Nucleic acid-polypeptide composition and use thereof

Abstract

To provide an oligonucleotide in production of pharmaceutical formulations for treating a subject suffering from cancer, neuromuscular disease, muscular dystrophy, muscular atrophy, muscle wasting, genetic disease, hereditary disease, or cardiovascular disease.SOLUTION: Provided is an oligonucleotide containing a compound of formula (IIa), and in the formula, R1 each are independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; L1, L2 and L3 are selected such that L1-L2-L3 may be equal to (a)-(CH2)3-, (b)-(CH2)4-, (c)-(CH2)5-, or (d)-(C2H2)(CH2)2-; and J is a binding group between nucleotides that binds to an adjacent nucleotide of the oligonucleotide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 858,285, filed June 6, 2019, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 6, 2020, is named 45532-734_601_SL.txt and is 3,143,382 bytes in size. [Background technology]

[0004] 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 that span 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

[0005] In certain embodiments, compositions and pharmaceutical formulations are disclosed herein that include a polynucleic acid molecule and a binding moiety conjugated to a polymer. Also described herein, in some embodiments, are methods of treating a disease or disorder (e.g., cancer) that utilize compositions and pharmaceutical formulations that include a polynucleic acid molecule and a binding moiety conjugated to a polymer.

[0006] In an embodiment, a compound of Formula (II) is disclosed herein:

[0007] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; R 2 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl; Or two R's 2 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 forming a heterocycloalkyl, L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0008] In some instances, L 2 is a bond, O, S, or NR3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C5-C8 arylene, phenylene, or cyclohexyl. 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene. 1 is C1-C5 alkylene, and L 3 is C1-C5 alkylene. In some embodiments, L 2 is methylene, a bond, O, S, or NR 3 is.

[0009] In some instances, R 1 are each independently substituted or unsubstituted C1-C6 alkyl. In some examples, R 2 are each independently substituted or unsubstituted C1-C6 alkyl, CH3, -CH2CH3, -CH2CH2CH3, or -CH2(CH3)2. In some examples, the compound is selected from the group consisting of:

[0010] [ka]

[0011] [ka] R xis H, halogen, unsubstituted or substituted C-C alkyl, unsubstituted or substituted C-C fluoroalkyl, unsubstituted or substituted C-C heteroalkyl, unsubstituted or substituted monocyclic carbocycle, unsubstituted or substituted monocyclic heterocycle, -CN, -OH, -O-alkyl, -COH, -CO-alkyl, -CHCOH, -CHCO-alkyl, -C(=O)NH, -C(=O)NH-alkyl, -CHC(=O)NH, -CHC(=O)NH-alkyl, NH, -NH-alkyl, -CHNH, -CHNH-alkyl, -NHC(=O)alkyl, -CHNHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SOH, -SO-alkyl, -SONH, or -SONH-alkyl.

[0012] Also disclosed is an oligonucleotide comprising at one of its termini a compound of formula (IIa):

[0013] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; J is an internucleotide linking group that connects adjacent nucleotides of the polynucleotide, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0014] In some examples, the oligonucleotide is an RNA oligonucleotide. In some examples, the oligonucleotide further comprises at least one modification or at least one 2'-modified nucleotide. In some embodiments, the oligonucleotide further comprises at least one 2'-modified nucleotide selected from 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 nucleotides. In some embodiments, the oligonucleotide further comprises at least one 2'-modified nucleotide selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA), and at least one modified internucleotide linkage. In some embodiments, the oligonucleotide further comprises at least one modified internucleotide linkage selected from a phosphorothioate linkage, a phosphorodithioate linkage, a methylphosphonate linkage, a phosphotriester linkage, or an amide linkage.

[0015] In some examples, the compound of formula (IIa) is located at the 5'-end of the oligonucleotide. In some examples, the oligonucleotide is conjugated to a binding moiety. In some embodiments, the compound of formula (IIa) is located at the 5'-end of the oligonucleotide and the binding moiety is conjugated to the 3'-end of the oligonucleotide.

[0016] In some examples, the binding moiety comprises an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof 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. In some embodiments, the binding moiety comprises a peptide, an aptamer, or a small molecule.

[0017] In some examples, the oligonucleotide comprises from about 8 to about 50 nucleotides, or from about 10 to about 30 nucleotides.

[0018] In some examples, the oligonucleotide is an RNA oligonucleotide, is conjugated to the binding site, is about 10 to about 30 nucleotides, comprises at least one 2'-modified nucleotide, and comprises at least one modified internucleotide linkage. In some examples, the oligonucleotide hybridizes to at least 8 adjacent bases of the target gene sequence. In some examples, the oligonucleotide mediates RNA interference. In some embodiments, the oligonucleotide is a sense strand. In some embodiments, the oligonucleotide 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 at least one 2'-modified nucleotide. In some embodiments, the second oligonucleotide comprises at least one 2'-modified nucleotide selected from 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 nucleotides. In some embodiments, the second oligonucleotide comprises at least one 2'-modified nucleotide selected from locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some embodiments, the second oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, the second oligonucleotide comprises at least one modified internucleotide linkage selected from a phosphorothioate linkage, a phosphorodithioate linkage, a methylphosphonate linkage, a phosphotriester linkage, or an amide linkage.

[0019] In some embodiments, the oligonucleotide comprises a polymer, hi some embodiments, the oligonucleotide comprises polyethylene glycol.

[0020] In some embodiments, the oligonucleotide comprises a first strand and a second strand, wherein the first strand 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, comprises at least one 2'-modified nucleotide, and comprises at least one modified internucleotide linkage, and the second strand is an antisense strand, is an RNA oligonucleotide, is about 10 to about 30 nucleotides, comprises at least one 2'-modified nucleotide, and comprises at least one modified internucleotide linkage.

[0021] In another embodiment, disclosed herein is an oligonucleotide conjugate of formula (I): AB Formula (I) During the ceremony, A is a binding moiety, B is an oligonucleotide comprising a nucleotide compound of formula (IIa),

[0022] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3, substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; J is an internucleotide linking group that connects adjacent nucleotides of the polynucleotide, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0023] In some examples, the oligonucleotide conjugate further includes C to form formula ABC (formula IA).

[0024] In certain embodiments, disclosed herein are methods for inhibiting expression of a target gene in primary cells of a patient, the method comprising administering to the primary cells a molecule as described above. In some embodiments, the method is an in vivo method. In some embodiments, the patient is human. Disclosed herein are methods for treating a subject with a disease or condition characterized by impaired protein expression, the method comprising administering to the subject an oligonucleotide as described herein to regulate expression of a gene encoding the protein, thereby treating the disease or condition characterized by impaired protein expression. Further disclosed herein are methods for treating a subject with a disease or condition characterized by overexpression of a protein, the method comprising administering to the subject an oligonucleotide 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 examples, the disease or condition is a neuromuscular disease, a genetic disease, a muscular dystrophy, muscle atrophy, muscle wasting, cancer, a genetic disease, or a cardiovascular disease in a human or mammal.

[0025] In certain embodiments, disclosed herein are immuno-oncology therapies comprising the above-described molecules for the treatment of a disease or disorder in a patient.

[0026] In certain embodiments, disclosed herein are kits comprising such molecules, oligonucleotides, or oligonucleotide conjugates. [Brief explanation of the drawings]

[0027] 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 1]1 shows a cartoon representation of a polynucleotide duplex comprising a compound of formula (II) attached to the 5' end of the guide (antisense) strand, and further comprising a linking moiety A that can be attached directly to the polynucleotide or via a linker to the 5' end of the passenger (sense) strand (top), the 3' end of the passenger (sense) strand (middle), or the 3' end of the guide (antisense) strand (bottom). [Figure 2A] Graphs of relative %SSB mRNA levels in various cell types are shown. Figure 2A shows a plot of concentration (nM) versus %SSB mRNA in HCT116 cells transfected with SSB-siRNA as described in Molecular Biology Example 1. [Figure 2B] 2A and 2B show graphs of relative %SSB mRNA levels in various cell types. Figure 2B shows a plot of concentration (nM) versus relative %SSB mRNA levels (% of untreated control) for DM1 control myoblasts transfected with SSB siRNA as described in Molecular Biology Example 2. [Figure 2C] Figure 2C shows a graph of relative %SSB mRNA levels in various cell types. Figure 2C shows a plot of concentration (nM) versus relative %SSB mRNA levels (% of untreated control) for SJCRH30 transfection with SSB siRNA as described in Molecular Biology Example 2. [Figure 3A] Graphs showing relative % MSTN mRNA levels in various cell types are shown. Figure 3A shows a plot of concentration (nM) versus relative MSTN mRNA levels (% of untreated control) for DM1 control myoblasts using MSTN siRNA as described in Molecular Biology Example 2. [Figure 3B] Figure 3B shows a graph of relative % MSTN mRNA levels in various cell types. Figure 3B shows a plot of concentration (nM) versus relative MSTN mRNA levels (% of untreated control) for SJCRH30 transfection with MSTN siRNA as described in Molecular Biology Example 2. [Figure 3C]1 shows a graph of relative %MSTN mRNA levels upon introduction of MSTN siRNA conjugates with various modified nucleotides into SJCRH30 cells. [Figure 3D] 1 shows a graph of relative %SSB mRNA levels upon transfection of SSB siRNA conjugates with various modified nucleotides into SJCRH30 cells. [Figure 4] 1 shows in vivo MSTN mRNA downregulation in (top left) gastrocnemius, (bottom left) quadriceps, (top right) diaphragm, and (bottom right) tibialis anterior muscles one week after intravenous administration of antibody-siRNA conjugates at 0.1, 0.3, 1.0, and 3.0 mg / kg as described in Molecular Biology Example 3. [Figure 5] 1 shows plots of siRNA concentration versus in vivo MSTN mRNA downregulation in (top left) gastrocnemius, (bottom left) quadriceps, (top right) diaphragm, and (bottom right) tibialis anterior muscles one week after intravenous administration of antibody-siRNA conjugates as described in Molecular Biology Example 3. [Figure 6] 1 shows in vivo MSTN mRNA downregulation in (top left) gastrocnemius, (bottom left) quadriceps, (top right) diaphragm, and (bottom right) tibialis anterior muscles one week after intravenous administration of antibody-siRNA conjugates as described in Molecular Biology Example 3. [Figure 7] As described in Molecular Biology Example 3, another in vivo dose-response study shows in vivo MSTN mRNA downregulation in (top left) gastrocnemius, (bottom left) quadriceps, (top right) diaphragm, and (bottom right) tibialis anterior muscles one week after intravenous administration of an antibody-siRNA conjugate. [Figure 8A] Graphs of in vivo MSTN mRNA downregulation and siRNA concentrations in gastrocnemius muscle in two separate studies are shown in Figures 8A and 8B. Figures 8A and 8B show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in gastrocnemius muscle in one in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 8B]Graphs of in vivo MSTN mRNA downregulation and siRNA concentrations in gastrocnemius muscle in two separate studies are shown in Figures 8A and 8B. Figures 8A and 8B show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in gastrocnemius muscle in one in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 8C] Figures 8C and 8D show graphs of in vivo MSTN mRNA downregulation and siRNA concentrations in gastrocnemius muscle in two separate studies. Figures 8C and 8D show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in gastrocnemius muscle in another in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 8D] Figures 8C and 8D show graphs of in vivo MSTN mRNA downregulation and siRNA concentrations in gastrocnemius muscle in two separate studies. Figures 8C and 8D show in vivo MSTN mRNA downregulation and MSTN siRNA concentrations in gastrocnemius muscle in another in vivo study measuring mRNA concentrations on days 7, 14, 21, 28, and 35. [Figure 9] 1 shows a dose-response graph of in vivo SSB mRNA downregulation in the liver by different types of SSB-siRNA, as described in Molecular Biology Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0028] Nucleic acid (e.g., RNAi) therapy is a targeted therapy that boasts high selectivity and specificity. However, in some cases, nucleic acid therapy is also hindered by poor cellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications of nucleic acid compositions have been 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.

[0029] In some embodiments, the arrangement or order of the various components comprising a nucleic acid composition further affects cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, when a 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 affects cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0030] In some embodiments, the present disclosure provides oligonucleotide conjugates in which the arrangement of the nucleic acid moiety results in cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. In some examples, the oligonucleotide conjugate comprises a binding moiety conjugated to a polynucleic acid molecule and a polymer. In some embodiments, the oligonucleotide conjugate comprises a compound of formula (II):

[0031] [ka]

[0032] In some embodiments, oligonucleotide conjugates comprising binding moieties conjugated to polynucleic acid molecules and polymers arranged as described herein enhance cellular 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 nonspecific immune stimulation. In some cases, the oligonucleotide conjugate comprises a compound of Formula (II).

[0033] [ka]

[0034] In additional embodiments, described herein are kits that include one or more of the molecules described herein.

[0035] Therapeutic Molecular Platforms In some embodiments, the oligonucleotide conjugates described herein (e.g., therapeutic oligonucleotide conjugates) comprise a linking moiety conjugated to a polynucleic acid molecule and a polymer comprising one or more modified nucleotides. In some embodiments, the oligonucleotide conjugate comprises a compound of Formula (I) or Formula (IA): AB Formula (I) (In the formula, A is a linking moiety, and B is an oligonucleotide comprising a compound of formula (II), ABC 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).

[0036] In some embodiments, the oligonucleotide comprises a compound of formula (II).

[0037] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; R 2 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl; Or two R's 2together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 forming a heterocycloalkyl, L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0038] In some embodiments of the compound of Formula (II), L 2 is a bond, O, S, or NR 3 In some embodiments, L 2 is O, S, or NR 3 In some embodiments, L 2 is NR 3 In some embodiments, L 2 is O. In some embodiments, L 2 is S. In some embodiments, L 2 is a bond.

[0039] In some embodiments of the compound of Formula (II), L 2 is a substituted or unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is a substituted or unsubstituted C5-C8 arylene. In some embodiments, L 2 is unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is unsubstituted C5-C8 arylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is cyclohexyl.

[0040] In some embodiments of the compound of Formula (II), L 1 is a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene, and L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene. 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene. In some embodiments, L 1 is C1-C5 alkylene, and L 3 is C1-C5 alkylene.

[0041] In some embodiments of the compound of Formula (II), L 1 , L 2 , and L 3 In some embodiments, at least two of L 1 is a bond. In some embodiments, L3 is a bond.

[0042] In some embodiments of the compound of Formula (II), R 1 are each independently substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 heteroalkyl. 1 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 1 are each independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R 1 are each independently -CH, -CHCHCH, or -CH(CH). In some embodiments, R 1 is -CH3.

[0043] In some embodiments of the compound of Formula (II), R 2 are each independently substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 heteroalkyl. 2 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 2 are each independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R 2 are each independently -CH, -CHCHCH, or -CH(CH). In some embodiments, R 2 are -CH2(CH3)2, respectively.

[0044] In some embodiments of the compound of Formula (II), two R 2 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 Forms a heterocycloalkyl.

[0045] In some embodiments of the compound of Formula (II), R 3is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, and unsubstituted or substituted C1-C6 heteroalkyl. 3 is selected from unsubstituted or substituted monocyclic carbocycles and unsubstituted or substituted monocyclic heterocycles. 3 is hydrogen.

[0046] In some embodiments of the compound of Formula (II), the compound is selected from the group consisting of:

[0047] [ka]

[0048] [ka] In the formula, R x is H, halogen, unsubstituted or substituted C-C alkyl, unsubstituted or substituted C-C fluoroalkyl, unsubstituted or substituted C-C heteroalkyl, unsubstituted or substituted monocyclic carbocycle, unsubstituted or substituted monocyclic heterocycle, -CN, -OH, -O-alkyl, -COH, -CO-alkyl, -CHCOH, -CHCO-alkyl, -C(=O)NH, -C(=O)NH-alkyl, -CHC(=O)NH, -CHC(=O)NH-alkyl, NH, -NH-alkyl, -CHNH, -CHNH-alkyl, -NHC(=O)alkyl, -CHNHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SOH, -SO-alkyl, -SONH, or -SONH-alkyl.

[0049] In some embodiments, R x is halogen, unsubstituted or substituted C-C alkyl, unsubstituted or substituted C-C fluoroalkyl, unsubstituted or substituted C-C heteroalkyl, unsubstituted or substituted monocyclic carbocycle, unsubstituted or substituted monocyclic heterocycle.x is H—CN, —OH, —O-alkyl, —COH, —CO-alkyl, —CHCOH, —CHCO-alkyl, —C(═O)NH, —C(═O)NH-alkyl, —CHC(═O)NH, —CHC(═O)NH-alkyl, NH, —NH-alkyl, —CHNH, —CHNH-alkyl, —NHC(═O)alkyl, —CHNHC(═O)alkyl, —SH, —S-alkyl, —S(═O)H, —S(═O)alkyl, —SOH, —SO-alkyl, —SONH, or —SONH-alkyl. In some embodiments, R x is halogen. In some embodiments, R x is H.

[0050] In some embodiments, the oligonucleotide comprises a compound of formula (III):

[0051] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; R 2 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl; Or two R's 2 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 forming a heterocycloalkyl, L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3, substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0052] In some embodiments, B is an oligonucleotide comprising a 3'-end and a 5'-end, one of the ends comprising a compound of Formula (IIa). In some embodiments, B is an oligonucleotide comprising a 3'-end and a 5'-end, the 5'-end comprising a compound of Formula (IIa).

[0053] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3, substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; J is an internucleotide linking group that connects adjacent nucleotides of the polynucleotide, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0054] In some embodiments of the compound of Formula (IIa), L 2 is a bond, O, S, or NR 3 In some embodiments, L 2 is O, S, or NR 3 In some embodiments, L 2 is NR 3 In some embodiments, L 2 is O. In some embodiments, L 2 is S. In some embodiments, L 2 is a bond.

[0055] In some embodiments of the compound of Formula (IIa), L 2 is a substituted or unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is a substituted or unsubstituted C5-C8 arylene. In some embodiments, L 2 is unsubstituted C4-C7 cycloalkylene. In some embodiments, L2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is unsubstituted C5-C8 arylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is cyclohexyl.

[0056] In some embodiments of the compound of Formula (IIa), L 1 is a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene, and L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene. 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene. In some embodiments, L 1 is C1-C5 alkylene, and L 3 is C1-C5 alkylene.

[0057] In some embodiments of the compound of Formula (IIa), L 1 , L 2 , and L 3 In some embodiments, at least two of L 1 is a bond. In some embodiments, L 3 is a bond.

[0058] In some embodiments of Formula (IIa), R 1 are each independently substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 heteroalkyl. 1are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 1 are each independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R 1 are each independently -CH, -CHCHCH, or -CH(CH). In some embodiments, R 1 is -CH3.

[0059] In some embodiments of Formula (IIa), R 2 are each independently substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 heteroalkyl. 2 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 2 are each independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R 2 are each independently -CH, -CHCHCH, or -CH(CH). In some embodiments, R 2 are -CH2(CH3)2, respectively.

[0060] In some embodiments of Formula (IIa), two R 2 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 Forms a heterocycloalkyl.

[0061] In some embodiments of Formula (IIa), R 3 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, and unsubstituted or substituted C1-C6 heteroalkyl. 3 is selected from unsubstituted or substituted monocyclic carbocycles and unsubstituted or substituted monocyclic heterocycles. 3 is hydrogen.

[0062] 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, 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.

[0063] 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, wherein 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 will contain about 12 nucleosides substituted with 12 compounds of Formula (II) (e.g., Formula IIa).

[0064] In some embodiments, the oligonucleotide conjugate is a molecule as exemplified.

[0065] [ka]

[0066] In some embodiments, the oligonucleotide conjugate is a molecule as exemplified.

[0067] [ka]

[0068] The above

[0069] [ka] are for illustrative purposes only and include a humanized antibody or binding fragment thereof, an anti-human antibody, an anti-murine antibody (e.g., an anti-mouse antibody, an anti-rat antibody), 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.

[0070] Additional Modifications In some examples, the additional modifications include synthetic or artificial nucleotide analogs or bases that contain modifications in one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or a combination thereof.

[0071] In some embodiments, modifications at the 2' hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluoro, 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).

[0072] In some embodiments, nucleotide analogs comprise modified bases, including 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)propionyl. propyluridine, 5-halocytidine, 5-halolidine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (such as 7-deaza-adenosine, 6-azolidine, 6-azacytidine, or 6-azothymidine), 5-methyl-2-thiazolinone, 5 ... These include auridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queusine, archaeosine, naphthyl, and substituted naphthyl groups, any O- 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 function as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. 5'-phosphonate modified nucleotides further include those nucleotides with modifications on the sugar moiety, as well as 5'-phosphonate 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 also includes what are known in the art as universal bases. Exemplary universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0073] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a 5'-phosphonate modified nucleotide nucleic acid having a modification at the 5' hydroxyl group of the ribose moiety. In some embodiments, the nucleotide analog or artificial nucleotide base comprises a 5'-vinylphosphonate modified nucleotide nucleic acid having a modification at the 5' hydroxyl group of the ribose moiety.

[0074] 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 its cellular uptake properties due to its zwitterionic properties.

[0075] In some instances, the 5'-phosphonate modified nucleotide is further modified at the 2' hydroxyl group in a locked or bridged ribose modified (e.g., locked nucleic acid or LNA), where 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. An exemplary representation of the chemical structure of a 5'-phosphonate modified LNA is illustrated below, where J is an internucleotide bond.

[0076] [ka]

[0077] In some embodiments, additional modifications include morpholinos, peptide nucleic acids (PNAs), methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or 1',5'-anhydrohexitol nucleic acids (HNAs). Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics that of natural nucleic acids but deviates from the normal sugar and phosphonate structures. 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 cases, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some cases, backbone changes remove all positive and negative charges, allowing the morpholino neutral molecules to cross cell membranes without the aid of cellular delivery agents, as used with charged oligonucleotides. Non-limiting examples of 5'-phosphonate modified morpholino oligonucleotides are illustrated below.

[0078] [ka]

[0079] In some embodiments, the 5'-phosphonate modified morpholino or PMO described above is a PMO that contains a positive charge or a cationic charge. In some examples, the PMO is PMOplus (Sarepta). PMOplus refers to a phosphorodiamidate morpholino oligomer that contains 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 the PMO described in U.S. Patent No. 7,943,762.

[0080] In some embodiments, the morpholino or PMO is PMO-X (Sarepta). In some instances, PMO-X refers to a phosphorodiamidate morpholino oligomer containing 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.

[0081] In some embodiments, the morpholino or PMO is a PMO as described in Table 5 of U.S. Patent Application Publication No. 2014 / 0296321.

[0082] 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 backbone charge.

[0083] [ka]

[0084] In some embodiments, one or more of the above modifications occur at the internucleotide bond. In some examples, the modified internucleotide bond includes, 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' linked boranophosphate and selenophosphate, phosphotriester, thionoalkylphosphotriester, hydrogen phosphonate bond, alkyl phosphonate, alkyl phosphonothioate, aryl phosphonothioate, phosphoroselenoate, phosphorodiselenoate, phosphinate, phosphoramidate, 3'-alkyl phosphoramidate, aminoalkyl phosphoramidate, thionophosphoamidate, phosphoropiper. These include 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, morpholino structure in which the base is bonded directly or indirectly to the aza nitrogen of the backbone, amide, or polyamide bond, and combinations thereof.

[0085] In some examples, the modification is a methyl or thiol modification, such as a methylphosphonate modification or a thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left), phosphorodithioates (center), and methylphosphonate nucleotides (right) are illustrated below.

[0086] [ka]

[0087] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, phosphoramidites exemplified as follows:

[0088] [ka]

[0089] In some instances, the modified internucleotide linkage is a phosphorodiamidate linkage. Non-limiting examples of phosphorodiamidate linkages with morpholino systems are shown below:

[0090] [ka]

[0091] In some instances, the modified internucleotide linkage is a methylphosphonate linkage. Non-limiting examples of methylphosphonate linkages are shown below:

[0092] [ka]

[0093] In some instances, the modified internucleotide bond is an amide bond. Non-limiting examples of amide bonds are shown below:

[0094] [ka]

[0095] 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 to 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.

[0096] In some embodiments, the further modified phosphate backbone comprises a methyl phosphonate, an ethyl phosphonate, a methylthiophosphonate, or a methoxyphosphonate. In some embodiments, the modified phosphate is a methyl phosphonate. In some embodiments, the modified phosphate is an ethyl phosphonate. In some embodiments, the modified phosphate is a methylthiophosphonate. In some embodiments, the modified phosphate is a methoxyphosphonate.

[0097] In some embodiments, the additional modified phosphate backbone comprises one of the following:

[0098] [ka]

[0099] In some embodiments, one or more modifications further optionally include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications of the 3'- or 5'-terminal nucleotide analog. For example, the 3'-terminus optionally includes a 3' cationic group, or the nucleoside is inverted at the 3'-terminus with a 3'-3' linkage. In another alternative, the 3'-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3'C5-aminoalkyl dT. In a further alternative, the 3'-terminus is optionally conjugated with an abasic site, e.g., an apurinic or apyrimidinic site.

[0100] In some embodiments, oligonucleotides comprising a compound of Formula (II) (e.g., Formula IIa) further comprise one or more artificial nucleotide analogs described herein. In some embodiments, oligonucleotides comprising a 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, methylphosphonate nucleotides, thiolphosphonate 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-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyloxyethyl ... and 2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5' phosphoramidite, or a combination thereof.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 2'-O-methyl 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 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 thiolphosphonate nucleotides.

[0101] In some examples, about 5 to about 100% of oligonucleotides comprising a compound of Formula (II) (e.g., Formula IIa) contain an artificial nucleotide analog 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 contain an artificial nucleotide analog 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) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof.

[0102] In some embodiments, the oligonucleotides described herein (or B in the AB formula) comprise RNA or DNA. In some cases, the oligonucleotide comprises RNA. In some examples, the RNA comprises small interfering RNA (siRNA), short 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.

[0103] In some embodiments, the oligonucleotide is about 10 to about 50 nucleotides in length, and 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.

[0104] In some embodiments, the oligonucleotide is about 50 nucleotides in length. In some instances, the oligonucleotide is about 45 nucleotides in length. In some instances, the oligonucleotide is about 40 nucleotides in length. In some instances, the oligonucleotide is about 35 nucleotides in length. In some instances, the oligonucleotide is about 30 nucleotides in length. In some instances, the oligonucleotide is about 25 nucleotides in length. In some instances, the oligonucleotide is about 20 nucleotides in length. In some instances, the oligonucleotide is about 19 nucleotides in length. In some instances, the oligonucleotide is about 18 nucleotides in length. In some instances, the oligonucleotide is about 17 nucleotides in length. In some instances, the oligonucleotide is about 16 nucleotides in length. In some instances, the oligonucleotide is about 15 nucleotides in length. In some instances, the oligonucleotide is about 14 nucleotides in length. In some instances, the oligonucleotide is about 13 nucleotides in length. In some instances, the oligonucleotide is about 12 nucleotides in length. In some instances, the oligonucleotide is about 11 nucleotides in length. In some instances, the oligonucleotide is about 10 nucleotides in length. In some instances, the oligonucleotide is about 10 to about 50 nucleotides in length. In some instances, the oligonucleotide is about 10 to about 45 nucleotides in length. In some instances, the oligonucleotide is about 10 to about 40 nucleotides in length. 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.

[0105] 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 passenger strand. In some examples, the oligonucleotide is an antisense strand or guide strand.

[0106] In some embodiments, the second oligonucleotide is about 10 to about 50 nucleotides in length, or 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.

[0107] 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 30 nucleotides in length.In some examples, the first oligonucleotide is about 19 to about 23 nucleotides in length, and in some examples, the second oligonucleotide is about 12 to about 30 nucleotides in length.

[0108] 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.

[0109] 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 a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule is 100% complementary to the target sequence described herein.

[0110] In some embodiments, the sequence of the polynucleic acid molecule has five or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has four or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has three or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has two or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has one or fewer mismatches to the target sequences described herein.

[0111] In some embodiments, the specificity of a polynucleic acid molecule that hybridizes 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 instances, hybridization is under high stringency hybridization conditions.

[0112] 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 a target sequence 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 an oncogene or any of its mRNAs, any gene associated with a genetic or inherited disease (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 polynucleic acid molecule hybridizes to at least 8 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 9 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 10 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 11 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 12 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 13 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 14 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 15 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 16 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 17 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 18 contiguous bases of a target sequence described herein.In some embodiments, the polynucleic acid molecule hybridizes to at least 19 contiguous bases of a target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 20 contiguous bases of a target sequence described herein.

[0113] 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 in which a polynucleic acid polymer directed to a specific target causes an unintended effect by directly or indirectly interacting with another mRNA sequence, DNA sequence, or cellular protein or other moiety. In some instances, an "off-target effect" occurs when there is simultaneous degradation of other transcripts due to partial homology or complementarity between the other transcripts and the sense and / or antisense strands of the polynucleic acid molecule.

[0114] In some cases, one or more of the artificial nucleotide analogs described herein are resistant to nucleases, such as ribonucleases such as RNase H, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases, compared to naturally occurring polynucleic acid molecules. In some examples, a compound 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) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or an artificial nucleotide analogue including a combination thereof, is RNase inhibitory. Resistance 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., resistant to RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases). In some examples, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease-resistant (e.g., resistant to RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases). In some examples, the 2'-O-aminopropyl modified polynucleic acid molecule is nuclease resistant (eg, RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, 2'-deoxy modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, 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., RNase H, 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., RNase H, 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., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, LNA-modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, ENA-modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance).In some examples, HNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). Morpholinos are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, PNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, methylphosphonate nucleotide-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, thiophosphonate nucleotide-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). 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.

[0115] In some embodiments, one or more of the artificially modified nucleotide analogs described herein, including compounds of Formula II (e.g., Formula IIa), have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleotide acid molecule. The compound of Formula II, one or more of the modified nucleotide analogs, including 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 LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, or 2'-fluoro N3-P5' phosphoramidite, can have increased binding affinity to its mRNA compared to the equivalent naturally occurring polynucleic acid molecule. In some instances, 2'-O-methyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-O-aminopropyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-deoxy modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-deoxy-2'-fluoro modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules.In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, LNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, ENA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, PNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, HNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, morpholino-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, methylphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, thiolphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules.In some cases, increased affinity is demonstrated by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0116] In some embodiments, modified nucleotide analogs comprising 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.

[0117] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugate moiety. In some examples, the aptamer-conjugate moiety is a DNA aptamer-conjugate moiety. In some examples, the aptamer-conjugate moiety is Alphamer (Centauri Therapeutics), which includes 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 an aptamer-conjugate moiety as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0118] 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 molecule is 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 a locked or bridged ribose structure (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is 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'-fluoroN3-P5'-phosphoramidite to increase its stability. In some examples, the polynucleic acid molecule is a chiral pure (or stereopure) polynucleic acid molecule. In some examples, the chiral pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications of RNA to increase the stability of delivery will be apparent to those skilled in the art.

[0119] In some embodiments, the polynucleic acid molecules described herein have RNAi activity that regulates the expression of 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, wherein one strand of the double-stranded siRNA molecule comprises a nucleotide sequence complementary to the nucleotide sequence of the gene or RNA encoded by the gene or a portion thereof, and the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the gene or 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, wherein each strand of the siRNA molecule comprises about 15-25, 18-24, or 19-23 nucleotides, and each strand comprises at least about 14, 17, or 19 nucleotides complementary to the nucleotides of the other strand. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate gene expression, wherein each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides complementary to nucleotides in the other strand.

[0120] 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).

[0121] One embodiment provides an oligonucleotide conjugate of formula (I) or formula (IA): AB Formula (I), ABC Formula (IA) During the ceremony, A is a binding moiety, C is optionally a polymer; B is a nucleotide compound of formula (II) or an oligonucleotide comprising a nucleotide compound derived from formula (II) (e.g., the phosphoramidite group of formula (II) is converted to a phosphate group in the oligonucleotide structure);

[0122] [ka] During the ceremony, R 1 are each independently substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, or substituted or unsubstituted C1-C6 heteroalkyl; R 2 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C6 alkyl, or substituted or unsubstituted C1-C6 heteroalkyl; Or two R's 2 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 forming a heterocycloalkyl, L 1 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene; L 2 is a bond, O, S, NR 3 , substituted or unsubstituted C4-C7 cycloalkylene, substituted or unsubstituted C4-C7 heterocycloalkylene, substituted or unsubstituted C5-C8 arylene, or substituted or unsubstituted C4-C8 heteroarylene; R 3 is, when present, selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, unsubstituted or substituted C1-C6 heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L 3is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene, and L 1 , L 2 , and L 3 At least two of these are not bonds.

[0123] In some embodiments of the compound of Formula (II), L 2 is a bond, O, S, or NR 3 In some embodiments, L 2 is O, S, or NR 3 In some embodiments, L 2 is NR 3 In some embodiments, L 2 is O. In some embodiments, L 2 is S. In some embodiments, L 2 is a bond.

[0124] In some embodiments of the compound of Formula (II), L 2 is a substituted or unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is a substituted or unsubstituted C5-C8 arylene. In some embodiments, L 2 is unsubstituted C4-C7 cycloalkylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is unsubstituted C5-C8 arylene. In some embodiments, L 2 is phenylene. In some embodiments, L 2 is methylene. In some embodiments, L 2 is cyclohexyl.

[0125] In some embodiments of the compound of Formula (II), L 1is a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C2-C5 alkenylene, or a substituted or unsubstituted C2-C5 alkynylene, and L 3 is a bond, substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C2-C5 alkenylene, or substituted or unsubstituted C2-C5 alkynylene. 1 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene, and L 3 is C1-C5 alkylene, C1-C3 alkenylene, or C1-C5 alkynylene. In some embodiments, L 1 is C1-C5 alkylene, and L 3 is C1-C5 alkylene.

[0126] In some embodiments of the compound of Formula (II), L 1 , L 2 , and L 3 In some embodiments, at least two of L 1 is a bond. In some embodiments, L 3 is a bond.

[0127] In some embodiments of Formula (II), R 1 are each independently substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 heteroalkyl. 1 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 1 are each independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R 1 are each independently -CH, -CHCHCH, or -CH(CH). In some embodiments, R 1 is -CH3.

[0128] In some embodiments of Formula (II), R 2are each independently substituted or unsubstituted C1-C6 alkyl or substituted or unsubstituted C1-C6 heteroalkyl. 2 are each independently substituted or unsubstituted C1-C6 alkyl. In some embodiments, R 2 are each independently -CH, -CHCH, -CHCHCH, or -CH(CH). In some embodiments, R 2 are each independently -CH, -CHCHCH, or -CH(CH). In some embodiments, R 2 are -CH2(CH3)2, respectively.

[0129] In some embodiments of Formula (II), two R 2 together with the nitrogen atom to which they are attached, form a substituted or unsubstituted C2-C 10 Forms a heterocycloalkyl.

[0130] In some embodiments of Formula (II), R 3 is selected from hydrogen, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted C1-C6 fluoroalkyl, and unsubstituted or substituted C1-C6 heteroalkyl. 3 is selected from unsubstituted or substituted monocyclic carbocycles and unsubstituted or substituted monocyclic heterocycles. 3 is hydrogen.

[0131] In some embodiments of the oligonucleotide conjugate of Formula (I), B is a compound having the structure of Formula (II).

[0132] One embodiment provides an oligonucleotide conjugate of formula (Xa): AX-B'-YC Formula (Xa) During the ceremony, A is a binding moiety, B' is a polynucleotide compound of formula (II), C is optionally a polymer; X is a single bond or a first linker, and Y is a single 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.

[0133] Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the polynucleotide further comprises at least one modified internucleotide linkage or at least one inverted abasic moiety.

[0134] Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein a compound of Formula (IIa) is positioned at an internucleotide linkage of the polynucleotide.

[0135] Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the compound of formula (IIa) is further modified at the 2' position.

[0136] Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein one or more additional unnatural 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-methylacetamido (2'-O-NMA) modified nucleotide.

[0137] Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the compound of formula (IIa) is selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

[0138] Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein at least one inverted abasic moiety is at least one terminus.

[0139] Another embodiment provides an oligonucleotide of formula (I) or (Xa), wherein the oligonucleotide is single-stranded.Another embodiment provides an oligonucleotide of formula (Xa), wherein the oligonucleotide is double-stranded.

[0140] Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 100 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 90 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 80 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 70 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 60 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 50 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 40 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 30 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 20 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 2 to about 10 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 8 to about 30 residues in length. Another embodiment provides an oligonucleotide of Formula (Xa), wherein the oligonucleotide is 10 to about 30 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 14 to about 30 residues in length. Another embodiment provides an oligonucleotide of Formula (Xa), wherein the oligonucleotide is 18 to about 30 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 22 to about 30 residues in length. Another embodiment provides an oligonucleotide of Formula (I) or (Xa), wherein the oligonucleotide is 26 to about 30 residues in length.

[0141] One embodiment provides a compound suitable for the synthesis of an oligonucleotide selected from the group consisting of:

[0142] [ka]

[0143] [ka] In the formula, R x is H, halogen, unsubstituted or substituted C-C alkyl, unsubstituted or substituted C-C fluoroalkyl, unsubstituted or substituted C-C heteroalkyl, unsubstituted or substituted monocyclic carbocycle, unsubstituted or substituted monocyclic heterocycle, -CN, -OH, -O-alkyl, -COH, -CO-alkyl, -CHCOH, -CHCO-alkyl, -C(=O)NH, -C(=O)NH-alkyl, -CHC(=O)NH, -CHC(=O)NH-alkyl, NH, -NH-alkyl, -CHNH, -CHNH-alkyl, -NHC(=O)alkyl, -CHNHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SOH, -SO-alkyl, -SONH, or -SONH-alkyl.

[0144] 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 include steroids, e.g., cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, polysaccharides, and the like. In some examples, the binding moiety is an antibody or binding fragment thereof. In some examples, the polynucleic acid molecule is further conjugated to a polymer and, optionally, to an endosomolytic moiety.

[0145] In some embodiments, the polynucleic acid molecule is conjugated to the binding moiety by a chemical ligation process. In some instances, the polynucleic acid molecule is conjugated to the binding moiety by native ligation. In some examples, the conjugation is as 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.

[0146] In some instances, polynucleic acid molecules are conjugated to binding moieties in a site-directed manner using "traceless" coupling technology (PhiloChem). In some instances, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group of a binding moiety that is conjugated to a polynucleic acid molecule containing an aldehyde group. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13): 5887-5892 (2012)).

[0147] In some examples, polynucleic acid molecules are conjugated to binding moieties by site-directed methods utilizing unnatural amino acids 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)).

[0148] In some instances, polynucleic acid molecules are conjugated to binding moieties by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Redwood). In some instances, SMARTag™ technology involves the generation of a formylglycine (FGly) residue from cysteine ​​by formylglycine generating enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent conjugation of FGly to an alkylhydrazine-functionalized polynucleic acid molecule via 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)).

[0149] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some examples, the polynucleic acid molecule is conjugated to a 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 glutamine in the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some examples, mTG is produced by Streptomyces mobaraensis. (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)).

[0150] In some examples, the polynucleic acid molecule is conjugated to the binding moiety by methods such as those described in PCT International Publication No. WO2014 / 140317, which utilize sequence-specific transpeptidases.

[0151] In some examples, the polynucleic acid molecule is conjugated to a binding moiety by methods such as those described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0152] 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 comprises a humanized 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 (e.g., an anti-human transferrin receptor antibody), 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 Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (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.

[0153] 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 Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (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 Fab2. In some examples, A is a single chain variable fragment (scFv).

[0154] In some embodiments, binding moiety A is a bispecific antibody or binding fragment thereof. In some examples, the bispecific antibody is a trispecific antibody or a bispecific miniantibody. In some cases, the bispecific antibody is a trispecific antibody. In some examples, the trispecific antibody is a full-length monoclonal antibody that contains binding sites for two different antigens. Exemplary trispecific 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), 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 mAb2 from F-star Biotechnology Ltd. In some examples, A is a bispecific trifunctional antibody.In some embodiments, A is selected from the group consisting of 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), RG7221 (RO5520985; angiopoietin The bispecific trifunctional antibodies were selected from the following: mAb 2 (targeting GD2 / 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 mAb 2 (from F-star Biotechnology Ltd.).

[0155] In some instances, the bispecific antibody is a bispecific miniantibody. In some examples, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE). 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).

[0156] In some embodiments, binding moiety A is a bispecific miniantibody. In some examples, A is a bispecific Fab2. In some examples, A is a bispecific F(ab)'3 fragment. Optionally, A is a bispecific bis-scFv. Optionally, A is a bispecific (scFv)2. In 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), such as DART (dual affinity retargeting platform; MacroGenics), blinatumomab (MT103, or AMG103; targets CD19 / CD3; Micromet), MT111 (targets CEA / CD3; Micromet / Amegen), MT112 (BAY2010112; targets PSMA / CD3; Micromet / Bayer), MT110 (AMG The bispecific mini-antibodies selected from the following: 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).

[0157] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody comprises a F(ab)'3 fragment or a triabody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, 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).

[0158] 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 cancer cells. Exemplary cancer antigens include, but are not limited to, alphafetoprotein, 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 (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), transferrin receptor, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), and the like. In some examples, the binding moiety is an α-transferrin receptor antibody or binding fragment thereof.In some examples, the binding moiety is an α-human transferrin receptor antibody. In some examples, the binding moiety is an α-human transferrin receptor antibody such as those described in PCT / US2019 / 068078, which is incorporated herein by reference.

[0159] 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.

[0160] 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. 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. PAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein (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.

[0161] 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, CD4 9f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-Select CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g. CD79a, CD79b), C The antibody or fragment thereof recognizes D90, 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.

[0162] In some embodiments, the antibody or binding fragment thereof 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 bogatox (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, GlaxoWellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblitutuzumab (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), dilentuximab (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), nesbacumab (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 (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 These include ublituximab (BMS-663513, Bristol-Myers Squibb), urelumab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), and zatuximab.

[0163] 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), asclinbacumab (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 bogatox (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, GlaxoWellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblitutuzumab (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), dilentuximab (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), nesbacumab (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 (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).

[0164] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. In some examples, the plasma protein includes albumin. In some examples, 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 a lysine bond.

[0165] In some examples, the linking 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 linking 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.

[0166] 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 the 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.

[0167] In some instances, the binding moiety A is a peptide. In some instances, the peptide comprises about 1 to about 3 kDa. In some instances, 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 instances, the peptide is a bicyclic peptide. In some instances, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the peptide moiety is a bicyclic peptide (e.g., Bicycle Therapeutics' Bicycle).

[0168] In further instances, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some instances, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, and the target-binding end can recognize and interact with a cell surface receptor. For example, in some instances, the target-binding end comprising a glutamic acid urea compound allows 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).

[0169] 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 a growth factor or mitogen, a receptor tyrosine kinase, a cytoplasmic tyrosine kinase, a cytoplasmic serine / threonine kinase, a regulatory GTPase, or a transcription factor. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of an oncogene selected from a growth factor or mitogen, a receptor tyrosine kinase, a cytoplasmic tyrosine kinase, a cytoplasmic serine / threonine kinase, a regulatory GTPase, or a transcription factor.

[0170] 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.

[0171] In some embodiments, the oncogenes described herein include KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or a β-catenin-related gene. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or a β-catenin-related gene. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of KRAS. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of EGFR. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of AR. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of CNNTB1 (β-catenin). In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of a CNNTB1 (β-catenin)-related gene. In some examples, 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.

[0172] Polynucleic acid molecules targeting Kirsten rat sarcoma viral oncogene homolog (KRAS) Kirsten rat sarcoma viral oncogene homolog (GTPase KRas, also known as V-Ki-ras Kirsten rat sarcoma viral oncogene homolog, or KRAS) is involved in the regulation of cell division. The K-Ras protein is a GTPase belonging to the Ras superfamily. In some instances, K-Ras regulates cell cycle progression and induces growth arrest, apoptosis, and replicative senescence under various environmental triggers (e.g., cellular stress, ultraviolet light, heat shock, or ionizing radiation). In some cases, mutations in the KRAS gene have been linked to cancer development, although wild-type KRAS genes have been shown to be frequently lost during tumor progression in various types of cancer. In some instances, KRAS amplification has also been implicated in cancer development (see, e.g., Valtorta et al., "KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy," Int. J. Cancer 133: 1259-1266 (2013)). In such cases, the cancer is associated with a refractory cancer in which the patient has developed resistance to a particular inhibitor or class of inhibitors.

[0173] In some embodiments, the KRAS gene is wild-type or contains mutations. In some examples, the KRAS mRNA is wild-type or contains mutations. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to the target region of wild-type KRAS DNA or RNA. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to the target region of KRAS DNA or RNA that contains mutations (e.g., substitutions, deletions, or additions).

[0174] In some embodiments, the KRAS DNA or RNA comprises one or more mutations. In some embodiments, the KRAS DNA or RNA comprises one or more mutations at codon 12 or 13 of exon 1. In some examples, the KRAS DNA or RNA comprises one or more mutations at codon 61, 63, 117, 119, or 146. In some examples, the KRAS DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or a combination thereof, of a KRAS polypeptide. In some embodiments, the KRAS DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a combination thereof in a KRAS polypeptide.

[0175] In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations at codon 12 or 13 in exon 1. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations at codon 61, 63, 117, 119, or 146. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA containing one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or a combination thereof, of a KRAS polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA that contains one or more mutations corresponding to an amino acid residue selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a combination thereof in a KRAS polypeptide.

[0176] In some embodiments, binding moiety A is conjugated to a polynucleic acid molecule (B) described herein and optionally a polymer (C) according to formula (I). In some examples, polymer C comprises a polyalkylene oxide (e.g., polyethylene glycol).

[0177] 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 binding fragment thereof.

[0178] In some embodiments, binding moiety A conjugates non-specifically to polynucleic acid molecule (B). In some examples, binding moiety A conjugates non-site-specifically to polynucleic acid molecule (B) via a lysine residue or a cysteine ​​residue. In some examples, binding moiety A conjugates non-site-specifically to polynucleic acid molecule (B) via a lysine residue. In some cases, binding moiety A conjugates non-site-specifically to polynucleic acid molecule (B) via a cysteine ​​residue. In some examples, binding moiety A is an antibody or binding fragment thereof.

[0179] In some embodiments, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner. In some examples, binding moiety A is conjugated to 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, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner via a lysine residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner via a cysteine ​​residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner at the 5'-end. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner at the 3'-end. In some examples, binding moiety A is conjugated to 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 binding fragment thereof.

[0180] 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 to the constant region, hinge region, or 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).

[0181] 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 examples, 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 binding fragment thereof.

[0182] 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 polynucleic acid molecule (B) to 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 polynucleic acid molecule (B) to binding moiety A is about 1 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 2 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 3 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 4 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 5 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 6 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 7 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 8 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 9 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 10 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 11 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 12 or greater.

[0183] In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A (e.g., 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 polynucleic acid molecule (B) to binding moiety A is about 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 3. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 5. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 7. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 9. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 10. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 11. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 12. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 13. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 14. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 15. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 16.

[0184] In some examples, the DAR ratio of polynucleic acid molecule (B) to 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 polynucleic acid molecule (B) to binding moiety A is 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 12.

[0185] In some embodiments, the antibody or binding fragment thereof is further modified using conventional techniques known in the art, e.g., by amino acid deletion, insertion, substitution, or addition, alone or in combination, and 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 an Fc receptor. In some examples, one or more modifications include, for example, those described in International Publication No. WO 97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into nucleic acid sequences underlying the amino acid sequence of an antibody or binding fragment thereof are well known to those of skill in the art.

[0186] In some instances, an antibody binding fragment further includes derivatives thereof, and comprises a polypeptide sequence comprising at least one CDR.

[0187] In some instances, the term "single-chain" as used herein means that the first and second domains of the 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.

[0188] In some instances, bispecific single-chain antibody constructs relate to constructs comprising binding domains derived from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some instances, the scFv comprises a VH and a VL domain connected by a linker peptide. In some instances, the linker is of sufficient length and sequence to allow each of the first and second domains to retain their distinct binding specificities independently of each other.

[0189] In some embodiments, as used herein, binding with or interaction by defines binding / interaction of at least two antigen-interaction sites with each other. In some examples, the antigen-interaction site defines a polypeptide motif that exhibits a specific interaction capacity with a specific antigen or a specific group of antigens. In some cases, binding / interaction is also understood to define specific recognition. In such cases, specific recognition refers to the ability of an antibody or binding fragment thereof to specifically interact and / or bind to at least two amino acids of each of the target molecules. For example, specific recognition relates to the specificity of the antibody molecule or its ability to distinguish a specific region of the target molecule. In additional examples, the specific interaction of the antigen-interaction site with the specific antigen results in the initiation of a signal, for example, by inducing a conformational change in the antigen, antigen oligomerization, etc. In further embodiments, binding is exemplified by the specificity of the "key-lock principle." Thus, in some examples, specific motifs in the amino acid sequences 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 the result of secondary modifications of said structure. In such cases, the specific interaction of the antigen interaction site with its specific antigen results in the site's easy binding to the antigen. In some instances, specific interaction further refers to a reduction in the cross-reactivity of the antibody or its binding fragment, or a reduction in off-target effects. For example, an antibody or its binding fragment that binds to a polypeptide / protein of interest but does not essentially bind to any other polypeptides is considered specific for the polypeptide / protein of interest. Examples of specific interactions of the 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 the antigen-binding site of an antibody.

[0190] Production of antibodies or binding fragments thereof In some embodiments, the polypeptides (e.g., antibodies and binding fragments thereof) described herein 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.

[0191] 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 synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0192] 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.

[0193] In some examples, the antibody or binding fragment 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 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) or an antibody library (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937) for clones of FAb fragments that bind to a specific antigen.

[0194] In some embodiments, techniques developed for producing "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., a humanized antibody.

[0195] In some embodiments, techniques described for the production of single-chain antibodies (U.S. 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).

[0196] In some embodiments, an expression vector containing an antibody nucleotide sequence or the antibody nucleotide sequence is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0197] In some embodiments, various host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent not only the vehicle in which the antibody coding sequence is produced and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequence, express the antibody or binding fragment thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment thereof coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment thereof coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody or binding fragment thereof 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 thereof coding sequence; and 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 mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0198] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing 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 a selectable marker. 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 on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or their binding fragments.

[0199] 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 is conferred by 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); and 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 that can 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).

[0200] In some instances, antibody expression levels are increased by vector amplification (for a 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 related to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0201] 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.

[0202] Polymer-conjugated moiety In some embodiments, 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, polymer moiety C is conjugated to a polynucleic acid molecule. In some cases, polymer moiety C is conjugated to a binding moiety. In other cases, polymer moiety C is conjugated to a polynucleic acid molecule-binding moiety molecule. In further cases, polymer moiety C is conjugated as discussed in the therapeutic molecular platform section.

[0203] In some examples, polymer moiety C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or a two- or three-dimensional crosslinked network of monomers. In some examples, polymer moiety C includes a polysaccharide, lignin, rubber, or polyalkylene oxide (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, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane, 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 comprises a polyalkylene oxide. In some examples, polymer portion C comprises PEG. In some examples, polymer portion C comprises polyethyleneimide (PEI) or hydroxyethyl starch (HES).

[0204] 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, 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 native ligation.

[0205] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some instances, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by average weight (weight-average) size and dispersity. In some instances, a monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight indicated represents the average molecular weight of the polyalkylene oxide, e.g., PEG, molecules.

[0206] 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.

[0207] 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, 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, , 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, 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, 8300, 8400, 850 In some examples, 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, C has a molecular weight of about 200 Da. In some examples, C has a molecular weight of about 300 Da. In some examples, C has a molecular weight of about 400 Da. In some examples, C has a molecular weight of about 500 Da. In some examples, C has a molecular weight of about 600 Da. In some examples, C has a molecular weight of 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.

[0208] In some embodiments, the polyalkylene oxide (e.g., PEG) is a dispersed PEG, which is a polymeric PEG containing more than one repeating ethylene oxide unit. In some examples, the dispersed PEG (dPEG) contains 2 to 60, 2 to 50, or 2 to 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 contains about 24 or more repeating ethylene oxide units. In some examples, dPEG contains about 26 or more repeating ethylene oxide units. In some examples, dPEG contains about 28 or more repeating ethylene oxide units. In some examples, dPEG contains about 30 or more repeating ethylene oxide units. In some examples, dPEG contains about 35 or more repeating ethylene oxide units. In some examples, dPEG contains about 40 or more repeating ethylene oxide units. In some examples, dPEG contains about 42 or more repeating ethylene oxide units. In some examples, dPEG contains about 48 or more repeating ethylene oxide units. In some examples, dPEG contains about 50 or more repeating ethylene oxide units. In some examples, dPEG is synthesized stepwise as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials. In some examples, the dPEG has a specific molecular weight rather than an average molecular weight. In some examples, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0209] In some embodiments, the polymer moiety C comprises a cationic mucic acid-based polymer (cMPA). In some examples, the cMPA comprises one or more subunits of at least one repeating subunit, the subunit structure being represented as formula (III):

[0210] [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.

[0211] In some examples, the cMAP is further conjugated to a PEG moiety to form 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.

[0212] In some examples, polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, polymer moiety C is a cMAP-PEG copolymer. In other cases, polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In further cases, polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0213] Endosomolytic or transmembrane moieties In some embodiments, the molecule of Formula (Xa):A-X1-B'-X2-C further comprises an additional conjugate moiety. In some examples, the additional conjugate moiety is an endosomolytic moiety and / or a cell membrane-spanning moiety. In some cases, the endosomolytic moiety is a compound capable of being released from any of the cell compartments known in the art, such as a cell compartment-releasing component, e.g., an endosome, a lysosome, an endoplasmic reticulum (ER), a Golgi apparatus, a microtubule, a peroxisome, or other endoplasmic reticulum comprising a cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer. In some cases, the cell membrane-spanning moiety comprises a cell-penetrating peptide (CPP). In other cases, the cell membrane-spanning moiety comprises a cell-penetrating lipid. In other cases, the cell membrane-spanning moiety comprises a cell-penetrating small molecule.

[0214] Endosomolytic polypeptides and transmembrane polypeptides In some embodiments, the molecule of formula (Xa):A-X1-B-X2-C 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 further 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 (derived from the Simian Virus 40 large antigen and NLS from HIV reverse transcriptase), Pvec (derived from VE-cadherin), VT5 (derived from a synthetic peptide), C105Y (derived from 1-antitrypsin), transportan (derived from galanin and mastoparan), TP10 (derived from galanin and mastoparan), MPG (derived from the hydrophobic domain of the fusion sequence of the NLS of HIV gp41 and SV40 T antigen), GH625 (HSV These include those derived from the type I glycoprotein gH), CADY (PPTG1 peptide), GALA (synthetic peptide), INF (influenza HA2 fusion peptide), HA2E5-TAT (influenza HA2 subunit of the influenza virus strain X31 fusion peptide), HA2-Penetratin (influenza HA2 subunit of the influenza virus strain X31 fusion peptide), HA-K4 (influenza virus strain X31 fusion peptide HA2 subunit), HA2E4 (influenza virus strain X31 fusion peptide HA2 subunit), H5WYG (HA2 analog), GALA-INF3-(PEG)6-NH (INF3 fusion peptide), or CM18-TAT11 (cecropin A-mellitin 2-12 (CM18) fusion peptide).

[0215] In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide that induces apoptosis through antagonism of inhibitory target such as Bcl-2 and / or Bcl-xL. 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).

[0216] In some examples, the endosomolytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) such as those described in PCT International Publication No. WO2013 / 166155 or WO2015 / 069587.

[0217] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the molecule of formula (Xa):A-X1-B'-X2-C is further conjugated to an endosomolytic lipid (e.g., 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 No. WO 09 / 126,933.

[0218] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, the molecule of Formula (Xa):A-X1-B'-X2-C is further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), ampiroquine, primaquine, mefloquine, nivaquines, halofantrine, quinoneimine, or a combination thereof. In some examples, quinoline endosomolytic moieties include, but are 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, -4-(4-diethylamino-1-butylamino)quinoline, 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-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline, 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, 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-diethylamino)-1-methylbutylamino]-6-methoxyquinoline, 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline, 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline, Examples of suitable endosomolytic moieties include (amino)-1-methylbutyl-amino]-6-methoxyquinoline, 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde, 1,1'-pentamethylenequinolinium diiodide, 8-quinolinol sulfate, and their amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs. In some examples, the endosomolytic moiety is a small molecule as described in 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-penetrating polypeptide comprises a short, positively charged peptide having 5 to 30 amino acids. In some embodiments, the cell-penetrating polypeptide comprises an arginine- or lysine-rich amino acid sequence. In some embodiments, the cell-penetrating polypeptide comprises any polypeptide or combination thereof, including Antennapedia Penetratin (43-58), HIV-1 TAT protein (48-60), pVEC cadherin (615-632), transportan galanin / 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.

[0219] 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 comprises a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group). In some examples, the linker comprises 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.

[0220] 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'-pyridyldithio)bis ... (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASE D), 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).

[0221] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional 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-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-hydroxysulfosuccinimide ester (sulfo-MB), N-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 ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl6-((((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 (MCH), 3-(2-pyridyldithio) (e) 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-NH-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 (sulfo-sANPAH), N -5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), 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), sulfosuccinimidyl7-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)propane (AsIB), and 2-(4-(ρ-azidosalicylamido)butyl)-3'-(2'-pyridyldithio)propane (AsIB). benzophenone-4-iodoacetamide (APDP), benzophenone-4-maleimidecarbonyl-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).

[0222] 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.

[0223] 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 comprises 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), described above.

[0224] 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 retro-Michael elimination. 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.

[0225] In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide comprises at least 1, 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 instances, the linker comprises Val-Cit. In some instances, the linker is Val-Cit.

[0226] 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).

[0227] 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 an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0228] 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 includes a linker described in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426.

[0229] 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.

[0230] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or 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 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.

[0231] In some examples, the linker comprises 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.

[0232] 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 described in "Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation," Chem. Commun. 49:8187-8189 (2013)."

[0233] In some examples, the linker may be a polymerizable compound, such as those 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 PCT Publication Nos. WO2015057699, WO2014080251, WO2014197854, WO2014145090, or WO2014177042.

[0234] 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.

[0235] 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 a C1-C6 alkyl group. In some cases, X is a C1-C6 alkyl group, such as, for example, a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. When used in the context of a linker, particularly in the context of X, alkyl refers to 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 comprises a homobifunctional linker or heterobifunctional linker described above. In some cases, X comprises a heterobifunctional linker. In some cases, X comprises sMCC. In other examples, X comprises a heterobifunctional linker optionally conjugated to a C1-C6 alkyl group. In other examples, X comprises sMCC optionally conjugated to a C1-C6 alkyl group. In some additional examples, X does not include a homobifunctional linker or a heterobifunctional linker described above.

[0236] 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 a C1-C6 alkyl group. In some examples, Y is a homobifunctional linker or a heterobifunctional linker described above. In some examples, Y is a homobifunctional linker described above. In some examples, Y is a heterobifunctional linker described above. In some examples, Y comprises a maleimide group, such as maleimidocaproyl (mc), or a self-stabilizing maleimide group, 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 a further example, Y comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In a further example, Y comprises an mc group. In a further example, Y comprises an mc-val-cit group. In a further example, Y comprises a val-cit-PABA group. In a further example, Y comprises a mc-val-cit-PABA group.

[0237] In some examples, L is a single bond or a linker. In some examples, L is a single bond. In some examples, L is a linker. In some embodiments, L is a C1-C6 alkyl group. In some examples, L is a homobifunctional linker or a heterobifunctional linker described above. In some examples, L is a homobifunctional linker described above. In some examples, L is a heterobifunctional linker described above. In some examples, L comprises a maleimide group, such as maleimidocaproyl (mc), or a self-stabilizing maleimide group, 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 a further example, L comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In a further example, L comprises an mc group. In a further example, L comprises an mc-val-cit group. In a further example, L comprises a val-cit-PABA group. In a further example, L comprises a mc-val-cit-PABA group.

[0238] How to use In some embodiments, the compositions or pharmaceutical preparations described herein, comprising a binding moiety and a polymer conjugated to a polynucleic acid molecule, are used to treat a disease or disorder. In some examples, the disease or disorder is muscular dystrophy, muscle atrophy, and / or muscle wasting. Muscular dystrophy refers to a loss of muscle mass and / or progressive muscle weakness and degeneration. In some cases, the loss of muscle mass and / or progressive muscle weakness and degeneration 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.

[0239] cancer In some embodiments, the compositions or pharmaceutical preparations 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 relapsed or refractory cancer or a metastatic cancer. In some examples, the solid tumor is a relapsed or refractory solid tumor or a metastatic solid tumor. In some cases, the hematological malignancy is a relapsed or refractory hematological malignancy or a metastatic hematological malignancy.

[0240] 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.

[0241] In some examples, the cancer is a hematological malignancy. In some examples, the compositions or pharmaceutical formulations described herein comprising an oligonucleotide, a polymer, or a combination thereof, optionally conjugated to a binding moiety, are used to treat a hematological malignancy. In some examples, the compositions or pharmaceutical formulations described herein comprising an oligonucleotide, a polymer, or a combination thereof, optionally conjugated to a binding moiety, are used to treat a leukemia, lymphoma, myeloma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma. In some instances, hematologic 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's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and idiopathic leukemia. leukemia, Burkitt's lymphoma, non-Burkitt's high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy or a metastatic hematologic malignancy.

[0242] In some examples, the cancer is a KRAS-related, EGFR-related, AR-related, HPRT1-related, or β-catenin-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, which include an oligonucleotide, a polymer, or a combination thereof, optionally conjugated to a binding moiety, 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, which include an oligonucleotide, a polymer, or a combination thereof, optionally conjugated to a binding moiety, are used to treat KRAS-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, which include an oligonucleotide, a polymer, or a combination thereof, optionally conjugated to a binding moiety, are used to treat EGFR-related cancer. In some examples, the compositions or pharmaceutical preparations described herein, which include an oligonucleotide, a polymer, or a combination thereof, optionally conjugated to a binding moiety, are used to treat AR-related cancer. In some examples, a composition or pharmaceutical formulation described herein comprising an oligonucleotide, polymer, or combination thereof, optionally conjugated to a binding moiety, is used to treat an HPRT1-associated cancer. In some examples, a composition or pharmaceutical formulation described herein comprising an oligonucleotide, polymer, or combination thereof, optionally conjugated to a binding moiety, is used to treat a β-catenin-associated cancer. 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.

[0243] 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 (TAAs) 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, e.g., 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 a 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.

[0244] In some embodiments, the compositions or pharmaceutical formulations 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 formulations described herein comprise a tumor targeting agent. In some examples, the tumor targeting agent is encompassed by binding moiety A. In other examples, the tumor targeting agent is an additional agent used in combination with a 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 through mechanisms such as direct killing (e.g., signaling-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.

[0245] 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 through 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.

[0246] In some embodiments, the compositions or pharmaceutical formulations described herein comprise 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 comprises a cytokine, a checkpoint inhibitor, or a combination thereof.

[0247] 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.

[0248] 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.

[0249] In some embodiments, exemplary checkpoint inhibitors include:

[0250] PD-L1 inhibitors, such as Genentech's MPDL3280A (RG7446), BioXcell's anti-mouse PD-L1 antibody clone 10F.9G2 (Cat# BE0101), Bristol-Myers Squibb's anti-PD-L1 monoclonal antibodies MDX-1105 (BMS-936559) and BMS-935559, MSB0010718C, mouse anti-PD-L1 clone 29E.2A3, and AstraZeneca's MEDI4736;

[0251] PD-L2 inhibitors, e.g., GlaxoSmithKline's AMP-224 (Amplimmune), and rHIgM12B7;

[0252] PD-1 inhibitors, such as BioXcell's anti-mouse PD-1 antibody clone J43 (Cat# BE0033-2), BioXcell's anti-mouse PD-1 antibody clone RMP1-14 (Cat# BE0146), mouse anti-PD-1 antibody clone EH12, Merck's MK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab, lambrolizumab), AnaptysBio's anti-PD-1 antibody known as ANB011, antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4 monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106), AstraZeneca's AMP-514 and AMP-224, and CureTech Ltd's pidilizumab (CT-011);

[0253] 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;

[0254] LAG3 inhibitors, such as eBioscience's anti-Lag-3 antibody clone eBioC9B7W (C9B7W), LifeSpan Biosciences' anti-Lag3 antibody LS-B2237, Immutep's IMP321 (ImmuFact), anti-Lag3 antibody BMS-986016, and LAG-3 chimeric antibody A9H12;

[0255] B7-H3 inhibitors, for example, MGA271;

[0256] KIR inhibitors, e.g., Lirilumab (IPH2101);

[0257] CD137 (41BB) inhibitors, such as urelumab (BMS-663513 Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

[0258] PS inhibitors, e.g., Bavituximab; and inhibitors, e.g., 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.

[0259] 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, such as 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, CD137, CD160, CD226, CD276, DR Binding moiety A comprising an inhibitor of 3, 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).

[0260] In some embodiments, a 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 selected from the group consisting 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 diseases or disorders (e.g., cancer).

[0261] 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 (or 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.

[0262] 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 approximately 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0263] 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.

[0264] Tumor necrosis factors (TNFs) are a group of cytokines that regulate apoptosis. In some instances, there are approximately 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).

[0265] 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).

[0266] In some embodiments, a composition or pharmaceutical formulation described herein comprises 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, a 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, a 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, a molecule of Formula (I) in combination with an antigen vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a 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, a molecule of Formula (I) in combination with a DNA vaccine is used to treat a disease or disorder (e.g., cancer). In some examples, a molecule of Formula (I) in combination with a vector-based vaccine is used to treat a disease or disorder (e.g., cancer).

[0267] 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.

[0268] In some embodiments, a molecule of Formula (I) in combination 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).

[0269] In some embodiments, a molecule of Formula (I) in combination with a natural killer (NK) T cell-based therapeutic agent is used to treat a disease or disorder (e.g., cancer). In some examples, the NK-based therapeutic agent is an activating NK agent that recognizes one or more of the CD4+ cell surface markers described above. In some cases, the NK-based therapeutic agent is 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. 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).

[0270] In some embodiments, a molecule of Formula (I) in combination with a CAR-T cell-based therapeutic is used to treat a disease or disorder (e.g., cancer).

[0271] In some embodiments, the molecule of formula (I) is used in combination with an additional agent that destabilizes the endosomal membrane (or disrupts 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.

[0272] 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).

[0273] 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 strength in a subject's diseased, damaged, or disused muscle tissue compared to the same muscle tissue in a control subject. In some embodiments, 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 in a control subject. In another embodiment, a significant decrease in muscle strength refers to a decrease in strength of disused muscle tissue compared to the muscle strength of the same muscle tissue in the same subject before a period of disuse. In some embodiments, a significant decrease in muscle strength refers to a decrease 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 in the same subject before a period of disuse.

[0274] In another embodiment, muscular dystrophy refers to a significant decrease in muscle mass. A significant decrease in muscle mass refers to a decrease in muscle volume in a subject's diseased, damaged, or disused muscle tissue compared to the same muscle tissue in a control subject. In one embodiment, a significant decrease in muscle volume refers to a decrease 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 in a control subject. In another embodiment, a significant decrease in muscle mass refers to a decrease in muscle volume in disused muscle tissue compared to the muscle volume in the same muscle tissue in the same subject before a period of disuse. In one embodiment, a significant decrease in muscle tissue refers to a decrease 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 volume in the same muscle tissue in 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).

[0275] Myotonic dystrophy is a multisystemic neuromuscular disease that includes two major types: 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 post-transcriptional splicing and polyadenylation site regulation, and loss of MBNL protein function causes downstream accumulation of nuclear foci, increasing mis-splicing events and subsequently leading to myotonia and other clinical symptoms. In some embodiments, methods for treating muscular dystrophy, muscle atrophy, and / or muscle wasting in a subject are described herein, the methods comprising providing a polynucleic acid molecule or a polynucleic acid molecule conjugate described herein to the subject and administering a therapeutically effective amount of the polynucleic acid molecule or polynucleic acid molecule conjugate to treat the 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.

[0276] 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 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.

[0277] 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.

[0278] 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 solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions. 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.

[0279] In some instances, the nanoparticles comprise a core or a core and a shell, such as core-shell nanoparticles.

[0280] 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, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some examples, the nanoparticles comprise graphene-coated nanoparticles.

[0281] In some cases, the nanoparticles have at least one dimension that is less than about 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

[0282] 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 nano-onion, a nanorod, a nanorope, or a quantum dot. In some examples, a 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.

[0283] In some embodiments, pharmaceutical formulations include 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, taurocholate, 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).

[0284] 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 within an acceptable range.

[0285] In some examples, pharmaceutical formulations 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.

[0286] In some instances, pharmaceutical formulations further include a diluent used to stabilize the compound, as the diluent can provide a more stable environment. Salts dissolved in buffers (which can further control or maintain pH) are utilized as diluents in the art, including, but not limited to, phosphate buffered saline solution. In some instances, the diluent increases the volume of the composition to facilitate compression or create sufficient volume for a homogeneous blend 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.

[0287] In some cases, pharmaceutical formulations contain disintegrants or disintegrants that promote the breakdown 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, e.g., corn starch or potato starch, pregelatinized starches, e.g., National 1551 or Amijel®, or sodium starch glycolate, e.g., Promogel® or Explotab®, cellulose, e.g., wood products, methyl crystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Min Examples of suitable crosslinking agents include Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or crosslinked celluloses such as crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®, crosslinked carboxymethylcellulose, or crosslinked croscarmellose sodium), crosslinked starches 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.

[0288] 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, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0289] 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.

[0290] Plasticizers include compounds used to soften microencapsulation materials or film coatings to reduce brittleness. 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 can also act as dispersing or wetting agents.

[0291] Solubilizing agents include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, docusate sodium, 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.

[0292] Stabilizers include any mixture of antioxidants, buffers, acids, preservatives, and the like.

[0293] Suspending agents include, for example, polyvinylpyrrolidone such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (for example, the polyethylene glycol may 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, cellulose 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.

[0294] 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, poloxamers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., Octoxynol 10, Octoxynol 40. Surfactants are often included to enhance physical stability or for other purposes.

[0295] Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0296] 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.

[0297] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic use. In some embodiments, the pharmaceutical compositions are administered once a day, twice a day, three times a day, or more. The pharmaceutical compositions are administered daily, 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 compositions are 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.

[0298] 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., the first administration of a first pharmaceutical composition is on day 1, followed by at least 1, 2, 3, 4, 5, or more days before the administration of at least a second pharmaceutical composition).

[0299] In some embodiments, two or more different pharmaceutical compositions are co-administered. In some instances, two or more different pharmaceutical compositions are co-administered. In some instances, two or more different pharmaceutical compositions are co-administered sequentially with no interval between administrations. In other instances, two or more different pharmaceutical compositions are co-administered sequentially with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or more between administrations.

[0300] If the patient's condition improves, at the physician's discretion, administration of the composition may continue, or the administered dose of the composition may be temporarily reduced or temporarily discontinued for a specified period of time (i.e., a "drug holiday"). In some examples, the length of the drug holiday may vary from 2 days to 1 year, and may include, 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%.

[0301] Once the patient's condition has improved, a maintenance dose is administered if necessary, after which the dosage and / or frequency of administration are optionally reduced, depending on the symptoms, to a level at which the improved disease, disorder, or condition is maintained.

[0302] 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 requiring treatment, and the like, but will nevertheless be routinely determined by methods 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, e.g., two, three, four or more sub-doses per day.

[0303] Because of the large number of variables associated with any particular treatment regimen, the foregoing ranges are only suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending upon many 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 practitioner.

[0304] In some embodiments, the toxicity and therapeutic efficacy of such therapeutic 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. 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. The dosage will vary within this range depending upon the dosage form used and the route of administration utilized.

[0305] Kits / Products In some embodiments, the present disclosure provides 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 divided to contain one or more containers, such as vials, tubes, and each container is equipped with 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, the container is made of various materials, such as glass or plastic.

[0306] 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 appropriate for the selected formulation and intended mode of administration and treatment.

[0307] 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 their use in the manner described herein.

[0308] Kits typically include 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.

[0309] 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, for example, 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, for example, in the manner described herein.

[0310] In some embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms comprising a compound 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 may be, for example, labeling approved by the U.S. Food and Drug Administration for prescription drugs or approved package inserts. In one embodiment, compositions comprising a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated disease.

[0311] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. It is understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used in 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 word "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0312] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" also 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.

[0313] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0314] 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 professional (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker).

[0315] chemical definition 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.

[0316] 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., -CH2O- is equivalent to -OCH2-.

[0317] 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 monovalent, divalent, and polyvalent radicals having the specified number of carbon atoms (i.e., C-C 10means 1 to 10 carbons). An 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, and homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those containing 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. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (—O—).

[0318] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from alkyl, as exemplified, but not limited to, by -CHCHCHCH-. 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 8 or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0319] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise specified, stable straight or branched chains, or combinations thereof, containing at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom can be optionally quaternized. The heteroatom (e.g., N, S, Si, or P) can 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-cyclizing chain. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH-2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. A heteroalkyl moiety may contain one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety can contain up to eight optionally different heteroatoms (e.g., O, N, S, Si, or P).

[0320] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified by, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for 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, the formula -C(O)R'- represents both -C(O)R'- and -R'C(O)-. As noted above, heteroalkyl groups as used herein include groups that are attached to the remainder of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -S0R'. When "heteroalkyl" is recited after a recitation of a specific heteroalkyl group, such as -NR'R'', it is understood that 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 specific heteroalkyl groups, such as -NR'R''.

[0321] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. 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. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. "Cycloalkyl" is also intended to refer to bicyclic and polycyclic hydrocarbon rings, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like.

[0322] 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 intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0323] 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.

[0324] The term "aryl," unless otherwise specified, refers to a polyunsaturated, aromatic, hydrocarbon substituent, which may be fused together (i.e., fused-ring aryl) or covalently linked, and may comprise one or more rings (preferably 1 to 3 rings). 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 five members and the other has six members, and where at least one ring is a heteroaryl ring. Similarly, a 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 a 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. The heteroaryl group can be attached to the rest 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.

[0325] Spirocycles are two or more rings in which adjacent rings are connected via one atom. The individual rings within a spirocycle can be the same or different. The individual rings in a spirocycle can be substituted or unsubstituted and can have different substituents than the other individual rings within a set of spirocycles. The possible substituents for the individual rings within a spirocycle are the possible substituents for the same ring when not part of a spirocycle (e.g., substituents for a cycloalkyl ring or heterocycloalkyl ring). The spirocycle can be a substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heterocycloalkylene, and the individual rings within the spirocycle group can be any of the rings in the list immediately above, including all rings of one type (e.g., all rings are substituted heterocycloalkylene, and each ring can 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 heterocyclic 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.

[0326] The following symbols

[0327] [ka] indicates the point of attachment of the chemical moiety to the rest of the molecule or chemical formula.

[0328] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0329] The term "alkylarylene" refers to an arylene moiety that is covalently linked to an alkylene moiety (also referred to herein as an alkylene linker). In certain embodiments, the alkylarylene group has the formula:

[0330] [ka]

[0331] The alkylarylene moiety 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) with halogen, oxo, -N, -CF, -CCl, -CBr, -CI, -CN, -CHO, -OH, -NH, -COOH, -CONH, -NO, -SH, -SOCH-SOH, -OSOH, -SONH, -NHNH, -ONH, -NHC(O)NHNH, substituted or unsubstituted C-C alkyl, or substituted or unsubstituted 2-5 membered heteroalkyl. In certain embodiments, the alkylarylene is unsubstituted.

[0332] 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.

[0333] 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', -COR', -CONR'R'', -OC(O)NR'R'', -NR' and m may be one or more of a variety of groups selected from: -C(O)R, -NR'-C(O)NR''R''', -NR''C(O)R, -NR-C(NR'R''R'')=NR'''', -NR-C(NR'R'')=NR'''', -S(O)R', -S(O)R', -S(O)NR'R'', -NRSOR', -NR'NR''R''', -ONR'R'', -NR'C(O)NR''NR'''R'''', -CN, -NO, -NR'SOR'', -NR'C(O)R'', -NR'C(O)-OR'', -NR'OR'', where m' is the total number of carbon atoms in such radical. R, R', R'', R''', and R'''' each preferably independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1 to 3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy group, or arylalkyl group. When the compounds described herein include more than one R group, for example, each R group is independently selected as the R', R'', R''', and R'''' group, 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 discussion of substituents above, one skilled in the art will understand that the term "alkyl" is intended to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0334] 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′, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′-C(O)NR″R′″, —NR″C(O)R′, —NR-C(NR′R″R′″)═NR′″, —NR-C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSOR′, —NR′NR″R′″, —ONR′R″, —NR′C(O)NR″NR 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 the R', R'', R''', and R'''' groups, respectively, when more than one of these groups is present.

[0335] Substituents for a ring (e.g., cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be represented as substituents on the ring rather than on a specific atom of the ring (commonly referred to as floating substituents). In such cases, the substituent may be attached to any of the ring atoms (according to the rules of chemical valency), and in the case of a fused or spiro ring, a substituent represented as associated with a member of the fused or spiro ring (a floating substituent on one ring) may be a substituent on either 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, or different spiro rings, and each substituent may optionally be different. When the point of attachment of a 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, or, in the case of a fused or spiro ring, any atom of either the fused or spiro ring, subject to the rules of chemovalency. When 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 points of attachment to the rest of the molecule), the floating substituents may be bonded to the heteroatoms. When a ring heteroatom is shown as bonded to one or more hydrogens (e.g., a nitrogen atom with two bonds to ring atoms and a third bond to a hydrogen) in a structure or formula having floating substituents, it is understood that when the heteroatom is bonded to the floating substituent, the substituent replaces the hydrogen, subject to the rules of chemovalency.

[0336] 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 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 form 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 form a spiro ring structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0337] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CRR') q A ring of the formula -U- may be formed, 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 be replaced with a substituent of the formula -A-(CH2)rB-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and 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 replaced with a substituent of the formula -(CRR')s-X'-(C''R''R'') d where s and d are independently integers from 0 to 3, and X' is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The 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.

[0338] As used herein, the term "heteroatom" or "ring heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0339] "Substituents," as used herein, means groups selected from the following moieties: (a) oxo, halogen, -CF, -CN, -OH, -NH, -COOH, -CONH, -NO, -SH, -SOH, -SOH, -SONH, -NHNH, -ONH, -NHC=(O)NHNH, -NHC=(O)NH, -NHSOH, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF, -OCHF, 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: (i) oxo, halogen, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, 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, -CF3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SON2NH2, -NHNH2, -ONH2, -NHC=(O)NHNH2, -NHC=(O)NH2, -NHSO2H, -NHC=(O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF2, 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, —CF3, —CN, —OH, —NH2, —COOH, —CONH2, —NO2, —SH, —S03H, —S04H, —SON2NH2, —NHNH2, —ONH2, —NHC═(O)NHNH2, —NHC═(O)NH2, —NHS02H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF3, —OCHF2, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0340] 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-C 20 each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl; each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl; each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10The aryl and the substituted or unsubstituted heteroaryl are each substituted or unsubstituted 5-10 membered heteroaryl.

[0341] As used herein, a "lower substituent" or "lower substituent group" refers to a group selected from all of the substituents described above for "substituent," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 7-membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 The aryl and the substituted or unsubstituted heteroaryl are each substituted or unsubstituted 5- to 9-membered heteroaryl.

[0342] In some specific embodiments, each substituent described in the compounds herein is substituted with at least one substituent.More specifically, in some specific 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 specific embodiments, at least one or all of these groups are substituted with at least one size-limited substituent.In other specific embodiments, at least one or all of these groups are substituted with at least one lower-rank substituent.

[0343] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C-C20 each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 20-membered heteroalkyl; each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl; each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3- to 8-membered heterocycloalkyl; each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10 In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C-C 20 Each of the substituted or unsubstituted heteroalkylenes is a substituted or unsubstituted 2- to 20-membered heteroalkylene; each of the substituted or unsubstituted cycloalkylenes is a substituted or unsubstituted C3-C8 cycloalkylene; each of the substituted or unsubstituted heterocycloalkylenes is a substituted or unsubstituted 3- to 8-membered heterocycloalkylene; each of the substituted or unsubstituted arylenes is a substituted or unsubstituted C6-C 10 The arylene and / or substituted or unsubstituted heteroarylene are each substituted or unsubstituted 5- to 10-membered heteroarylene.

[0344] In some particular embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2-8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3-7 membered heterocycloalkyl, and each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C 10In some particular embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2-8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3-7 membered heterocycloalkylene, and each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C 10 arylene, and / or the substituted or unsubstituted heteroarylene is each 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.

[0345] Certain compounds of the present invention possess 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 can be prepared using chiral synthons or chiral reagents or resolved 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.

[0346] As used herein, the term "isomers" refers to compounds that have the same number and kind of atoms, and hence the same molecular weight, but that differ with respect to the structural arrangement or configuration of the atoms.

[0347] 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.

[0348] It will be apparent to one skilled in the art that certain compounds of this invention exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention.

[0349] 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.

[0350] 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 by 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 this invention.

[0351] 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.

[0352] Note that throughout this application, options are written in Markush groups, e.g., each amino acid position containing more than one possible amino acid. It is specifically contemplated that each member of a Markush group should be considered separately, thereby comprising alternative embodiments, and that the Markush group is not to be read as a single unit.

[0353] "Analog" or "analogous" is used according to its simple 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.

[0354] 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 specified group can be substituted with one or more of any or all of the specified substituents. For example, when a group such as an alkyl group or heteroaryl group is substituted with an unsubstituted C-C 20 When substituted with alkyl or unsubstituted 2-20 membered heteroalkyl, the group may also contain one or more unsubstituted C-C 20 The alkyl may include one or more unsubstituted 2- to 20-membered heteroalkyl groups.

[0355] 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, and each R substituent is optionally different. When a particular R group occurs in a description of a chemical genus (such as formula (I)), a roman alphabet symbol may be used to distinguish between occurrences of that particular R group. For example, multiple R 13When substituents are present, each R 13 The substituents are R 13A , R 13B , R 13C , R 13D etc., where R 13A , R 13B , R 13C , R 13D Each of these is R 13 and optionally defined differently.

[0356] 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 with one or more of many substituents, such substitutions are selected to comply with the principles of chemical bonding and to result in a compound that is not inherently unstable and / or known by 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 is bonded to the rest of the molecule through a ring heteroatom according to the principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.

[0357] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When a compound of the present invention contains a relatively acidic functionality, a base addition salt can be obtained by contacting the neutral form of such a compound 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 a compound of the present invention contains a relatively basic functionality, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid in a neat or suitable inert solvent. Examples of pharmaceutically 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 acid, as well as those derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, sublingual, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, or methanesulfonic acid. Also included are salts of amino acids, such as arginine 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.

[0358] Therefore, the compounds of the present invention may exist as salts with pharmaceutically acceptable acids, etc. 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 a mixture thereof, including a racemic mixture), 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.

[0359] The neutral forms of the compounds are 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 certain embodiments, the compounds of the present invention contain both basic and acidic functionalities that allow the compound to be converted into either a base or an acid addition salt. The neutral forms of the compounds 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 purposes of the present invention, unless otherwise specified.

[0360] In addition to salt forms, the present invention also provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily 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 a suitable enzyme or chemical reagent.

[0361] Certain compounds of the present invention can exist in unsolvated and solvated forms, including hydrated forms. Generally, 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. Generally, all physical forms are applicable to the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0362] "Contacting" is used according to its simple 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.

[0363] 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 signaling pathway.

[0364] 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 of, or the activation, sensitization, or upregulation of signal transduction or enzymatic activity, or the amount of a protein that is decreased in disease.

[0365] 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, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more over 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.

[0366] 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 disease symptom. In certain embodiments, inhibition refers to a decrease in the activity of a specific 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 enzymatic activity, or the amount of a protein. In certain embodiments, inhibition refers to a decrease in the activity of a target protein due to a direct interaction (e.g., an inhibitor binding to the target protein). In certain embodiments, inhibition refers to a decrease in the activity of a target protein from an indirect interaction (e.g., an inhibitor binding to a protein that activates the target protein, thereby preventing activation of the target protein).

[0367] The terms "inhibitor," "suppressor," "antagonist," or "downregulator" refer interchangeably to a substance capable of detectably reducing the expression or activity of a given gene or protein. Antagonists 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, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or even less than expression or activity without the antagonist. Antagonists prevent, reduce, inhibit, or neutralize the activity of an agonist, and antagonists 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 decreases, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates, for example, a gene, protein, ligand, receptor, or cell. An inhibitor may also be defined as a molecule that decreases, blocks, or inactivates constitutive activity. An "antagonist" is a molecule that opposes the action of an agonist.

[0368] 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.

[0369] The term "treating" or "treatment" refers to any indication of success in curing or ameliorating 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; or improving the patient's physical or mental well-being. 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 its conjugations can include prevention of an injury, condition, illness, or illness. In some embodiments, treating is preventing. In some embodiments, treating does not include preventing.

[0370] "Treating" or "treatment," as used herein (and as well understood in the art), broadly includes any approach for obtaining 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 the disease state; prevention of the transmission or spread of the disease; delay or slowing of disease progression; improvement or reduction of the 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.

[0371] "Treating" and "treatment," as used herein, may also include prophylactic treatment. A 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 an agent used for treatment or prevention may be increased or decreased during a particular treatment or prevention regimen. Changes in dosage may occur and be evident by standard diagnostic assays known in the art. In some cases, long-term administration may be required. For example, a composition is administered to a subject in an amount and for a period sufficient to treat the patient.

[0372] The term "prevent" 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 its progression to an adverse or otherwise undesirable state. [Example]

[0373] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. Chemical synthesis examples The compounds in Table 1 are prepared as described in the Examples below.

[0374] [Table 1-1]

[0375] [Table 1-2]

[0376] [Table 1-3]

[0377] [Table 1-4]

[0378] Example 1: 2-cyanoethyl(3-(dimethoxyphosphoryl)propyl)diisopropylphosphoramidite

[0379] [ka]

[0380] Step 1A: 3-((tert-butyldimethylsilyl)oxy)propan-1-ol

[0381] [ka]

[0382] To a solution of propane-1,3-diol (1.00 equiv.) in DMF was added DIPEA (10.0 equiv.) and TBSCl (1.05 equiv.). The mixture was stirred at 25° C. for 16 hours. TLC (petroleum ether / ethyl acetate=5 / 1, R f = 0.43), which indicated that propane-1,3-diol was completely consumed. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (100 mL), and washed with water (200 mL × 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 100 / 1 to 1 / 1).

[0383] Step 1B: tert-butyl(3-iodopropoxy)dimethylsilane

[0384] [ka]

[0385] Imidazole (1.50 equiv.) and iodine (1.35 equiv.) were added to a solution of PPh3 (1.20 equiv.) in DCM. 3-((tert-butyldimethylsilyl)oxy)propan-1-ol (1.00 equiv.) in DCM was added dropwise to the mixture, which was stirred at 25° C. for 16 hours. TLC (petroleum ether / ethyl acetate=5 / 1, Compound 3B:R f =0.89), which indicated that tert-butyl(3-iodopropoxy)dimethylsilane was completely consumed. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 1 / 0 to 100 / 1).

[0386] Step 1C: Dimethyl (3-((tert-butyldimethylsilyl)oxy)propyl)phosphonate

[0387] [ka]

[0388] To a solution of dimethylphosphonate (1.30 equiv.) in THF was added sodium hydride (1.30 equiv.) at 0° C. The mixture was stirred at 0° C. for 0.5 h and then at 70° C. for 1.5 h. The mixture was then cooled to 0° C., and tert-butyl(3-iodopropoxy)dimethylsilane (1.00 equiv.) in THF was added to the mixture and stirred at 25° C. for 24 h. TLC (petroleum ether:ethyl acetate=1:1, product: R f= 0.38), which indicated complete consumption of the starting material. The reaction mixture was quenched by the addition of NH4Cl, then diluted with ethyl acetate and washed with NH4Cl. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by column chromatography (SiO2, PMA, petroleum ether:ethyl acetate = 1:0 to 1:1).

[0389] Step 1D: Dimethyl(3-hydroxypropyl)phosphonate

[0390] [ka]

[0391] To a solution of dimethyl(3-((tert-butyldimethylsilyl)oxy)propyl)phosphonate (1.00 equiv.) in MeOH was added NHF (3.00 equiv.). The mixture was stirred at 65° C. for 3 hours. TLC (petroleum ether / ethyl acetate=0 / 1) showed that the product R f = 0.09), which indicated that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO2, KMnO4, petroleum ether / ethyl acetate = 10 / 1 to 0:1).

[0392] Step 1E: 2-cyanoethyl(3-(dimethoxyphosphoryl)propyl)diisopropylphosphoramidite

[0393] [ka]

[0394] To a solution of dimethyl(3-hydroxypropyl)phosphonate (1.00 equiv.) in DCM was added DIPEA (4.00 equiv.) and 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (1.20 equiv.) at 0° C. The mixture was stirred at 25° C. for 1 hour. TLC (petroleum ether / ethyl acetate=0 / 1) showed product R f =0.43), which indicated that the compound was completely consumed. The reaction mixture was quenched by the addition of NaHCO3, diluted with DCM, and extracted with DCM (5 mL × 2). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / TEA / DCM = 10 / 1 / 0.5% / 25% to 3 / 1 / 0.5% / 25%). 1 H NMR:400MHz,CD3CN:δ ppm 3.51-3.75(m,2H),3.55-3.70(m,10H),2.64(t,J=6.02 Hz,2H),1.76-1.83(m,4H),1.16-1.18(m,12H) 31 P NMR:162MHz,CD3CN:δ ppm 147.19(s,1P),34.03(s,1P).

[0395] Example 2: 2-cyanoethyl(4-(dimethoxyphosphoryl)butyl)diisopropylphosphoramidite

[0396] [ka]

[0397] Step 2A: 4-((tert-butyldimethylsilyl)oxy)butan-1-ol

[0398] [ka]

[0399] To a solution of butane-1,4-diol (10.0 g, 110 mmol, 9.80 mL, 1.00 equiv.) in DMF (70.0 mL), DIPEA (143 g, 1.11 mol, 193 mL, 10.0 equiv.) and TBSCl (17.5 g, 116 mmol, 14.3 mL, 1.05 equiv.) were added. The mixture was stirred at 25 °C for 16 h. TLC (petroleum ether / ethyl acetate = 5 / 1, R f =0.43), indicating complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure to give a residue, which was then diluted with ethyl acetate (100 mL) and washed with water (200 mL × 3). The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 100 / 1 to 1 / 1). 4-((tert-butyldimethylsilyl)oxy)butan-1-ol (112 g, 58.7 mmol, 52.9% yield) was obtained as a white solid. 1 H NMR:400MHz, CDCl3:δ ppm 3.51-3.65(m,4H),1.43-1.66(m,4H),0.80-0.86(m,9H),-0.06-0.03(m,6H).

[0400] Step 2B: tert-butyl(4-iodobutoxy)dimethylsilane

[0401] [ka]

[0402] To a solution of PPh3 (7.70 g, 29.3 mmol, 1.20 equiv.) in DCM (20.0 mL) was added imidazole (2.50 g, 36.7 mmol, 1.50 equiv.) and iodine (8.38 g, 33.0 mmol, 6.65 mL, 1.35 equiv.). 4-((tert-butyldimethylsilyl)oxy)butan-1-ol (5.00 g, 24.4 mmol, 1.00 equiv.) in DCM (15.0 mL) was added dropwise to the mixture. The mixture was stirred at 25 °C for 16 h. TLC (petroleum ether / ethyl acetate = 5 / 1, R f =0.89), indicating complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (SiO2, PMA petroleum ether / ethyl acetate = 1 / 0 to 100 / 1). tert-Butyl(4-iodobutoxy)dimethylsilane (6.70 g, 21.32 mmol, 87.1% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CDCl3:δ ppm 3.64(t,J=6.2 Hz,2H),3.23(t,J=7.0 Hz,2H),1.92(q,J=7.2 Hz,2H),1.58-1.68(m,2H),0.89-0.93(m,9H),0.03-0.08(m,6H).

[0403] Step 2C: Dimethyl (4-((tert-butyldimethylsilyl)oxy)butyl)phosphonate

[0404] [ka]

[0405] To a solution of dimethylphosphonate (1.46 g, 13.2 mmol, 1.21 mL, 1.30 equiv.) in THF (15.0 mL) was added NaH (529 mg, 13.2 mmol, 60.0% purity, 1.30 equiv.) at 0 °C, and the mixture was stirred at 0 °C for 0.5 h and at 70 °C for 1.5 h. The reaction mixture was then cooled to 0 °C, and tert-butyl(4-iodobutoxy)dimethylsilane (3.20 g, 10.2 mmol, 2.64 mL, 1.00 equiv.) in THF (5.00 mL) was added to the mixture, which was then stirred at 25 °C for 24 h. TLC (petroleum ether:ethyl acetate=1:1, product: R f = 0.38), indicating complete consumption of the starting material. The reaction mixture was quenched by the addition of NH4Cl (100 mL), then diluted with ethyl acetate (100 mL) and washed with NH4Cl (100 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue that was purified by column chromatography (SiO2, PMA, petroleum ether:ethyl acetate = 1:0 to 1:1). Dimethyl (4-((tert-butyldimethylsilyl)oxy)butyl)phosphonate (2.20 g, 7.42 mmol, 72.9% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CDCl3:δ ppm 3.74(d,J=10.8 Hz,6H),3.62(t,J=6.0 Hz,2H),1.57-1.83(m,7H),0.89(s,9H),0.05(s,6H) 31 P NMR:162MHz CDCl3:δ ppm 34.93(s,1P).

[0406] Step 2D: Dimethyl(4-hydroxybutyl)phosphonate

[0407] [ka]

[0408] To a solution of dimethyl (4-((tert-butyldimethylsilyl)oxy)butyl)phosphonate (2.10 g, 7.08 mmol, 1.00 equiv.) in MeOH (21.0 mL) was added NHF (787 mg, 21.2 mmol, 3.00 equiv.). The mixture was stirred at 65 °C for 3 h. TLC (petroleum ether / ethyl acetate = 0 / 1, R f = 0.09), which indicated that the starting material had been completely consumed. The reaction mixture was concentrated under reduced pressure and purified by column chromatography (SiO2, KMnO4, petroleum ether / ethyl acetate = 10 / 1 to 0:1). Dimethyl (4-hydroxybutyl)phosphonate (1.3 g, crude) was obtained as a colorless oil. 1 H NMR:400MHz, CDCl3:δ ppm 3.74(d,J=10.8 Hz,6H),3.66(t,J=6.0 Hz,2H),1.98(s,1H),1.62-1.85(m,6H).

[0409] Step 2E: 2-cyanoethyl(4-(dimethoxyphosphoryl)butyl)diisopropylphosphoramidite

[0410] [ka]

[0411] To a solution of dimethyl(4-hydroxybutyl)phosphonate (1.30 g, 7.14 mmol, 1.00 equiv.) in DCM (13.0 mL) was added DIPEA (3.69 g, 28.5 mmol, 4.97 mL, 4.00 equiv.) and 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile (2.03 g, 8.56 mmol, 1.20 equiv.) at 0° C. The mixture was stirred at 25° C. for 1 h. TLC (petroleum ether / ethyl acetate=0 / 1, product: R f=0.43), indicating complete consumption of the compound. The reaction mixture was quenched by the addition of NaHCO3 (20 mL), then diluted with DCM (10 mL) and extracted with DCM (5 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / TEA / DCM = 10 / 1 / 0.5% / 25% to 3 / 1 / 0.5% / 25%). 2-Cyanoethyl (4-(dimethoxyphosphoryl)butyl)diisopropylphosphoramidite (0.173 g, 452 μmol, 6.34% yield) was obtained as a colorless oil. 1 H NMR:400MHz,CD3CN:δ ppm 3.71-3.84(m,2H),3.56-3.70(m,10H),2.64(t,J=6.02 Hz,2H),1.58-1.79(m,7H),1.13-1.20(m,14H). 31 P NMR:162MHz,CD3CN:δ ppm 147.03(s,1P),34.12(s,1P).

[0412] Example 3: (Dimethyl (E)-(5-((2-cyanoethoxy)(diisopropylamino)phosphanyl)pent-1-en-1-yl)phosphonate)

[0413] [ka]

[0414] Step 3A: 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol

[0415] [ka]

[0416] To a solution of propane-1,3-diol (9.0 g, 100 mmol, 1 equiv.) in DMF (90.0 mL) was added DIPEA (143 g, 1.11 mol, 193 mL, 10.0 equiv.) and TBDPSCl (30.4 g, 110 mmol). The mixture was stirred at 25 °C for 16 h. TLC (petroleum ether / ethyl acetate = 5 / 1) revealed the product R f =0.55), indicating complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (100 mL), and washed with HO (200 mL × 3). The combined organic layers were washed with brine (200 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, PMA petroleum ether / ethyl acetate = 100 / 1 to 1 / 1). 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol (16.4 g, 50% yield) was obtained as a white solid.

[0417] Step 3B: 3-((tert-butyldiphenylsilyl)oxy)propanal

[0418] [ka]

[0419] A solution of 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol (16.4 g, 50 mmol, 1 equiv.) in dichloromethane (200 mL) was cooled in an ice bath under an inert atmosphere. PCC (11.8 g, 55 mmol) was added in five portions, and the reaction mixture was allowed to warm to RT over 1 h and stirred at RT for an additional 2 h. The reaction mixture was filtered, and the filtrate was washed with ether. The combined organic layers were washed with brine (200 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue that was used in the next reaction without further purification.

[0420] Step 3C: Dimethyl-(4-((tert-butyldiphenylsilyl)oxy)but-1-en-1-yl)phosphonate

[0421] [ka]

[0422] A solution of tetramethyl methylene diphosphonate in dichloromethane (200 mL) was cooled in an ice bath under an inert atmosphere, and NaH was added in several portions under an argon atmosphere. 3-((tert-butyldiphenylsilyl)oxy)propanal was introduced via syringe, and the reaction mixture was slowly brought to RT over 2 h and stirred at RT for an additional 3 h. The reaction mixture was quenched with NH4Cl solution and then washed with NH4Cl solution (3 × 50 mL). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a sticky residue, which was purified by silica gel chromatography (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1). Dimethyl (E)-(4-((tert-butyldiphenylsilyl)oxy)but-1-en-1-yl)phosphonate (4.1 g, 22% yield from compound 2) was obtained as a white solid.

[0423] Step 3D: Dimethyl-(4-hydroxybut-1-en-1-yl)phosphonate

[0424] [ka]

[0425] To a solution of dimethyl (E)-(4-((tert-butyldiphenylsilyl)oxy)but-1-en-1-yl)phosphonate (4 g) in methanol (20.0 mL) was added NHF (644 mg, 2 equiv.). The mixture was stirred at 70 °C for 2 h. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO, KMnO, petroleum ether / ethyl acetate = 10 / 1 to 0:1). Dimethyl-(4-hydroxybut-1-en-1-yl)phosphonate (1.06 g) was obtained as a colorless oil.

[0426] Step 3E: 2-cyanoethyl (4-(dimethoxyphosphoryl)but-3-en-1-yl)diisopropylphosphoramidite

[0427] [ka]

[0428] To a solution of dimethyl-(4-hydroxybut-1-en-1-yl)phosphonate (0.7 g, 3.8 mmol, 1 equiv.) in DCM (10 mL) was added diisopropylammonium tetrazolide (0.84 g) and 2-cyanoethyl N,N,N,N-tetraisopropylphosphorodiamidite (1.9 g). The mixture was stirred at 0 °C for 1 h, quenched with NaHCO (20 mL), diluted with DCM (10 mL), and then extracted with DCM (10 mL × 2). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that was purified by column chromatography (SiO, petroleum ether / ethyl acetate / TEA / DCM = 10 / 1 / 0.5% / 25% to 3 / 1 / 0.5% / 25%). 2-Cyanoethyl (4-(dimethoxyphosphoryl)but-3-en-1-yl)diisopropylphosphoramidite (0.3) was obtained as a colorless oil. 1 H NMR:400MHz,CD3CN:δ ppm 6.7-7.9(m,1H),5.75(dd,1H),4.1-4.25(m,2H),3.8-3.95(m,4H),3.7 0-3.85(m,6H),3.50-3.70(m,4H),2.55-2.85(m,6H),1.20-1.50(m,6H) 31 P NMR:162MHz, CDCl3:δ ppm 147.81(s,1P),31.39(s,1P).

[0429] Example 4: 2-cyanoethyl(5-(dimethoxyphosphoryl)pentyl)diisopropylphosphoramidite

[0430] [ka]

[0431] Step 4A: 5-((tert-butyldimethylsilyl)oxy)pentan-1-ol

[0432] [ka]

[0433] To a solution of pentane-1,5-diol (10.0 g, 96.0 mmol, 10.1 mL, 1.00 equiv.) in DMF (100 mL) was added DIPEA (124 g, 960 mmol, 167 mL, 10.0 equiv.) and TBSCl (15.2 g, 100 mmol, 12.3 mL, 1.05 equiv.). The mixture was stirred at 25 °C for 16 h. TLC (petroleum ether / ethyl acetate = 5 / 1, product: R f = 0.43), indicating complete consumption of the starting material. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate (200 mL), and washed with water (200 mL × 3). The combined organic layers were washed with brine (400 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 1 / 1). 5-((tert-butyldimethylsilyl)oxy)pentan-1-ol (11.5 g, 52.6 mmol, 54.8% yield) was obtained as a colorless oil. 1 H NMR:400MHz, CDCl3:δ ppm 3.55-3.61(m,4H...

Claims

1. A compound suitable for the synthesis of oligonucleotides, said compound comprising formula (II): 【Chemistry 1】 During the ceremony, R １ each independently represents a substituted or unsubstituted C １ -C ６ Alkyl, substituted or unsubstituted C １ -C ６ fluoroalkyl, or substituted or unsubstituted C １ -C ６ is heteroalkyl, R ２ are each independently hydrogen, deuterium, substituted or unsubstituted C １ -C ６ Alkyl, or substituted or unsubstituted C １ -C ６ is heteroalkyl, Or, two R's ２ are, together with the nitrogen atom to which they are attached, substituted or unsubstituted C ２ -C １０ forming a heterocycloalkyl, L １ is a bond, a substituted or unsubstituted C １ -C ５ Alkylene, substituted or unsubstituted C ２ -C ５ Alkenylene, or substituted or unsubstituted C ２ -C ５ is alkynylene, L ２ is a bond, O, S, NR ３ , substituted or unsubstituted C ４ -C ７ Cycloalkylene, substituted or unsubstituted C ４ -C ７ Heterocycloalkylene, substituted or unsubstituted C ５ -C ８ Arylene or substituted or unsubstituted C ４ -C ８ is heteroarylene, R ３ When present, is hydrogen, unsubstituted or substituted C １ -C ６ Alkyl, unsubstituted or substituted C １ -C ６ Fluoroalkyl, unsubstituted or substituted C １ -C ６ selected from heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L ３ is a bond, a substituted or unsubstituted C １ -C ５ Alkylene, substituted or unsubstituted C ２ -C ５ Alkenylene, or substituted or unsubstituted C ２ -C ５ alkynylene, and L １ , L ２ , and L ３ At least two of the following are not bonds: compound.

2. L ２ The compound of claim 1 , wherein is a bond.

3. L ２ is O, S, or NR ３ 2. The compound of claim 1 ,

4. L ２ is a substituted or unsubstituted C ４ -C ７ The compound of claim 1 which is a cycloalkylene.

5. L ２ is a substituted or unsubstituted C ５ -C ８ The compound of claim 1 which is an arylene.

6. L ２ The compound of claim 1 , wherein is phenylene.

7. L ２ The compound of claim 1 , wherein is cyclohexyl.

8. L １ is C １ -C ５ Alkylene, C １ -C ３ Alkenylene, or C １ -C ５ alkynylene, and L ３ is C １ -C ５ Alkylene, C １ -C ３ Alkenylene, or C １ -C ５ The compound of claim 1 which is an alkynylene.

9. L １ is C １ -C ５ alkylene, and L ３ is C １ -C ５ The compound of claim 1 which is an alkylene.

10. L ２ The compound of claim 9 , wherein is methylene.

11. L ２ is a bond, O, S, or NR ３ 10. The compound of claim 9,

12. R １ Each independently represents a substituted or unsubstituted C １ -C ６ The compound of any one of claims 1-11, which is alkyl.

13. R １ Each independently represents -CH ３ , -CH ２ CH ３ , -CH ２ CH ２ CH ３ , or -CH ２ (CH ３ ) ２ The compound according to any one of claims 1 to 11,

14. R １ are -CH ３ The compound according to any one of claims 1 to 11,

15. R ２ each independently represents a substituted or unsubstituted C １ -C ６ The compound of any one of claims 1-14, which is alkyl.

16. R ２ Each independently represents -CH ３ , -CH ２ CH ３ , -CH ２ CH ２ CH ３ , or -CH ２ (CH ３ ) ２ The compound according to any one of claims 1 to 14,

17. R ２ are -CH ２ (CH ３ ) ２ The compound according to any one of claims 1 to 14,

18. 【Chemical 2】 【Chemistry 3】 Selected from the group consisting of In the formula, R ｘ is H, halogen, unsubstituted or substituted C １ -C ６ Alkyl, unsubstituted or substituted C １ -C ６ Fluoroalkyl, unsubstituted or substituted C １ -C ６ Heteroalkyl, unsubstituted or substituted monocyclic carbocycle, unsubstituted or substituted monocyclic heterocycle, -CN, -OH, -O-alkyl, -CO ２ H, -CO ２ -Alkyl, -CH ２ CO ２ H, -CH ２ CO ２ -Alkyl, -C(=O)NH ２ , —C(═O)NH-alkyl, —CH ２ C(=O)NH ２ , -CH ２ C(=O)NH-alkyl, NH ２ , -NH-alkyl, -CH ２ N.H. ２ , -CH ２ NH-alkyl, -NHC(=O)alkyl, -CH ２ NHC(=O)alkyl, -SH, -S-alkyl, -S(=O)H, -S(=O)alkyl, -SO ２ H, -SO ２ -Alkyl, -SO ２ N.H. ２ , or -SO ２ The compound of any one of claims 1-17, which is NH-alkyl.

19. An oligonucleotide comprising a compound of formula (IIa): 【Chemistry 4】 During the ceremony, R １ each independently represents a substituted or unsubstituted C １ -C ６ Alkyl, substituted or unsubstituted C １ -C ６ fluoroalkyl, or substituted or unsubstituted C １ -C ６ is heteroalkyl, L １ is a bond, a substituted or unsubstituted C １ -C ５ Alkylene, substituted or unsubstituted C ２ -C ５ Alkenylene, or substituted or unsubstituted C ２ -C ５ is alkynylene, L ２ is a bond, O, S, NR ３ , substituted or unsubstituted C ４ -C ７ Cycloalkylene, substituted or unsubstituted C ４ -C ７ Heterocycloalkylene, substituted or unsubstituted C ５ -C ８ Arylene or substituted or unsubstituted C ４ -C ８ is heteroarylene, R ３ When present, is hydrogen, unsubstituted or substituted C １ -C ６ Alkyl, unsubstituted or substituted C １ -C ６ Fluoroalkyl, unsubstituted or substituted C １ -C ６ selected from heteroalkyl, unsubstituted or substituted monocyclic carbocycle, and unsubstituted or substituted monocyclic heterocycle; L ３ is a bond, a substituted or unsubstituted C １ -C ５ Alkylene, substituted or unsubstituted C ２ -C ５ Alkenylene, or substituted or unsubstituted C ２ -C ５ is alkynylene, J is an internucleotide linking group that links adjacent nucleotides of a polynucleotide, and L １ , L ２ , and L ３ At least two of the following are not bonds: Oligonucleotides.

20. 20. The oligonucleotide of claim 19, wherein the oligonucleotide is an RNA oligonucleotide.

21. 21. The oligonucleotide of claim 19 or 20, further comprising at least one modification.

22. The oligonucleotide of any one of claims 19 to 21, further comprising at least one 2' modified nucleotide.

23. 23. The oligonucleotide of any one of claims 19-22, further comprising at least one 2' modified nucleotide 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'-O-N-methylacetamide (2'-O-NMA) modified nucleotide.

24. The oligonucleotide of any one of claims 19-23, further comprising at least one 2' modified nucleotide selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

25. 25. The oligonucleotide of any one of claims 19-24, further comprising at least one modified internucleotide linkage.

26. 26. The oligonucleotide of any one of claims 19-25, further comprising at least one modified internucleotide linkage selected from a phosphorothioate linkage, a phosphorodithioate linkage, a methylphosphonate linkage, a phosphotriester linkage, or an amide linkage.

27. The oligonucleotide of claim 19, wherein the compound of formula (IIa) is located at the 5'-end of the oligonucleotide.

28. 20. The oligonucleotide of claim 19, wherein the oligonucleotide is conjugated to a binding moiety.

29. 29. The oligonucleotide of claim 28, wherein the compound of formula (IIa) is located at the 5'-end of the oligonucleotide and the linking moiety is conjugated to the 3'-end of the oligonucleotide.

30. The oligonucleotide of any one of claims 28-29, wherein the binding moiety comprises an antibody or a binding fragment thereof.

31. The oligonucleotide of claim 30, wherein the antibody or binding fragment thereof 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.

32. 30. The oligonucleotide of claim 28, wherein the binding moiety comprises a peptide or a small molecule.

33. 30. The oligonucleotide of claim 28, wherein the binding moiety comprises an aptamer.

34. 33. The oligonucleotide of any one of claims 19-32, comprising from about 8 to about 50 nucleotides.

35. 34. The oligonucleotide of any one of claims 19-33, comprising from about 10 to about 30 nucleotides.

36. 34. The oligonucleotide of any one of claims 19-33, comprising from about 15 to about 25 nucleotides.

37. The oligonucleotide is is an RNA oligonucleotide, Conjugated to a binding moiety, about 10 to about 30 nucleotides, at least one 2' modified nucleotide; and 37. The oligonucleotide of any one of claims 19-36, comprising at least one modified internucleotide linkage.

38. The oligonucleotide of any one of claims 19-37, wherein the oligonucleotide hybridizes to at least 8 contiguous bases of the target gene sequence.

39. The oligonucleotide of any one of claims 19-38, wherein the oligonucleotide mediates RNA interference.

40. The oligonucleotide of any one of claims 19-37, wherein the oligonucleotide is a sense strand.

41. The oligonucleotide of claim 40, wherein the oligonucleotide hybridizes to a second oligonucleotide to form a double-stranded oligonucleic acid molecule.

42. 42. The oligonucleotide of claim 41, wherein the second oligonucleotide is an antisense strand.

43. 43. The oligonucleotide of claim 41 or 42, wherein the second oligonucleotide is an RNA oligonucleotide.

44. The oligonucleotide of any one of claims 41-43, wherein the second oligonucleotide comprises at least one modification.

45. 45. The oligonucleotide of claim 44, wherein the second oligonucleotide comprises at least one 2' modified nucleotide.

46. 46. ​​The oligonucleotide of claim 44 or 45, wherein the second oligonucleotide comprises at least one 2' modified nucleotide 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'-O-N-methylacetamide (2'-O-NMA) modified nucleotide.

47. The oligonucleotide of any one of claims 44-46, wherein the second oligonucleotide comprises at least one 2' modified nucleotide selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

48. The oligonucleotide of any one of claims 44-47, wherein the second oligonucleotide comprises at least one modified internucleotide bond.

49. The oligonucleotide of any one of claims 44-48, wherein the second oligonucleotide comprises at least one modified internucleotide bond selected from a phosphorothioate bond, a phosphorodithioate bond, a methylphosphonate bond, a phosphotriester bond, or an amide bond.

50. The oligonucleotide of any one of claims 19-37 or 40-49, wherein the oligonucleotide comprises a polymer.

51. 51. The oligonucleotide of claim 50, wherein the oligonucleotide comprises polyethylene glycol.

52. the oligonucleotide comprises a first strand and a second strand; The first strand is is the sense strand, 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 bond, and the second strand comprises is the antisense strand, is an RNA oligonucleotide, about 10 to about 30 nucleotides, at least one 2' modified nucleotide; and 52. The oligonucleotide of any one of claims 19-37 or 40-51, comprising at least one modified internucleotide linkage.

53. A method of treating a subject having a disease or disorder characterized by impaired expression of a protein, the method comprising the step of administering to the subject an oligonucleotide of claims 19-52 to regulate expression of a gene encoding the protein, thereby treating the disease or disorder characterized by impaired expression of the protein.

54. A method of treating a subject having a disease or condition characterized by overexpression of a protein, the method comprising the step of administering to the subject an oligonucleotide of claims 19-52 to modulate expression of a gene encoding the protein, thereby treating the disease or condition characterized by overexpression of the protein.

55. 55. The method of claim 53 or 54, wherein the disease or condition is cancer.

56. 55. The method of claim 53 or 54, 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.

57. The method of any one of claims 53-56, wherein the subject is a human.

58. A kit comprising a compound of any one of claims 1-18 or an oligonucleotide of any one of claims 19-52.

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