." When a moiety is R-substituted, the moiety is substituted with at least one R substituent, each of which is optionally different. When a particular R group occurs within a description of a chemical genus (such as formula (II)), roman alphabet symbols may be used to distinguish each occurrence of that particular R group. For example, multiple R 13 When substituents are present, each R 13 The substituent is R 13A , R 13B , R 13C , R 13D etc., where R 13A , R 13B , R 13C , R 13D Each of the above is R 13 and optionally differently defined.

[0375] The description of the compounds of the present invention is limited by the principles of chemical bonding known to those skilled in the art.Therefore, when a group can be substituted by one or more of many substituents, such substitutions are selected to comply with the principles of chemical bonding and to bring about compounds that are not inherently unstable and / or are known to those skilled in the art as possibly unstable under ambient conditions, such as aqueous, neutral, and various known physiological conditions.For example, heterocycloalkyl or heteroaryl are bonded to the rest of the molecule through ring heteroatoms according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0376] The term "pharmaceutically acceptable salts" is meant to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base in a neat or suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid in a neat or suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous acids, as well as those derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, or methanesulfonic acid. Also included are salts of amino acids, such as arginic acid, and salts of organic acids, such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19).Certain compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0377] Therefore, the compounds of the present invention may exist as salts with pharma- ceutically acceptable acids and the like. The present invention includes such salts. Non-limiting examples of such salts include hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinate, benzoate, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, etc.). Such salts can be prepared by methods known to those skilled in the art.

[0378] The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents. In some embodiments, the compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. The neutral form of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. Although the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents, the salts disclosed herein are equivalent to the parent form of the compound for the purposes of the present invention, unless otherwise indicated.

[0379] In addition to salt forms, the present invention provides compounds in prodrug form.Prodrugs of the compounds described herein are compounds that easily undergo chemical changes under physiological conditions to provide the compounds of the present invention.Prodrugs of the compounds described herein can be converted in vivo after administration.In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment, for example, when contacted with appropriate enzymes or chemical reagents.

[0380] Certain compounds of the present invention can exist in unsolvated as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention. Certain compounds of the present invention can exist in multiple crystalline or amorphous forms. In general, all physical forms are applicable to the applications contemplated by the present invention and are intended to be within the scope of the present invention.

[0381] "Contacting" is used according to its plain and ordinary meaning to refer to a process that allows at least two distinct species (e.g., chemical compounds, including biological molecules or cells) to be in sufficient proximity to react, interact, or come into physical contact. However, it should be understood that the resulting reaction product may be produced directly from the reaction between the additional reagents or may be produced from an intermediate from one or more of the additional reagents that may be produced in the reaction mixture.

[0382] The term "contacting" includes allowing two species to react, interact, or come into physical contact, where the two species can be a compound described herein and a protein or enzyme. In some embodiments, contacting includes allowing a compound described herein to interact with a protein or enzyme involved in a signal pathway.

[0383] As defined herein, the terms "activation," "activate," "activating," and the like, with respect to proteins, refer to the conversion of a protein from an initial inactive or inactivated state to a biologically active derivative. These terms refer to the activation, or activating, sensitizing, or upregulating signaling or enzymatic activity, or the amount of a protein that is reduced in a disease.

[0384] The terms "agonist", "activator", "upregulator" and the like refer to a substance that can detectably increase the expression or activity of a given gene or protein. An agonist can increase expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control in the absence of the agonist. In some instances, the expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more over the expression or activity in the absence of the agonist. In some embodiments, an agonist is a molecule that interacts with a target to cause or promote increased activation of the target. In some embodiments, an activator is a molecule that increases, activates, promotes, enhances activation, sensitizes, or upregulates, for example, a gene, protein, ligand, receptor, or cell.

[0385] As defined herein, with respect to protein-inhibitor interactions, the terms "inhibit," "inhibit," "inhibiting," and the like refer to negatively affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition refers to negatively affecting (e.g., decreasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to a reduction in a disease or a symptom of a disease. In some embodiments, inhibition refers to a decrease in the activity of a particular protein target. Thus, inhibition includes at least partially, partially, or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signal transduction, or enzyme activity, or the amount of a protein. In some embodiments, inhibition refers to a decrease in the activity of a target protein due to a direct interaction (e.g., an inhibitor binds to a target protein). In some embodiments, inhibition refers to a decrease in the activity of a target protein from an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein).

[0386] The terms "inhibitor", "suppressor" or "antagonist" or "downregulator" refer interchangeably to a substance that can detectably reduce the expression or activity of a given gene or protein. An antagonist can reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control without the antagonist. In some instances, the expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or less than the expression or activity without the antagonist. An antagonist prevents, reduces, inhibits or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit or reduce the constitutive activity of a target, e.g., a target receptor, even in the absence of an identified agonist. In some embodiments, an inhibitor is a molecule that reduces, blocks, prevents, slows activation, inactivates, desensitizes, or downregulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity. An "antagonist" is a molecule that opposes the action of an agonist.

[0387] The term "disease" or "condition" refers to a state or condition of a patient or subject that can be treated with the compounds or methods provided herein. The disease can be cancer.

[0388] The term "treating" or "treatment" refers to any indication of success in the treatment or amelioration of an injury, disease, condition, or illness, including any objective or subjective parameter, such as remission; reducing symptoms or making the injury, condition, or illness more tolerable to the patient; slowing the rate of degeneration or decline; making the end point of degeneration less debilitating; improving the physical or mental health of the patient. The treatment or amelioration of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric testing, and / or psychiatric evaluation. The term "treating" and conjugations thereof may include prevention of an injury, condition, illness, or illness. In some embodiments, treating is preventing. In some embodiments, treating does not include preventing.

[0389] "Treating" or "treatment", as used herein (and as well understood in the art), broadly includes any approach to obtain beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether partial or total, and whether detectable or undetectable, reduction in the extent of disease, stabilization (i.e., not worsening) of a disease state, prevention of disease transmission or spread, delay or slowing of disease progression, improvement or reduction of a disease state, and remission. In other words, "treatment", as used herein, includes any cure, amelioration, or prevention of a disease. Treatment can prevent a disease from occurring; inhibit the spread of a disease; sufficiently alleviate symptoms of a disease, completely or partially eliminate the underlying cause of a disease, shorten the duration of a disease, or a combination of these.

[0390] "Treat" and "treatment" as used herein may also include prophylactic treatment. The treatment method includes administering a therapeutically effective amount of a compound described herein to a subject. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on various factors, such as the severity of the disease, the age of the patient, the concentration of the compound, the activity of the composition used in the treatment, or a combination thereof. It will be understood that the effective amount of the agent used for treatment or prevention may be increased or decreased during a particular treatment or prevention regimen. The change in dosage may result and be evident by standard diagnostic assays known in the art. In some instances, long-term administration may be required. For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient.

[0391] The term "preventing" refers to a reduction in the occurrence of symptoms in a patient. As noted above, prevention can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed compared to those that would occur in the absence of treatment. In certain embodiments, preventing refers to slowing the progression of a disease, disorder, or condition, or inhibiting progression to a harmful or otherwise undesirable state. EXAMPLES

[0392] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0393] Chemical synthesis examples The compounds in Tables 1-3 are prepared in the Examples below.

[0394] [Table 1-1]

[0395] [Table 1-2]

[0396] [Table 1-3]

[0397] [Table 1-4]

[0398] [Table 2]

[0399] [Table 3]

[0400] Example 1. Compounds 1-13, General Synthetic Scheme for Preparation of UNA 2' Derivatives Compounds 1 to 15 are synthesized according to the following scheme.

[0401] [ka]

[0402] Example 2. General synthetic scheme for UNA 2'fluoro compounds Compounds 2 and 8 are synthesized according to the following scheme.

[0403] [ka]

[0404] Example 3. General synthetic scheme for solid phase synthesis of oligonucleotides

[0405] [ka]

[0406] Example 4. UNA key

[0407] [Table 4]

[0408] Example 5. Synthesis of uridine-UNA-2'MOE,3' phosphoramidite The uridine-UNA-2'MOE,3' phosphoramidite is synthesized according to the following scheme.

[0409] [ka]

[0410] Example 6. Synthesis of cytosine-UNA-2'MOE,3' phosphoramidite Cytosine-UNA-2'MOE,3' phosphoramidite is synthesized according to the following scheme.

[0411] [ka]

[0412] Example 7. Synthesis of adenine-UNA-2'MOE,3' phosphoramidite Adenine-UNA-2'MOE,3' phosphoramidite is synthesized according to the following scheme.

[0413] [ka]

[0414] Example 8: Synthesis of Guanosine-UNA-2'MOE,3' Phosphoramidite The guanosine-UNA-2'MOE,3' phosphoramidite is synthesized according to the following scheme.

[0415] [ka]

[0416] Example 9: General procedure for the preparation of compound 2

[0417] [ka]

[0418] To a solution of compound 1 (350 g, 1.43 mol, 1.00 equiv.) in Py (2.1 L) was added DMTCl (500.2 g, 1.48 mol, 1.03 equiv.) at 0° C. The reaction mixture was stirred at 25° C. for 3 h. TLC (dichloromethane:methanol=10:1, R f =0.51) showed no starting material. The mixture was diluted with NaHCO 3 Dilute with aqueous solution (2.0 L), extract with DCM (2.0 L x 2), and add anhydrous Na 2 SO 4 The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with petroleum ether / ethyl acetate=10 / 1 to 0 / 1 and 0.5% TEA to obtain compound 2 (620 g, 1.13 mol, 79.2 yield) as a yellow solid.

[0419] Example 10: General procedure for the preparation of compound 3

[0420] [ka]

[0421] To a solution of compound 2 (310 g, 567.2 mmol, 1.00 equiv) in dioxane (1.5 L) was added NaIO 4 (121.3 g, 567.2 mmol, 31.4 mL, 1.00 equiv.) and H 2 O (600 mL) was added at 25° C., and the reaction mixture was stirred at 25° C. for 2 h. TLC (dichloromethane:methanol=10:1, R f=0.64) indicated complete consumption of the starting material. The two reactions were combined for workup. The reaction mixture was filtered. The crude product was used in the next step without further purification. Compound 3 (600 g, crude) was obtained as a colorless oil.

[0422] Example 11. General procedure for the preparation of compound 4

[0423] [ka]

[0424] To a solution of compound 3 (300 g, 550.9 mmol, 1 equiv.) in dioxane (1.8 L) was added NaBH 4 (20.8 g, 550.9 mmol, 1.00 equiv) was added at 0° C. and the mixture was stirred at 20° C. for 1 h. TLC (dichloromethane:methanol=10:1, R f = 0.43) showed no starting material. The two reactions were combined for workup and the reaction mixture was quenched with acetone, neutralized with 20% aqueous acetic acid, and concentrated to give a residue. The residue was diluted with DCM (1.8 L) and diluted with H 2 Wash with O (1.8 L) and add anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure to give compound 4 (600 g, 1.09 mol, 99.3% yield) as a white solid.

[0425] Example 12. General procedure for the preparation of compound 5

[0426] [ka]

[0427] To a solution of compound 4 (300 g, 546.9 mmol, 1.00 equiv.) in Tol (600 mL) was added lipozyme TL IM (60 g, 0.60 equiv.) and benzoyl benzoate (124.9 g, 552.3 mmol, 104.1 mL, 1.01 equiv.) at 20° C. The reaction mixture was stirred at 40° C. for 4 h. TLC (dichloromethane:methanol=10:1, R f = 0.53) showed no starting material. For workup, the two reactions were combined, the reaction mixture was filtered, and the filtrate was diluted with NaHCO 3 The organic phase was separated and washed with brine (600 mL x 3) and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure to give compound 5 (620 g, crude) as a white solid.

[0428] Example 13. General procedure for the preparation of compound 6

[0429] [ka]

[0430] To a solution of compound 5 (310 g, 475.0 mmol, 1.00 equiv) in Py (1.8 L) was added TBSCl (85.9 g, 569.9 mmol, 69.8 mL, 1.20 equiv). The reaction mixture was stirred at 25° C. for 8 h. The two batches were combined for workup and purification. NaHCO 3 Aqueous solution (1.8 L) was added and the mixture was extracted with DCM (1.8 L) and concentrated in vacuo to give a residue. The residue was dissolved in Py (1.2 L) and MeOH (5.4 L), then 1M NaOH in MeOH (300 mL) was added dropwise to the mixture at 0° C., then stirred at 0° C. for 1 h. TLC (petroleum ether / ethyl acetate=1:1, R f =0.43) showed no starting material. Saturated NH 4 Aqueous Cl solution (1.2 L) was added to the mixture, which was stirred for 10 min and H 2O (1.2 L) was added and the mixture was extracted with DCM (3 L). 2 SO 4 The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with petroleum ether / ethyl acetate = 10 / 1 to 0 / 1 and 0.5% TEA) to obtain compound 6 (280 g, 422.4 mmol, 44.5% yield) as a yellow oil.

[0431] Example 14. General procedure for the preparation of compound 7

[0432] [ka]

[0433] To a solution of NaH (27.9 g, 697.0 mmol, 60% purity, 3.30 equiv.) in DMF (850 mL), compound 6 (140 g, 211.2 mmol, 1.00 equiv.) was added at 0° C. and the reaction mixture was stirred at 0° C. for 1 h, then compound a (44.0 g, 316.8 mmol, 29.8 mL, 1.50 equiv.) was added at 0° C. and the reaction mixture was stirred at 10° C. for 16 h. TLC (petroleum ether:ethyl acetate=1:2, R f =0.43) showed no starting material. The two batches were combined for workup and purification. NH 4 The residue was diluted with aqueous Cl (4.2 L), extracted with EtOAc (1.0 L x 2), and the combined organic layers were washed with brine (1.0 L) and anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate and filtered. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with petroleum ether / ethyl acetate = 10 / 1 to 0:1 and 0.5% TEA) to obtain compound 7 (110 g, 152.6 mmol, 36.1% yield) as a yellow oil.

[0434] 1 H NMR:ET32436-23-P1A(400MHz,DMSO-d6 )

[0435] δ 11.37(s,1H),7.70(d,J=8.0 Hz,1H),7.33-7.30(m,4H),7.19-7.16(m,5H),6.86-6.84(m,4H),6.00(t,J=4.8 Hz,1H),5.55(d,J=8.0 Hz,1H),3.73(s,6H),3.71-3.69(m,2H),3.65-3.63(m,2H),3.56-3.55(m,3H),3 .41-3.39(m,2H),3.21(s,3H),2.97-2.95(m,2H),-0.76(s,9H),-0.04(d,J=7.6 Hz,6.0H)

[0436] Example 15. General procedure for the preparation of compound 11

[0437] [ka]

[0438] To a solution of compound 7 (15 g, 20.8 mmol, 1.00 equiv) in MeOH (120 mL) was added NH 4 F (6.17 g, 166.5 mmol, 8.00 equiv.) was added at 20° C., and the reaction mixture was stirred at 40° C. for 16 h. TLC (petroleum ether / ethyl acetate=0:1, R f =0.13) showed no starting material. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with petroleum ether / ethyl acetate = 10 / 1 to 0:1 and 0.5% TEA) to obtain compound 11 (7.50 g, 12.4 mmol, 59.4% yield) as a yellow oil.

[0439] 1 H NMR:ET32436-28-P1A(400MHz,DMSO-d 6 )

[0440] δ 11.39(s,1H),7.68(d,J=8.0 Hz,1H),7.32-7.28(m,4H),7.20-7.16(m,5H),6.87-6.85(m,4H),5.98(t,J=6.0 Hz,1H),5.54(d,J=4.0 Hz,1H),4.74(t,J=5.2 Hz,1H),3.73(s,7H),3.71(s,1H),3.63-3.59(m,1),3.51-3.55(m,2),3.42-3.41(m,4H),3.21(s,3H),3.00-2.94(m,2H).

[0441] Example 16. General procedure for the preparation of U-UNA-2'MOE 3' phosphoramidites

[0442] [ka]

[0443] To a solution of compound 11 (8.0 g, 13.2 mmol, 1.00 equiv) in DCM (48.0 mL) was added 1H-imidazole-4,5-dicarbonitrile (2.34 g, 19.8 mmol, 1.50 equiv) at 0° C., followed by compound b (6.76 g, 22.4 mmol, 7.12 mL, 1.70 equiv). The reaction mixture was stirred at 25° C. for 3 h. TLC (dichloromethane:MeOH=10:1, R f =0.53) showed no starting material. The reaction mixture was purified by column chromatography (SiO 2 The mixture was purified by elution with 2:1 to 1:2 petroleum ether / ethyl acetate, 1% TEA. Combine with (ET32436-32-P1) for purification: Column: Agela DuraShell C18 250 * 80mm * 10um; Mobile phase: [water (10mM NH 4 HCO 3 )-ACN]; B%: 40%-80%, 20 min. Compound U-UNA-2'MOE 3' phosphoramidite was obtained as a yellow solid.

[0444] 1H NMR: (400MHz, DMSO-d 6 ):δ 11.40(br s,1H),7.68(d,J=8.0 Hz,1H),7.32-7.29(m,4H),7.20-7.16(m,5H),6.86-6.84(m,4H),5.99-5.96(m,1H),5.56(d,J=8.0 Hz,1H),3.73(s,7H),3.70-3.65(m,4H),3.56-3.55(m,5H),3.40-3.39(m,2 H),3.21(s,3H),3.09-2.94(m,2H),2.72-2.69(m,2H),1.17-0.96(m,12H).

[0445] 31 PNMR: (162MHz, DMSO-d 6 ): δ 147.46

[0446] HPLC: 97.3% purity

[0447] MS:M-1=805.3

[0448] Example 17. General procedure for the preparation of compound 8

[0449] [ka]

[0450] To a solution of compound 7 (50 g, 69.4 mmol, 1.00 equiv.) in DCM (300 mL) and TEA (17.6 g, 173.4 mmol, 24.13 mL, 2.50 equiv.) was added DMAP (8.47 g, 69.4 mmol, 1.00 equiv.) and TPSCl (51.1 g, 173.4 mmol, 2.50 equiv.). The reaction mixture was stirred at 25° C. for 1 h and then NH 3 .H 2 2H2O (81.03 g, 693.6 mmol, 89.0 mL, 30% purity, 10.0 equiv.) was added and stirred at 25° C. for 2 h. TLC (dichloromethane:methanol=10:1, R f= 0.23) showed no starting material. The reaction mixture was extracted with EtOAc (300 mL) and saturated NaHCO 3 and brine (200 mL each). The organic layer was washed with anhydrous Na 2 SO 4 The residue was dried at 40° C., filtered and concentrated under reduced pressure to give compound 8 (50 g, crude) as a yellow oil.

[0451] Example 17. General procedure for the preparation of compound 9

[0452] [ka]

[0453] To a solution of compound 8 (40 g, 55.6 mmol, 1.00 equiv.) in Py (240 mL), BzCl (11.7 g, 83.3 mmol, 9.68 mL, 1.50 equiv.) was added at 0° C., and the reaction mixture was stirred at 25° C. for 2 h. TLC (dichloromethane:methanol=10:1, R f =0.43) showed no starting material. The mixture was diluted with NaHCO 3 Dilute with aqueous solution (500 mL), extract with DCM (500 mL x 2), and add anhydrous Na 2 SO 4 The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with petroleum ether / ethyl acetate = 10 / 1 to 0 / 1 and 0.5% TEA) to obtain compound 9 (34.0 g, 41.3 mmol, 74.3% yield) as a yellow oil.

[0454] 1 H NMR: (400MHz, DMSO-d 6)δ 11.36(s,1H),8.58-8.57(m,1H),8.20-8.18(m,1H),8.04-8.02(m,2H), 7.64-7.51(m,3H),7.38-7.15(m,12H),6.86-6.83(m,4H),6.16(t,J=5.2 Hz,1H),3.73-3.69(m,4H),3.67(s,6H),3.60-3.52(m,4H),3.42-3.39(m,2H ),3.21-3.19(m,3H),3.00-2.99(m,2H),0.77(s,9H),-0.03(d,J=8.0Hz,6H).

[0455] Example 18. General procedure for the preparation of compound 10

[0456] [ka]

[0457] To a solution of compound 9 (30 g, 36.4 mmol, 1.00 equiv) in THF (300 mL), TBAF (1 M, 40.1 mL, 1.10 equiv) was added at 0° C., and the reaction mixture was stirred at 25° C. for 16 h. TLC (dichloromethane:methanol=10:1, R f =0.24) showed no starting material. The mixture was diluted with NaHCO 3 Dilute with aqueous solution (600 mL), extract with DCM (500 mL x 2), and add anhydrous Na 2 SO 4 The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with petroleum ether / ethyl acetate = 10 / 1 to 0:1 and 0.5% TEA to obtain compound 10 (16.5 g, 23.3 mmol, 63.9% yield) as a yellow oil.

[0458] 1 H NMR: (400MHz, DMSO-d 6)δ 11.36(s,1H),8.20(d,J=7.2 Hz,1H),8.03(d,J=7.6 Hz,2H),7.64(t,J=7.4 Hz,1H),7.55-7.51(m,2H),7.37(d,J=7.2 Hz,1H),7.31-7.29(m,4H),7.17(d,J =8.8 Hz,5H),6.86(d,J=8.8 Hz,4H),6.17(t,J=4.8Hz,1H),4.77(t,J=5.4Hz,1H),3.76-3.74(m,2H),3.68 (s,7H),3.60-3.58(m,2H),3.46-3.39(m,4H),3.20(s,3H),3.05-2.96(m,2H).

[0459] Example 19. General procedure for the preparation of C-UNA-2'MOE 3' phosphoramidites

[0460] [ka]

[0461] To a solution of compound 10 (13.3 g, 18.7 mmol, 1.00 equiv) in DCM (130 mL) was added 1H-imidazole-4,5-dicarbonitrile (3.32 g, 28.1 mmol, 1.50 equiv) at 0° C., followed by compound b (9.60 g, 31.9 mmol, 10.1 mL, 1.70 equiv). The reaction mixture was stirred at 25° C. for 1 h. TLC (petroleum ether / ethyl acetate=1:1, R f =0.43) showed no starting material. The reaction mixture was purified by column chromatography (SiO 2 The mixture was purified by elution with 5% TEA (petroleum ether / ethyl acetate=1 / 1 to 0 / 1, 5% TEA) to obtain compound C-UNA-2'MOE 3' phosphoramidite as a white solid.

[0462] 1 H NMR: (400MHz, DMSO-d 6 )

[0463] δ 11.37(s,1H),8.19(d,J=6.8 Hz,1H),8.03(d,J=7.6 Hz,2H),7.64(t,J=7.4 Hz,1H),7.53(t,J=7.6 Hz,2H),7.36(d,J=7.2 Hz,1H),7.29-7.26(m,4H),7.18-7.15(m,5H),6.85(d,J=8.4 Hz,4H),6.15(s,1H),3.87-3.68(m,11H),3.64-3.57(m,4H),3.54-3.44(m,2H),3.40(s,2H) ),3.20(s,3H),3.12-2.30(m,2H),2.74-2.69(m,2H),1.12-1.10(m,6H),1.05-1.01(m,6H)

[0464] 31 PNMR: (162MHz, DMSO-d 6 ): δ 147.57

[0465] HPLC: 98.4% purity

[0466] MS:M-1=909.3

[0467] Example 20. General procedure for the preparation of compound 13

[0468] [ka]

[0469] To a solution of compound 12 (200 g, 539 mmol, 1.00 equiv.) in Py (2.00 L) was added DMTCl (200 g, 592 mmol, 1.10 equiv.), followed by stirring at 25° C. for 2 h. TLC (dichloromethane:methanol=20:1, R f =0.44) showed no starting material. MeOH (150 mL) was added to the reaction mixture, which was then concentrated in vacuo. The residue was dissolved in EtOAc (2.00 L) and washed with aqueous acetic acid (500 mL x 2) and brine (500 mL), and concentrated in anhydrous Na 2 SO 4The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated in vacuo. The residue was washed with MTBE (1.00 L x 2). Compound 13 (280 g, crude) was obtained as a yellow solid.

[0470] 1 H NMR: (400MHz, DMSO-d 6 ):δ 11.18(s,1H),8.68(s,1H),8.58(s,1H)8.04(d,J=7.2 Hz,1H),7.65-7.63(m,1H),7.57-7.53(m,2H),7.37-7.36(m,2H)7.20-7.28(m,10H),7.07-7.09(m,2H),6.85-6.82(m,6H),6.07(d,J=4.0 Hz,1H),5.61(d,J=4.0 Hz,1H),5.24(d,J=4.0 Hz,1H),4.79-4.78(m,1H),4.34-4.32(m,1H),4.12-4.11(m,1H),3.74-3.72(m,9H),3.26-3.25(m,2H).

[0471] Example 21. General procedure for the preparation of compound 14

[0472] [ka]

[0473] To a solution of compound 13 (80.0 g, 119 mmol, 1.00 equiv) in dioxane (1.20 L) was added H 2 NaIO in O (400 mL) 4 (27.9 g, 130 mmol, 7.24 mL, 1.10 equiv.) was added at 25° C., and then stirred at 25° C. for 1 h. TLC (dichloromethane:methanol=15:1, R f =0.32) showed no starting material. For workup and purification, the three batches were combined. The reaction mixture was filtered and the filtrate was used directly in the next step. Compound 14 (240 g, crude) was obtained as a yellow oil. The crude was used directly in the next step.

[0474] Example 22. General procedure for the preparation of compound 15

[0475] [ka]

[0476] To a solution of compound 14 (80.0 g, 119 mmol, 1.00 equiv) in dioxane (1.20 L) was added NaBH 4 (4.96 g, 131 mmol, 1.10 equiv.) was added at 25° C., and then stirred at 25° C. for 1 hour. TLC (dichloromethane:methanol=15:1, R f =0.34) showed no starting material. The reaction mixture was added with AcOH / pyridine (1 / 1, 50.0 mL) and then concentrated in vacuo. The three batches were combined with ET32454-7-P1A for work-up and purification. The residue was extracted with EtOAc (1.00 l x 2) and washed with NaHCO 3 Wash with aqueous solution (600 mL) and brine (600 mL) and add anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO 2 The residue was purified by elution with 50:1 to 10:1 DCM:MeOH, 1% TEA) to give compound 15 (230.0 g, 46.4% yield over three steps) as a yellow solid.

[0477] LCMS: RT=2.213 min, M-1=674.3.

[0478] 1 H NMR: (400MHz, DMSO-d 6 )

[0479] δ 11.25(s,1H),8.75(s,1H),8.67(s,1H),8.07-80.5(m,2H),7.67-7.63(m,1H),7.57-7.53(m, 2H),7.22-7.12(m,3H),7.07-7.05(m,1H),6.97-6.93(m,4H),6.79-6.77(m,4H),6.05(t,J=12 Hz,1H),5.31-5.26(m,1H),4.83(t,J=8.0 Hz,1H),4.14-4.04(m,2H),3.84(s,1H),3.69(s,6H),3.49-3.47(m,2H),2.89-2.78(m,1H),2.77-2.75(m,1H).

[0480] Example 22. General procedure for the preparation of compound 16

[0481] [ka]

[0482] To a solution of compound 15 (145 g, 214 mmol, 1.00 equiv) in DCM (2.00 L) was added DBU (65.3 g, 429 mmol, 64.7 mL, 2.00 equiv), then cooled to -70°C, BzCl (33.2 g, 236 mmol, 27.4 mL, 1.10 equiv) was added dropwise at -70°C, and then stirred at -70°C for 1 h. TLC (dichloromethane:methanol = 10:1, R f =0.43) showed no starting material. EtOH (300 mL) was added to the reaction mixture at -70 °C, then brought to 25 °C and added NaHCO 3 Wash with aqueous solution (2.00 L x 2) and brine (1.00 L), and add anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo to give compound 16 (160 g, crude) as a yellow solid which was used directly in the next step.

[0483] Example 23. General procedure for the preparation of compound 17

[0484] [ka]

[0485] To a solution of compound 16 (160 g, 205 mmol, 1.00 equiv.) in DCM (960 mL) was added imidazole (21.0 g, 308 mmol, 1.50 equiv.) and TBSCl (40.2 g, 267 mmol, 32.7 mL, 1.30 equiv.), then stirred at 25° C. for 1 h. TLC (dichloromethane:methanol=10:1, R f =0.51) showed no starting material. The reaction mixture was diluted with NaHCO 3 Wash with aqueous solution (800 mL x 2) and brine (800 mL x 2) and add anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo to give compound 17 (160 g, crude) as a yellow solid which was used directly in the next step.

[0486] Example 23. General procedure for the preparation of compound 18

[0487] [ka] To a solution of compound 17 (200 g, 224 mmol, 1.00 equiv) in Py (1.20 L) and MeOH (480 mL), NaOH (1 M, 224 mL, 1.00 equiv) was added dropwise at 0° C., then stirred at 0° C. for 0.5 h. TLC (dichloromethane:methanol=10:1, R f =0.42) showed no starting material. The reaction mixture was extracted with EtOAc (1.00 L x 2) and NH 4 Wash with aqueous Cl (1.00 L x 2) and brine (800 mL), and then with anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated in vacuo. For purification, ET32454-18-P1A was combined with ET32454-19-P1A. The residue was purified by column chromatography (SiO 2 The mixture was purified by elution with 10:1 petroleum ether / ethyl acetate to 20:1 DCM:MeOH, 1% TEA) to give compound 18 (58 g, 21.7% yield over three steps) as a white solid.

[0488] 1 H NMR: (400MHz, DMSO-d 6 )δ 11.24(s,1H),8.75(s,1H),8.70(s,1H),8.07-8.05(m,2H),7.69-7.65(m,1H),7.59-7.56(m,2H),7.24-7.20(m,2H) ,7.17-7.13(m,1H),7.08-7.06(m,1H),6.99-6.94(m,4H),6.81-6.78(m,4H),6.07-6.04(m,1H),5.30-5.27(t,J=12 Hz,1H),4.20-4.14(m,1H),4.06-3.99(m,1H),3.88-3.85(m,1H),3.76-3.70(m,7H),3.61-3.57(m,1H),0.80(s,9H),0.03(d,J=12 Hz,6H).

[0489] Example 24. General procedure for the preparation of compound 19

[0490] [ka] To a solution of NaH (9.52 g, 238 mmol, 60% purity, 3.30 equiv.) in DMF (300 mL), a solution of compound 18 (57.0 g, 72.2 mmol, 1.00 equiv.) was added dropwise at 0° C., and then stirred at 0° C. for 1 h. To the mixture, compound a (15.0 g, 108 mmol, 10.2 mL, 1.50 equiv.) was added dropwise at 0° C., and then stirred at 15° C. for 15 h. TLC (dichloromethane:methanol=10:1, R f =0.43) showed no starting material. The reaction mixture was diluted with NH 4 Pour into aqueous Cl (2.00 L), extract with EtOAc (800 mL x 2), wash with brine (500 mL x 2), and add anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo to give compound 19 (60.0 g, crude) as a yellow oil which was used directly in the next step.

[0491] LCMS:RT=3.219 min, MS-1=846.4.

[0492] Example 25. General procedure for the preparation of compound 20

[0493] [ka]

[0494] To a solution of compound 19 (60.0 g, 70.8 mmol, 1.00 equiv) in MeOH (360 mL) was added NH 4 F (21.0 g, 566 mmol, 8.00 equiv.) and TEA (14.3 g, 142 mmol, 19.7 mL, 2.00 equiv.) were added at 15° C., followed by stirring at 15° C. for 16 h. TLC (dichloromethane:methanol=10:1, R f = 0.33) showed no starting material. The reaction mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO 2 The residue was purified by elution with 100:1 to 10:1 DCM:MeOH, 1% TEA) to give compound 20 (16.5 g, 22.5 mmol, 28.6% yield) as a white solid.

[0495] 1 H NMR: (400MHz, DMSO-d 6 )δ 11.24(s,1H),8.76(s,1H),8.71(s,1H),8.07-8.05(m,2H),7.69-7.65(m,1H),7.59-7.56(m,2H),7.24-7.20(m,2H) ),7.17-7.13(m,1H),7.08-7.06(m,1H),6.99-6.94(m,4H),6.81-6.78(m,4H),6.25-6.22(m,1H),4.84-4.82(t,J=6 Hz,1H),4.30-4.25(m,1H),4.09-4.05(m,1H),3.87-3.85(m,1H),3.69(d,J=2 Hz,7H),3.62-3.60(m,2H),3.50-3.47(m,2H),3.39-3.37(m,2H),3.17(s,3H),2.90-2.86(m,1H),2.77-2.73(m,1H).

[0496] Example 26. General procedure for the preparation of A-UNA-2'MOE 3' phosphoramidites

[0497] [ka]

[0498] To a solution of compound 20 (15.0 g, 20.4 mmol, 1.00 equiv.) in dry DCM (90.0 mL), DCI (3.62 g, 30.7 mmol, 1.50 equiv.) and compound b (10.5 g, 34.8 mmol, 11.0 mL, 1.70 equiv.) were added at 0° C., followed by stirring at 15° C. for 2 h. TLC (dichloromethane:acetone=3:1, R f =0.53) showed no starting material. The solution was poured directly onto a silica gel column. The reaction mixture was purified by column chromatography (SiO 2 The residue was purified by elution with 5 / 1 to 0 / 1 petroleum ether / ethyl acetate, 1% TEA) to give A-UNA-2'MOE 3' phosphoramidite (7.00 g, 34.2% yield) as a white solid.

[0499] 1 H NMR: (400MHz, DMSO-d 6 )δ 11.26(s,1H),8.77-8.72(m,2H),8.06(d,J=7.6 Hz,2H),7.63(t,J=14.8 Hz,1H),7.56(t,J=15.2 Hz,2H),7.20-7.12(m,3H),7.08-7.04(m,2H),6.98-6.92(m,4H),6.79-6.76(m,4H),6.22(t,J=12 Hz,1H),4.27-4.24(m,1H),4.04-3.98(m,2H),3.69-3.66(m,10H),3.63-3.62(m,2H),3. 62-3.60(m,2H),3.39-3.37(m,2H),3.17(s,3H),2.96-2.71(m,4H),1.19-1.03(m,12H). 31 P NMR: (162MHz, DMSO-d6 ) δ 147.62,147.46 HPLC: 97.8% purity

[0500] MS:M-1=932.2

[0501] Example 27. General procedure for the preparation of compound 22

[0502] [ka]

[0503] To a solution of compound 21 (150.0 g, 424.5 mmol, 1.00 equiv.) in Py (900 mL), DMTCl (158.2 g, 467.0 mmol, 1.10 equiv.) was added at 0° C., then stirred at 15° C. for 2 h. TLC (dichloromethane:methanol=20:1, R f =0.38) showed no starting material. MeOH (150 mL) was added to the reaction mixture, which was then concentrated in vacuo. The residue was dissolved in EtOAc (1000 mL), washed with aqueous acetic acid (500 mL x 2) and brine (500 mL), and concentrated in anhydrous Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated in vacuo. The residue was washed with MTBE (1000 mL×2). Compound 22 (284.0 g, crude) was obtained as a yellow solid.

[0504] 1 H NMR: (400MHz, DMSO-d 6)δ 12.10(s,1H),11.66(s,1H),8.58(d,J=4.0 Hz,1H),8.12(s,1H),7.38-7.35(m,3H),7.33-7.25(m,8H),7.23-7.20(m,1H),6.84-6.80(m,4H),5.86(d,J=4.8 Hz,1H),5.62(d,J=5.6 Hz,1H),5.19(d,J=5.6 Hz,1H),4.54-4.53(m,1H),4.22-4.20(m,1H),3.74-3.72(m,7H),3.25 -3.18(m,1H),2.78-2.76(m,1H),2.76-2.75(m,1H),1.13-1.10(m,6H).

[0505] Example 28. General procedure for the preparation of compound 23

[0506] [ka]

[0507] Three batches were carried in parallel. A solution of compound 22 (80.0 g, 122.0 mmol, 1.00 equiv) in dioxane (1600 mL) was added with H 2 NaIO in O (640 mL) 4 (26.1 g, 122.0 mmol, 6.76 mL, 1.00 equiv) was added at 15° C., and then stirred at 15° C. for 1 h. TLC (dichloromethane:methanol=15:1, R f =0.28) showed no starting material. The reaction mixture was filtered and the filtrate was carried forward directly. Compound 23 (239.3 g, crude) was obtained as a yellow oil. The crude was carried forward directly.

[0508] Example 29. General procedure for the preparation of compound 24

[0509] [ka]

[0510] To a solution of compound 23 (79.8 g, 122.0 mmol, 1.00 equiv) in dioxane (1400 mL) was added NaBH 4 (4.62 g, 122.0 mmol, 1.00 equiv) was added at 15° C., and then the...

Claims

1. A compound having the structure of formula (II), or a pharma- ceutically acceptable salt or solvate thereof: 【Chemistry 1】 During the ceremony, Ring G is 【Chemistry 2】 and X is O, NH, or C(=O); R １ is hydrogen, deuterium, halogen, substituted or unsubstituted C １ -C １０ Alkyl, substituted or unsubstituted C ２ -C １０ Alkenyl, substituted or unsubstituted C ２ -C １０ Alkynyl, substituted or unsubstituted C １ -C １０ Fluoroalkyl, substituted or unsubstituted C １ -C ２０ Heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted -O-aryl, -OR ６ , -C(=O)R ６ , -C(=O)OR ６ , -C(=O)NR ７ R ７ , -OCH ２ C(=O)R ６ , -OCH ２ C(=O)OR ６ , or -OCH ２ C(=O)NR ７ R ７ and R ２ are each independently hydrogen, deuterium, or substituted or unsubstituted C １ -C １０ is alkyl, Or, two R's ２ are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C ３ -C １０ Forming a heterocycloalkyl; R ３ is hydrogen, 4,4'-dimethoxytrityl (DMT), -R ６ , -OR ６ or -NR ７ R ７ and R ４ each independently represents hydrogen, substituted or unsubstituted C １ -C ６ alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R ５ is -C(=O)R ６ and R ６ and R ７ each independently represents hydrogen, substituted or unsubstituted C １ -C ６ Alkyl, substituted or unsubstituted C １ -C ６ Fluoroalkyl, substituted or unsubstituted C ３ -C ６ cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; The compound, or a pharma- ceutically acceptable salt or solvate thereof.

2. R １ is halogen, substituted or unsubstituted C １ -C １０ Alkyl, substituted or unsubstituted C １ -C １０ Fluoroalkyl, substituted or unsubstituted C １ -C ２０ Heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, or substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted -O-aryl, -OH, -OR ６ , -C(=O)R ６ , -C(=O)OR ６ , -C(=O)NR ７ R ７ , -OCH ２ C(=O)R ６ , or -OCH ２ C(=O)OR ６ , or -OCH ２ C(=O)NR ７ R ７ 2. The compound of claim 1, wherein:

3. R １ is halogen, substituted or unsubstituted C １ -C １０ Alkyl, substituted or unsubstituted C １ -C ２０ Heteroalkyl, substituted or unsubstituted -O-aryl, -OCH ２ C(=O)R ６ , or -OCH ２ C(=O)OR ６ , or -OCH ２ C(=O)NR ７ R ７ 3. The compound of claim 1 or 2, wherein:

4. R １ is halogen or substituted or unsubstituted C １ -C ２０ 4. The compound of any one of claims 1-3, or a pharma- ceutically acceptable salt or solvate thereof, which is heteroalkyl.

5. R １ teeth 【Chemistry 3】 and n is 0, 1, 2, 3, 4, or 5; and R ８ is hydrogen or a substituted or unsubstituted C １ -C ３ 5. The compound of any one of claims 1-4, or a pharma- ceutically acceptable salt or solvate thereof, wherein R is an alkyl group.

6. R ８ is a substituted or unsubstituted C １ -C ３ 6. The compound of claim 5, or a pharma- ceutically acceptable salt or solvate thereof, wherein: R is an alkyl group;

7. R ８ is methyl or ethyl, or a pharma- ceutically acceptable salt or solvate thereof.

8. 8. The compound of any one of claims 5-7, wherein n is 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof.

9. R １ Ha-OCH ２ C(=O)NR ７ R ７ and R ７ Each independently represents hydrogen or a substituted or unsubstituted C １ -C ３ 4. The compound of any one of claims 1-3, or a pharma- ceutically acceptable salt or solvate thereof, wherein R is an alkyl group.

10. Ring G is 【Chemistry 4】 and R ４ The compound of any one of claims 1-9, or a pharma- ceutically acceptable salt or solvate thereof, wherein each is independently hydrogen or substituted or unsubstituted benzoyl.

11. Ring G is 【Chemistry 5】 and R ４ are each independently hydrogen or substituted or unsubstituted benzoyl, and R ５ is -C(=O)R ６ 10. The compound of any one of claims 1-9, which is: or a pharma- ceutically acceptable salt or solvate thereof.

12. Ring G is 【Chemistry 6】 and R ５ -C(=O)CH(CH ３ ) ２ 12. The compound of any one of claims 1-9 or 11, which is: or a pharma- ceutically acceptable salt or solvate thereof.

13. R ２ are each independently hydrogen, deuterium, or substituted or unsubstituted C １ -C １０ 13. The compound of any one of claims 1-12, or a pharma- ceutically acceptable salt or solvate thereof, wherein: R is an alkyl group;

14. R ２ each independently represents a substituted or unsubstituted C １ -C １０ 14. The compound of any one of claims 1-13, or a pharma- ceutically acceptable salt or solvate thereof, wherein R is an alkyl group.

15. R ２ or a pharma- ceutically acceptable salt or solvate thereof. The compound of any one of claims 1-14, wherein each is independently isopropyl.

16. Two R's ２ are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C ３ -C １０ 13. The compound of any one of claims 1-12, or a pharma- ceutically acceptable salt or solvate thereof, which forms a heterocycloalkyl.

17. X is O and R ３ The compound of any one of claims 1-16, or a pharma- ceutically acceptable salt or solvate thereof, wherein is 4,4'-dimethoxytrityl (DMT).

18. A compound having the structure of formula (IIa), or a pharma- ceutically acceptable salt or solvate thereof: 【Chemistry 7】 During the ceremony, Ring G is 【Chemistry 8】 and X is O, NH, or C(=O); n is 0, 1, 2, 3, or 4; R ８ is a substituted or unsubstituted C １ -C ６ is alkyl, R ２ are each independently hydrogen, deuterium, or substituted or unsubstituted C １ -C １０ is alkyl, Or, two R's ２ are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C ３ -C １０ Forming a cycloalkyl; R ３ is hydrogen, 4,4'-dimethoxytrityl (DMT), -R ６ , -OR ６ or -NR ７ R ７ and R ４ each independently represents hydrogen, substituted or unsubstituted C １ -C ６ alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R ５ is -C(=O)R ６ and R ６ and R ７ each independently represents hydrogen, substituted or unsubstituted C １ -C ６ Alkyl, substituted or unsubstituted C １ -C ６ Fluoroalkyl, substituted or unsubstituted C ３ -C ６ cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; The compound, or a pharma- ceutically acceptable salt or solvate thereof.

19. Ring G is 【Chemistry 9】 and R ４ 20. The compound of claim 18, or a pharma- ceutically acceptable salt or solvate thereof, wherein each is independently hydrogen, acetylisobutyl, or substituted or unsubstituted benzoyl.

20. Ring G is 【Chemistry 10】 20. The compound of claim 18 or 19, which is: or a pharma- ceutically acceptable salt or solvate thereof.

21. Ring G is 【Chemistry 11】 and R ４ are each independently hydrogen or substituted or unsubstituted benzyl, and R ５ is -C(=O)R ６ 20. The compound of claim 18, wherein:

22. Ring G is 【Chemistry 12】 and R ５ -C(=O)CH(CH ３ ) ２ 22. The compound of claim 18 or 21, which is: or a pharma- ceutically acceptable salt or solvate thereof.

23. R ６ is a substituted or unsubstituted C １ -C ６ 23. The compound of claim 22, or a pharma- ceutically acceptable salt or solvate thereof, wherein: R is an alkyl group;

24. R ８ is methyl or ethyl, or a pharma- ceutically acceptable salt or solvate thereof.

25. 25. The compound of any one of claims 18-24, wherein n is 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof.

26. 26. The compound of any one of claims 18-25, wherein n is 1, or a pharma- ceutically acceptable salt or solvate thereof.

27. R ２ are each independently hydrogen, deuterium, or substituted or unsubstituted C １ -C １０ 27. The compound of any one of claims 18-26, or a pharma- ceutically acceptable salt or solvate thereof, wherein: R is an alkyl group;

28. R ２ each independently represents a substituted or unsubstituted C １ -C １０ 28. The compound of any one of claims 18-27, or a pharma- ceutically acceptable salt or solvate thereof, wherein: R is an alkyl group;

29. R ２ or a pharma- ceutically acceptable salt or solvate thereof. The compound of any one of claims 18-28, wherein each is independently isopropyl.

30. Two R's ２ are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C ３ -C １０ 27. The compound of any one of claims 18-26, or a pharma- ceutically acceptable salt or solvate thereof, which forms a heterocycloalkyl.

31. R ３ The compound of any one of claims 18-30, or a pharma- ceutically acceptable salt or solvate thereof, wherein is 4,4'-dimethoxytrityl (DMT).

32. 【Chemical 13】 【Chemistry 14】 32. The compound of any one of claims 1-31, selected from: or a pharma- ceutically acceptable salt or solvate thereof.

33. 33. The compound of any one of claims 1-32, or a pharma- ceutically acceptable salt or solvate thereof, wherein said compound is suitable for the synthesis of oligonucleotides.

34. 【Catalogue 15】 【Chemistry 16】 A compound suitable for the synthesis of an oligonucleotide selected from:

35. An oligonucleotide conjugate of formula (I), A-B Formula (I) During the ceremony, A is a linking moiety, B is a compound of formula (II-1), (II-2), or (II-3), or an oligonucleotide comprising a compound derived from formula (II-1), (II-2), or (II-3), 【Chemistry 17】 During the ceremony, Ring G is 【Chemistry 18】 and X is O, NH, or C(=O); L １ and L ２ are each independently absent or a linking moiety selected from a phosphodiester bond, an amide bond, and an ester bond; R １ is hydrogen, deuterium, halogen, substituted or unsubstituted C １ -C １０ Alkyl, substituted or unsubstituted C ２ -C １０ Alkenyl, substituted or unsubstituted C ２ -C １０ Alkynyl, substituted or unsubstituted C １ -C １０ Fluoroalkyl, substituted or unsubstituted C １ -C ２０ Heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted -O-aryl, -OR ６ , -C(=O)R ６ , -C(=O)OR ６ , -C(=O)NR ７ R ７ , -OCH ２ C(=O)R ６ , -OCH ２ C(=O)OR ６ , or -OCH ２ C(=O)NR ７ R ７ and R ２ are each independently hydrogen, deuterium, or substituted or unsubstituted C １ -C １０ is alkyl, Or, two R's ２ are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C ３ -C １０ Forming a heterocycloalkyl; R ３ is hydrogen, 4,4'-dimethoxytrityl (DMT), -R ６ , -OR ６ or -NR ７ R ７ and R ４ each independently represents hydrogen, substituted or unsubstituted C １ -C ６ alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted benzoyl; R ５ is -C(=O)OR ６ and R ６ and R ７ each independently represents hydrogen, substituted or unsubstituted C １ -C ６ Alkyl, substituted or unsubstituted C １ -C ６ Fluoroalkyl, substituted or unsubstituted C ３ -C ６ The oligonucleotide conjugate is cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

36. The oligonucleotide conjugate of claim 35, wherein B is an oligonucleotide comprising a compound of formula (II-1).

37. The oligonucleotide conjugate of claim 35, wherein B is an oligonucleotide comprising a compound of formula (II-2).

38. The oligonucleotide conjugate of claim 35, wherein B is an oligonucleotide comprising a compound derived from formula (II-3).

39. The oligonucleotide conjugate of any one of claims 35-38, wherein the oligonucleotide is an RNA oligonucleotide.

40. 40. The oligonucleotide conjugate of any one of claims 35-39, comprising at least one modification.

41. The oligonucleotide conjugate of any one of claims 35-40, comprising at least one 2' modified nucleotide.

42. 42. The oligonucleotide conjugate of any one of claims 35-41, comprising at least one 2' modified nucleotide selected from a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleotide.

43. 43. The oligonucleotide conjugate of any one of claims 35-42, comprising at least one 2' modified nucleotide selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

44. 44. The oligonucleotide conjugate of any one of claims 35-43, comprising at least one modified internucleotide bond.

45. 44. The oligonucleotide conjugate of any one of claims 35-43, comprising at least three modified internucleotide linkages.

46. 45. The oligonucleotide conjugate of any one of claims 35-44, comprising at least one modified internucleotide bond selected from a phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, or amide bond.

47. The oligonucleotide conjugate of claim 35, wherein the compound of formula (II-2) is located at the 3'-end of the oligonucleotide.

48. The oligonucleotide conjugate of claim 35, wherein the compound of formula (II-1) is located at an internal position within the oligonucleotide.

49. The oligonucleotide conjugate of any one of claims 35-48, wherein formula (I) further comprises C to form ABC (formula IA), and A and C are not attached to B at the same end.

50. 50. The oligonucleotide conjugate of claim 49, wherein the compound of formula (II-2) is located at the 5'-end of the oligonucleotide and the linking moiety is conjugated to the 3'-end of the oligonucleotide.

51. 50. The oligonucleotide conjugate of claim 49, wherein the compound of formula (II-2) is located at the 3'-end of the oligonucleotide and the binding moiety is conjugated to the 5'-end of the oligonucleotide.

52. The oligonucleotide conjugate of claim 49, wherein the compound of formula (II-1) is located internally of the oligonucleotide and the linking moiety is conjugated to the 5'-end or 3'-end of the oligonucleotide.

53. The oligonucleotide conjugate of any one of claims 49-52, wherein the binding moiety comprises an antibody or a binding fragment thereof.

54. 54. The oligonucleotide conjugate of any one of claims 49-53, wherein the binding moiety comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

55. 50. The oligonucleotide conjugate of claim 49, wherein the binding moiety comprises a peptide or a small molecule.

56. The oligonucleotide conjugate of any one of claims 35-55, comprising from about 8 to about 50 nucleotides.

57. The oligonucleotide conjugate of any one of claims 35-56, comprising from about 10 to about 30 nucleotides.

58. B is, comprises an RNA oligonucleotide; having a length of about 10 to about 30 nucleotides; at least one 2' modified nucleotide; and 58. The oligonucleotide of any one of claims 35-57, comprising at least one modified internucleotide linkage.

59. The oligonucleotide of any one of claims 35-58, wherein B hybridizes to at least 8 contiguous bases of the target gene sequence.

60. 60. The oligonucleotide of any one of claims 35-59, wherein B mediates RNA interference.

61. The oligonucleotide of any one of claims 35-60, wherein B is the sense strand.

62. The oligonucleotide of claim 61, wherein B hybridizes to a second oligonucleotide to form a double-stranded oligonucleic acid molecule.

63. 63. The oligonucleotide of claim 62, wherein the second oligonucleotide is an antisense strand.

64. 64. The oligonucleotide of claim 62 or 63, wherein the second oligonucleotide is an RNA oligonucleotide.

65. The oligonucleotide of any one of claims 58-64, wherein the second oligonucleotide comprises at least one modification.

66. The oligonucleotide of any one of claims 58-65, wherein the second oligonucleotide comprises a compound of formula (II) or a compound of formula (IIa).

67. 67. The oligonucleotide of claim 66, wherein the second oligonucleotide comprises at least one 2' modified nucleotide.

68. 68. The oligonucleotide of claim 66 or 67, wherein the second oligonucleotide comprises at least one 2' modified nucleotide selected from a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleotide.

69. The oligonucleotide of any one of claims 66-68, wherein the second oligonucleotide comprises at least one 2' modified nucleotide selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

70. The oligonucleotide of any one of claims 66-69, wherein the second oligonucleotide comprises at least one modified internucleotide bond.

71. The oligonucleotide of any one of claims 66-70, wherein the second oligonucleotide comprises at least one modified internucleotide bond selected from a phosphorothioate bond or a phosphorodithioate bond.

72. 50. The oligonucleotide of claim 49, wherein C is a polymer.

73. 73. The oligonucleotide of claim 72, wherein the polymer comprises polyethylene glycol.

74. The oligonucleotide comprises a sense strand and an antisense strand, The sense strand is is an RNA oligonucleotide, conjugated to a binding moiety, polymer, or combination thereof; about 10 to about 30 nucleotides, at least one 2' modified nucleotide; and at least one modified internucleotide linkage, and The antisense strand is is an RNA oligonucleotide, about 10 to about 30 nucleotides, at least one 2' modified nucleotide; and 74. The oligonucleotide of any one of claims 65-73, comprising at least one modified internucleotide linkage.

75. 11. A method of treating a subject having a disease or disorder characterized by impaired expression of a protein, the method comprising administering to the subject an oligonucleotide conjugate of claims 35-74 to regulate expression of a gene encoding the protein, thereby treating the disease or disorder characterized by impaired expression of the protein.

76. 11. A method of treating a subject having a disease or condition characterized by overexpression of a protein, the method comprising administering to the subject an oligonucleotide conjugate of claims 35-74 to modulate expression of a gene encoding the protein, thereby treating the disease or condition characterized by overexpression of the protein.

77. 77. The method of claim 75 or 76, wherein the disease or condition is cancer.

78. 77. The method of claim 75 or 76, wherein the disease or condition is a neuromuscular disease, a muscular dystrophy, muscle atrophy, muscle wasting, a genetic disease, cancer, a genetic disease, or a cardiovascular disease.

79. The method of any one of claims 75-78, wherein the subject is a human.

80. A kit comprising a compound of any one of claims 1-32 or an oligonucleotide conjugate of any one of claims 35-74.

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