Nucleic Acid Polypeptide Compositions and Uses Thereof

Abstract

To provide compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a modified polynucleic acid molecule, and a polymer; and also, to provide methods for treating a cancer which utilize a composition and a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule, and a polymer.SOLUTION: Provided is a polynucleotide molecule comprising at least one 5'-vinylphosphonate modified non-natural nucleotide, for example, having the following structure in which B is a heterocyclic base moiety, R1, R2, and R3 are independently selected from hydrogen, halogen, alkyl, alkoxy, or aminoalkyl, and J is an internucleotide linking group that binds to adjacent nucleotides of the polynucleotide molecule.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] cross reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 568,238, filed October 4, 2017, which is incorporated by reference herein in its entirety. [Background technology]

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

[0004] In certain embodiments, disclosed herein are compositions and pharmaceutical formulations comprising a binding moiety and a polymer conjugated to a polynucleic acid molecule. Also described herein in some embodiments are methods of treating a disease or condition (e.g., cancer) utilizing compositions and pharmaceutical formulations comprising a binding moiety and a polymer conjugated to a polynucleic acid molecule.

[0005] In certain embodiments, the compound of formula (I): AXBYC Formula I Disclosed herein is a molecule of During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a single bond or a first linker; and Y is a bond or a second linker; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide.

[0006] In some embodiments, the polynucleotide comprises a single strand. In some embodiments, the polynucleotide comprises two or more strands. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some embodiments, the second polynucleotide comprises at least one modification.

[0007] In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide and the second polynucleotide are siRNA molecules.

[0008] In some embodiments, the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242. In some embodiments, the first polynucleotide consists of a sequence selected from SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242.

[0009] In some embodiments, the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242. In some embodiments, the second polynucleotide consists of a sequence selected from SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242.

[0010] In some embodiments, X and Y are independently a single bond or a non-polymeric linker group. In some embodiments, X is a single bond. In some embodiments, X is C 1 -C 6 In some embodiments, Y is an alkyl group. 1 -C 6 In some embodiments, X is optionally C 1 -C 6 In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker attached to an alkyl group.

[0011] In some embodiments, the binding moiety is an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , single chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb), or camelid antibody, or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof is an anti-EGFR antibody or binding fragment thereof.

[0012] In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 5,000 Da.

[0013] In some embodiments, AX is attached to the 5' end of B and YC is attached to the 3' end of B. In some embodiments, YC is attached to the 5' end of B and AX is attached to the 3' end of B. In some embodiments, AX, YC, or a combination thereof are attached to an internucleotide linkage group.

[0014] In some embodiments, the molecule further comprises D. In some embodiments, D is attached to C or A. In some embodiments, D is attached to a molecule of Formula (I) according to Formula (II): (AXBYC n )-LD Formula II During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and n is an integer between 0 and 1; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide; and D is linked to any of A, B, or C.

[0015] In some embodiments, D is INF7 or melittin.

[0016] In some embodiments, D is an endosomolytic polymer.

[0017] In some embodiments, L is C 1 -C 6 is an alkyl group. In some embodiments, L is a homobifunctional linker or a heterobifunctional linker.

[0018] In some embodiments, the molecule further comprises at least a second binding moiety, A. In some embodiments, at least the second binding moiety, A, binds to A, B, or C. In some embodiments, at least the second binding moiety, A, is cholesterol.

[0019] In some embodiments, the molecule further comprises at least one additional polynucleotide B. In some embodiments, the at least one additional polynucleotide B binds to A, B, or C.

[0020] In some embodiments, the molecule further comprises at least one additional polymer C. In some embodiments, the at least one additional polymer C is attached to A, B, or C.

[0021] In certain embodiments, disclosed herein is a molecule of formula (I):AXBYC (Formula I), where A is an antibody or binding fragment thereof; B is a polynucleotide; C is a polymer; X is a single bond or a first non-polymeric linker; and Y is a single bond or a second linker; the polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide; and A and C are not attached to B at the same end. In some embodiments, at least one modified internucleotide bond comprises a phosphorothioate bond or a phosphorodithioate bond. In some embodiments, at least one inverted abasic moiety is at at least one end. In some embodiments, the polynucleotide comprises a single strand. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some embodiments, the second polynucleotide comprises at least one modification. In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242. In some embodiments, the second polynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242. In some embodiments, Y is a non-polymeric linker group. In some embodiments, X is a single bond. In some embodiments, X is a C 1 -C 6 In some embodiments, Y is an alkyl group. 1 -C 6 In some embodiments, X is optionally C1 -C 6 In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker attached to an alkyl group. In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or binding fragments thereof. In some embodiments C is polyethylene glycol. In some embodiments C has a molecular weight of about 1000 Da, 2000 Da, or 5000 Da. In some embodiments, AX is attached to the 5' end of B and YC is attached to the 3' end of B. In some embodiments, YC is attached to the 5' end of B and AX is attached to the 3' end of B. In some embodiments, the molecule further comprises D. In some embodiments D is attached to C or A. In some embodiments, D is attached to a molecule of formula (I) according to formula (II) and is selected from the group consisting of (AXBYC c )-LD (Formula II) where A is an antibody or binding fragment thereof; B is a polynucleotide; C is a polymer; X is a single bond or a first non-polymeric linker; Y is a single bond or a second linker; L is a single bond or a third linker; D is an endosomolytic moiety; and c is an integer between 0 and 1; the polynucleotide comprises at least one 2' modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; A and C are not attached to B at the same terminus; and D is attached anywhere in A or C or to the terminus of B. In some embodiments, D is INF7 or melittin. In some embodiments, D is an endosomolytic polymer. In some embodiments, L is C 1 -C 6is an alkyl group. In some embodiments, L is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the molecule further comprises at least a second binding moiety. In some embodiments, the at least second binding moiety binds to A, B, or C. In some embodiments, the at least second binding moiety A is cholesterol. In some embodiments, the molecule further comprises at least one additional polynucleotide B. In some embodiments, the at least one additional polynucleotide B binds to A, B, or C. In some embodiments, the molecule further comprises at least one additional polymer C. In some embodiments, the at least one additional polymer C binds to A, B, or C.

[0022] In certain embodiments, disclosed herein is a pharmaceutical composition comprising the above-described molecule and a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

[0023] In certain embodiments, disclosed herein are methods of treating a disease or condition in a patient, comprising administering to the patient a composition comprising the above-mentioned molecule. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer comprises a KRAS-associated cancer, an EGFR-associated cancer, an AR-associated cancer, a β-catenin-associated cancer, a PIK3C-associated cancer, or a MYC-associated cancer. In some embodiments, the cancer comprises bladder cancer, breast cancer, colon cancer, endometrial cancer, esophageal cancer, glioblastoma multiforme, head and neck cancer, renal cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, or thyroid cancer. In some embodiments, the cancer comprises acute myeloid leukemia, CLL, DLBCL, or multiple myeloma. In some embodiments, the method is an immuno-oncology treatment.

[0024] In certain embodiments, disclosed herein is a method of inhibiting expression of a target gene in a primary cell of a patient, comprising administering to the primary cell a molecule as described above. In some embodiments, the method is an in vivo method. In some embodiments, the patient is a human.

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

[0026] In certain embodiments, disclosed herein are kits that contain the above-described molecules. [Brief description of the drawings]

[0027] [Figure 1A] 1 shows a pictorial representation of the structure of a 21-mer duplex, with 19 bases of complementarity and a 3' dinucleotide overhang, as described in Molecular Biology Example 10. [Figure 1B] 1 shows a pictorial representation of a blunt-ended duplex structure with 19 bases of complementarity and a single 3' dinucleotide overhang, as described in Molecular Biology Example 10. [Diagram 2] 1 shows a plot of log (siRNA in nM) versus relative HPRT mRNA (%) for HCT transfections with HPRT siRNA as described in Molecular Biology Example 11. [Diagram 3] 1 shows a plot of log (siRNA in nM) versus relative MSTN mRNA levels (% of untreated control) for SJCRH30 transfection with MSTN siRNA as described in Molecular Biology Example 12. [Figure 4] 1 shows downregulation of MSTN and HPRT mRNA in vivo in gastrocnemius muscle following IV administration of antibody-siRNA conjugates as described in Molecular Biology Example 13. [Diagram 5]1 shows in vivo MSTN mRNA downregulation in (A) gastrocnemius, (B) quadriceps, and (C) cardiac muscle following IV administration of antibody-siRNA conjugates as described in Molecular Biology Example 14. [Figure 6] 1 shows in vivo MSTN mRNA downregulation in (A) gastrocnemius muscle and (B) liver following IV administration of antibody-siRNA conjugates as described in Molecular Biology Example 15. [Figure 7] 1 shows a plot of concentration vs. %SSB mRNA (nM) as described in Molecular Biology Example 16. [Figure 8] 8A and 8B show plots of siRNA concentration (nM) for SJCRH30 transfection versus relative MSTN (FIG. 8A) and SSB (FIG. 8B) mRNA expression, as described in Molecular Biology Example 17. [Figure 9] As described in Molecular Biology Example 18, plots of siRNA concentration (nM) for SJCRH30 versus relative MSTN (FIG. 8A) and SSB (FIG. 8B) mRNA expression are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 intracellular uptake, limited blood stability, and non-specific immune stimulation. To address these issues, various modifications of nucleic acid compositions are being explored, such as novel linkers for better stabilization and / or lower toxicity, optimization of binding moieties for increased target specificity and / or targeted delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0029] In some embodiments, the arrangement or order of the various components that make up the nucleic acid composition further provides for cellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation. For example, when the nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide), the order or arrangement of the binding moiety, polymer, and / or polynucleic acid molecule (or polynucleotide) (e.g., binding moiety polynucleic acid molecule polymer, binding moiety polymer polynucleic acid molecule, or polymer binding moiety polynucleic acid molecule) further provides for cellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation.

[0030] In some embodiments, the molecules of the nucleic acid component configuration described herein provide cellular uptake, stability, toxicity, efficacy, and / or non-specific immune stimulation. In some examples, the molecules include a linking moiety attached to a polynucleic acid molecule and a polymer. In some embodiments, the molecules include a molecule of formula (I): AXBYC; where A is a linking moiety, B is a polynucleotide comprising at least one 5'-vinylphosphonate modified nucleotide, C is a polymer, X is a single bond or a first linker, and Y is a single bond or a second linker. In some examples, the polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide. In some examples, the molecules of formula (I) include D, an endosomolytic moiety.

[0031] In some embodiments, molecules comprising a linking moiety attached to the polynucleic acid molecules and polymers described herein enhance cellular uptake, stability, and / or efficacy. In some examples, molecules comprising a linking moiety attached to the polynucleic acid molecules and polymers arranged as described herein reduce toxicity and / or non-specific immune stimulation. In some cases, the molecule comprises a molecule of formula (I): AXBYC; where A is a linking moiety, B is a polynucleotide comprising at least one 5'-vinylphosphonate modified nucleotide, C is a polymer, X is a single bond or a first linker, and Y is a single bond or a second linker. In some examples, the polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide. In some examples, the molecule of formula (I) comprises D, an endosomolytic moiety.

[0032] In some embodiments, the molecules described herein are further used to treat disease or illness. In some examples, the molecule for treating disease or illness is a molecule of formula (I): AXBYC; where A is a linking moiety, B is a polynucleotide comprising at least one 5'-vinylphosphonate modified nucleotide, C is a polymer, X is a single bond or a first linker, and Y is a single bond or a second linker. In some examples, the polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide. In some examples, the molecule of formula (I) comprises D, an endosomolytic moiety.

[0033] In some embodiments, the molecules described herein are further used to inhibit the expression of target genes in primary cells of patients.In such examples, the molecules for such use are of formula (I):AXBYC;, where A is a linking moiety, B is a polynucleotide comprising at least one 5'-vinylphosphonate modified nucleotide, C is a polymer, X is a single bond or a first linker, and Y is a single bond or a second linker.In some examples, the polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide, at least one modified internucleotide bond, or at least one inverted abasic moiety.In some examples, the molecule of formula (I) comprises D, an endosomolytic moiety.

[0034] In some embodiments, the molecules described herein are further used as immuno-oncological therapy for the treatment of disease or illness. In some examples, the molecule is of formula (I): AXBYC; where A is a linking moiety, B is a polynucleotide comprising at least one 5'-vinylphosphonate modified nucleotide, C is a polymer, X is a single bond or a first linker, and Y is a single bond or a second linker. In some examples, the polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide, at least one modified internucleotide bond, or at least one inverted abasic moiety. In some examples, the molecule of formula (I) comprises D, an endosomolytic moiety.

[0035] In additional embodiments, kits are described herein that include one or more of the molecules described herein.

[0036] Therapeutic Molecular Platforms In some embodiments, the molecules (e.g., therapeutic molecules) described herein comprise a linking moiety attached to a polynucleic acid molecule comprising at least one 5'-vinylphosphonate modified nucleotide and polymer. In some embodiments, the molecule (e.g., therapeutic molecule) comprises a conjugate having the formula (I): AXBYC Formula I containing a molecule of During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; and Y is a bond or a second linker; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide.

[0037] In some examples, the molecule of formula (I) includes D, an endosomolytic moiety.

[0038] In some embodiments, at least one A and / or at least one C is attached to the 5' end of B, the 3' end of B, an internal site of B, or any combination thereof. In some examples, at least one A is attached at one end of B while at least one C is attached at the opposite end of B. In some examples, at least one A is attached at one end of B while at least one C is attached at an internal site of B.

[0039] In some cases, A and C are not conjugated or attached to B at the same end. In some cases, A is conjugated or attached to B at a first end of B. In some cases, C is conjugated to B at a second end of B, and the second end of B is different from the first end. In some cases, A is conjugated to B at the 5' end of B, and C is conjugated to B at the 3' end of B. In other cases, A is conjugated to B at the 3' end of B, and C is conjugated to B at the 5' end of B.

[0040] In some embodiments, A is an antibody or binding fragment thereof. Optionally, C is a polymer. Optionally, A and C are not attached to B at the same end. Optionally, A is attached to B at a first end of B. Optionally, C is attached to B at a second end of B, and the second end of B is different from the first end. Optionally, A is attached to B at the 5' end of B, and C is attached to B at the 3' end of B. In other cases, A is attached to B at the 3' end of B, and C is attached to B at the 5' end of B. Optionally, X connecting A to B is a single bond or a non-polymeric linker. Optionally, X is a non-peptide linker (or a linker that does not include an amino acid residue). Optionally, Y connecting B to C is a single bond or a second linker. In some examples, X connects A to the 5' end of B, and Y connects C to the 3' end of B. In other examples, X connects A to the 3' end of B, and Y connects C to the 5' end of B.

[0041] In some embodiments, XB binds to the N-terminus, C-terminus, constant region, hinge region, or Fc region of A. In some examples, XB binds to the N-terminus of A. In some examples, XB binds to the C-terminus of A. In some examples, XB binds to the hinge region of A. In some examples, XB binds to a constant region of A. In some examples, XB binds to an Fc region of A.

[0042] In some examples, at least one B and / or at least one C, and optionally at least one D, are bound to the first A. In some examples, at least one B is bound to the first A at a terminal end (e.g., at the 5' or 3' end) or via an internal site. In some cases, at least one C is bound to the first A directly or indirectly via two or more Bs. When indirectly bound via two or more Bs, the two or more Cs are bound at the same end on B as the first A, at the opposite end from the first A, or independently at an internal site. In some examples, at least one additional A is further bound to the first A, B, or C. In further examples, at least one D is optionally bound to the first A, at least one B, or at least one C, directly or indirectly. When directly bound to the first A, the at least one D is also optionally bound to at least one B to form an ADB conjugate, or is optionally bound to at least one B and at least one C to form an ADBC ​​conjugate. Optionally, the at least one additional A is different from the first A.

[0043] In some cases, two or more Bs and / or two or more Cs are linked to the first A. In some examples, two or more Bs are linked to the first A at a terminal end (e.g., at the 5' or 3' end) or via an internal site. In some examples, two or more Cs are linked directly to the first A or indirectly via two or more Bs. When indirectly linked via two or more Bs, two or more Cs are linked at the same end on B as the first A, at an opposite end from the first A, or independently at an internal site. In some examples, at least one additional A is further linked to the first A, two or more Bs, or two or more Cs. In a further example, at least one D is optionally linked directly or indirectly to the first A, two or more Bs, or two or more Cs. When indirectly bound to the first A, at least one D is bound to the first A by two or more Bs, by two or more Cs, in a BC orientation to form an ABCD type conjugate, or by a CB orientation to form an ACBD type conjugate. In some cases, at least one additional A is different from the first A. In some cases, two or more Bs are different. In other cases, two or more Bs are the same. In some instances, two or more Cs are different. In other instances, two or more Cs are the same. In further instances, two or more Ds are different. In further instances, two or more Ds are the same.

[0044] In other cases, two or more Bs and / or two or more Ds, optionally two or more Cs, are linked to the first A. In some examples, two or more Bs are linked to the first A at a terminal end (e.g., at the 5' or 3' end) or to the first A via an internal site. In some examples, two or more Ds are linked directly to the first A or indirectly via two or more Bs. When linked indirectly via two or more Bs, the two or more Ds are linked at the same end on B as the first A, at an opposite end from the first A, or independently at an internal site. In some examples, at least one additional A is further linked to the first A, two or more Bs, or two or more Ds. In further examples, two or more Cs are optionally linked directly or indirectly to the first A, two or more Bs, or two or more Ds. In some cases, at least one additional A is different from the first A. In some cases, two or more Bs are different. In other cases, two or more Bs are the same. In some examples, two or more Cs are different. In other examples, two or more Cs are the same. In a further example, two or more D are different. In a further example, two or more D are the same.

[0045] In some embodiments, a molecule described herein (e.g., a therapeutic molecule) comprises a molecule according to formula (II): (AXBYC c )-LD Formula II During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer between 0 and 1; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide; and D is linked to any of A, B, or C.

[0046] In some embodiments, a molecule described herein (e.g., a therapeutic molecule) comprises a molecule according to formula (III): A a -XB b -YC c -LD n Formula III During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; D is an endosomolytic moiety; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; a and b are independently integers between 1 and 3; c is an integer between 0 and 3; and n is an integer between 0 and 10; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide; A is attached anywhere on B, C, or D; B is attached anywhere on A, C, or D; C is attached anywhere on A, B, or D; and D is attached anywhere on A, B, or C.

[0047] In some embodiments, a molecule described herein (eg, a therapeutic molecule) comprises a molecule according to formula (IIIa): AXBLDYC.

[0048] In some embodiments, a molecule described herein (e.g., a therapeutic molecule) has the formula (IIIb): a -XB b -LD n The present invention includes molecules according to the following formula:

[0049] In some embodiments, a molecule described herein (e.g., a therapeutic molecule) comprises a molecule according to formula (IV): AX-(B b-YC c -LD n ) m During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; D is an endosomolytic moiety; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; a and b are independently integers between 1 and 3; c is an integer between 0 and 3; n is an integer between 0 and 10; and m is an integer between 1 and 3; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide; C is attached at any position on B or D; and D is attached at any position on B or C.

[0050] In some embodiments, the molecules (e.g., therapeutic molecules) described herein have the formula (IVa): AX-(B b -LD n -YC c ) m The present invention includes molecules according to the following formula:

[0051] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0052] [ka]

[0053] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0054] [ka]

[0055] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0056] [ka]

[0057] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0058] [ka]

[0059] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0060] [ka]

[0061] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0062] [ka]

[0063] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0064] [ka]

[0065] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0066] [ka]

[0067] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0068] [ka]

[0069] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0070] [ka]

[0071] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0072] [ka]

[0073] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0074] [ka]

[0075] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0076] [ka]

[0077] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0078] [ka]

[0079] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0080] [ka]

[0081] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0082] [ka]

[0083] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0084] [ka]

[0085] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0086] [ka]

[0087] In some embodiments, the molecule (e.g., a therapeutic molecule) is a molecule as exemplified:

[0088] [ka]

[0089] [ka] are for illustrative purposes only and include humanized antibodies or binding fragments thereof, chimeric antibodies or binding fragments thereof, monoclonal antibodies or binding fragments thereof, monovalent Fab', bivalent Fab 2 , single chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb) or camelid antibody or binding fragment thereof.

[0090] Polynucleic acid molecular target In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule (or polynucleotide) that hybridizes to a target region on an oncogene. In some examples, oncogenes are further classified into several categories: growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, and transcription factors. Exemplary growth factors include c-Sis. Exemplary receptor tyrosine kinases include epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and HER2 / neu. Exemplary cytoplasmic tyrosine kinases include the Src family tyrosine kinases, the Syk-ZAP-70 family of tyrosine kinases, the BTK family of tyrosine kinases, and the Abl gene in CML. Exemplary cytoplasmic serine / threonine kinases include Raf kinases and cyclin-dependent kinases. Exemplary regulatory GTPases include the Ras family of proteins, such as KRAS. Exemplary transcription factors include the MYC gene. In some examples, the oncogenes described herein include oncogenes selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, or transcription factors. In some embodiments, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of an oncogene selected from growth factors or mitogens, receptor tyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmic serine / threonine kinases, regulatory GTPases, or transcription factors.

[0091] In some embodiments, the oncogene described herein 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, LMO 1, 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. A polynucleic acid molecule that hybridizes to a target region of 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.

[0092] In some embodiments, the oncogenes described herein include KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related genes. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-catenin-related genes. In some embodiments, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to 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 CNNTB1 (β-catenin)-related genes. In some examples, the β-catenin related genes include PIK3CA, PIK3CB, and Myc. In some examples, polynucleic acid molecule B is a polynucleic acid molecule that hybridizes to a target region of HPRT1.

[0093] Polynucleic acid molecules targeting Kirsten rat sarcoma viral oncogene homolog (KRAS) Kirsten rat sarcoma viral oncogene homolog (GTPase KRas, also known as ∨-Ki-ras Kirsten rat sarcoma viral oncogene homolog, or KRAS) is involved in the regulation of cell division. K-Ras protein is a GTPase that belongs 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.

[0094] In some embodiments, the KRAS gene is wild type or contains a mutation. In some examples, the KRAS mRNA is wild type or contains a mutation. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of wild type KRAS DNA or RNA. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of KRAS DNA or RNA that contains a mutation (e.g., a substitution, deletion, or addition).

[0095] 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 combinations 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 combinations thereof in a KRAS polypeptide.

[0096] In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA that contains one or more mutations at codons 12 or 13 in exon 1. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA that contains one or more mutations at codons 61, 63, 117, 119, or 146. In some embodiments, the polynucleic acid molecule hybridizes to a target region of KRAS DNA or RNA that contains 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 combinations 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.

[0097] Polynucleic acid molecules targeting epidermal growth factor receptor (EGFR) The epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is a transmembrane tyrosine kinase receptor and a member of the ErbB family of receptors, which further includes HER2 / c-neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In some instances, EGFR mutations drive downstream activation of the RAS / RAF / MAPK, PI3K / AKT, and / or JAK / STAT pathways, resulting in inhibition of mitosis, cell proliferation, and apoptosis. In addition, amplification of the wild-type EGFR gene has been implicated in the development of cancers such as glioblastoma and non-small cell lung cancer (Talasila, et al., “EGFR Wild-type Amplification and Activation Promote Invasion and Development of Glioblastoma Independent of Angiogenesis,” Acta Neuropathol. 125(5): 683-698(2013); Bell et al., “Epidermal Growth Factor Receptor Mutations and Gene Amplification in Non-Small-Cell Lung Cancer: Molecular Analysis of the IDEAL / INTACT Gefitinib Trials,” J. Clinical Oncology 23(31): 8081-8092(2005)).

[0098] In some embodiments, the EGFR DNA or RNA is wild-type EGFR or EGFR that contains a mutation. In some examples, the EGFR is wild-type EGFR. In some examples, the EGFR DNA or RNA contains a mutation. In some examples, the polynucleic acid molecule hybridizes to a target region of wild-type EGFR DNA or RNA. In some examples, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains a mutation (e.g., a substitution, deletion, or addition).

[0099] In some examples, the EGFR DNA or RNA comprises one or more mutations. In some embodiments, the EGFR DNA or RNA comprises one or more mutations in one or more exons. In some examples, the one or more exons include exon 18, exon 19, exon 20, exon 21, or exon 22. In some examples, the EGFR DNA or RNA comprises one or more mutations in exon 18, exon 19, exon 20, exon 21, exon 22, or a combination thereof.

[0100] In some embodiments, the EGFR DNA or RNA may be a sequence similar to amino acid residues 34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 3 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 733, 73 9-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765, 767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774, 774-775, 776, 779, 783, 784, 786, 790, 792, In some embodiments, the EGFR DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues 747, 761, 790, 854, 858, or a combination thereof of an EGFR polypeptide.In some embodiments, the EGFR DNA or RNA comprises one or more mutations at a position corresponding to amino acid residue 761, 790, 858, or a combination thereof, of an EGFR polypeptide. In some embodiments, the EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 747 of an EGFR polypeptide. In some embodiments, the EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 761 of an EGFR polypeptide. In some embodiments, the EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 790 of an EGFR polypeptide. In some embodiments, the EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 854 of an EGFR polypeptide. In some embodiments, the EGFR DNA or RNA comprises a mutation at a position corresponding to amino acid residue 858 of an EGFR polypeptide.

[0101] In some embodiments, the EGFR DNA or RNA comprises one or more of the following mutations in the EGFR polypeptide: T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense mutation), D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288 (frameshift), A289D, A289V, A289T, A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T363I, P373Q, R337S, S380 (frameshift), T384S, D393Y, R427L, G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R 527L, R531 (nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C 628Y, C628F, C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G7 19A, G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L, K739-I744 (insertion), V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion), S752-I759 (deletion), A750P, D761-E762 (e.g., residues EAFQ insertion (SEQ ID NO:2110), D761N, D761Y, A763V, V765A, A767-S768 (e.g., residue TLA insertion), A767-V769 (e.g., residue ASV insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y insertion), 770-771 (e.g., residue GL insertion), 770-771 (e.g., residue G insertion), 770-771 (e.g., residue CV insertion), 770-771 (e.g., residue SVD insertion), P772R, 773-774 (e.g., residue NPH insertion), H773R, H773L, V774M, 774-775 (e.g., residue HV insertion), R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790 M, L792P, P794H, L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N, V851A , I853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y, T1141S, S1153I, Q1164R, L1167M, or a combination thereof.

[0102] In some examples, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations, hi some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations in exon 18, exon 19, exon 20, exon 21, exon 22, or a combination thereof.

[0103] In some embodiments, the polynucleic acid molecule is selected from the group consisting of amino acid residues 34, 38, 45, 62, 63, 77, 78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220, 222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276, 282, 288, 289, 301, 303, 304, 309, 314, 315, 316, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 326, 331, 354, 363, 373, 337, 380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531, 536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636, 641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720, 721, 731, 7 33, 739-744, 742, 746-750, 746-752, 746, 747, 747-749, 747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765, 767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774, 774-775, 776, 779, 783, 784, 786, 790, 7 92, 794, 798, 803, 805, 807, 810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850, 851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042, 1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations at positions corresponding to amino acid residues 747, 761, 790, 854, 858, or combinations thereof of an EGFR polypeptide.In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations at a position corresponding to amino acid residue 761, 790, 858, or a combination thereof, of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains a mutation at a position corresponding to amino acid residue 747 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains a mutation at a position corresponding to amino acid residue 761 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains a mutation at a position corresponding to amino acid residue 790 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains a mutation at a position corresponding to amino acid residue 854 of the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains a mutation at a position corresponding to amino acid residue 858 of the EGFR polypeptide.

[0104] In some embodiments, the polynucleic acid molecule comprises a nucleotide sequence encoding the following mutations in the EGFR polypeptide: T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K, R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T, D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G, R252C, R252P, F254I, R255 (nonsense mutations) , D256Y, T263P, Y270C, T273A, Q276 (nonsense), E282K, G288 (frameshift), A289D, A289V, A289T, A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R, T354M, T363I, P373Q, R337S, S380 (frameshift), T384S, D393Y, R427L, G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C5 26S, R527L, R531 (nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S, P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F, C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A, E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A, G719C, G719D, G719S, S720C, S720F, G721V, W731Stop, P733L, K739-I744 (insertion), V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion), L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I, S752Y, K754 (deletion), S752-I759 (deletion), A750P, D761-E762 (e.g., residues EAFQ insertion (SEQ ID NO:2110), D761N, D761Y, A763V, V765A, A767-S768 (e.g., residue TLA insertion), A767-V769 (e.g., residue ASV insertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Y insertion), 770-771 (e.g., residue GL insertion), 770-771 (e.g., residue G insertion), 770-771 (e.g., residue CV insertion), 770-771 (e.g., residue SVD insertion), P772R, 773-774 (e.g., residue NPH insertion), H773R, H773L, V774M, 774-775 (e.g., residue HV insertion), R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H, L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C, R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L, T847K, T847I, H850N, V851A, I In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations selected from 853T, F856L, L858R, L858M, L861Q, L861R, G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y, T1141S, S1153I, Q1164R, L1167M, or a combination thereof. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations selected from L747S, D761Y, T790M, T854A, L858R, or a combination thereof in an EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations selected from D761Y, T790M, L858R, or combinations thereof in the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that contains one or more mutations selected from D761Y, T790M, L858R, or combinations thereof in the EGFR polypeptide.The polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that comprises a mutation D761Y in the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that comprises a mutation T790M in the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that comprises a mutation T854A in the EGFR polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of EGFR DNA or RNA that comprises a mutation L858R in the EGFR polypeptide.

[0105] Polynucleic acid molecules targeting the androgen receptor (Ar) The androgen receptor (AR) (also known as NR3C4, nuclear receptor subfamily 3, group C, gene 4) belongs to the steroid hormone group of the nuclear receptor superfamily, together with related members: estrogen receptor (ER), glucocorticoid receptor (GR), progesterone receptor (PR), and mineralocorticoid receptor (MR). Androgens or steroid hormones regulate protein synthesis and tissue remodeling through the androgen receptor. The AR protein is a ligand-inducible zinc finger transcription factor that regulates target gene expression. The presence of mutations in the AR gene has been observed in several types of cancer (e.g., prostate, breast, bladder, or esophageal cancer) and, in some cases, has been implicated in metastatic progression.

[0106] In some embodiments, the AR DNA or RNA is wild type or contains one or more mutations and / or splice variants. In some examples, the AR DNA or RNA contains one or more mutations. In some examples, the AR DNA or RNA contains one or more splice variants selected from AR splice variants including, but not limited to, AR1 / 2 / 2b, ARV2, ARV3, ARV4, AR1 / 2 / 3 / 2b, ARV5, ARV6, ARV7, ARV9, ARV10, ARV11, ARV12, ARV13, ARV14, ARV15, ARV16, and ARV(v567es). In some examples, the polynucleic acid molecule hybridizes to a target region of the AR DNA or RNA that contains a mutation (e.g., a substitution, deletion, or addition) or splice variant.

[0107] In some embodiments, the AR DNA or RNA comprises one or more mutations. In some embodiments, the AR DNA or RNA comprises one or more mutations in one or more exons. In some examples, the one or more exons include exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8. In some embodiments, the AR DNA or RNA comprises one or more mutations in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or a combination thereof. In some examples, the AR DNA or RNA comprises one or more mutations in one or more exons. The DNA or RNA may be modified to cleave the amino acid residues 2, 14, 16, 29, 45, 54, 57, 64, 106, 112, 176, 180, 184, 194, 198, 204, 214, 221, 222, 233, 243, 252, 255, 266, 269, 287, 288, 334, 335, 340, 363, 368, 369, 390, 403, 443, 491, 505, 513, 524, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 691, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 11 524, 528, 533, 547, 548, 564, 567, 568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579, 580, 581, 582, 585, 586, 587, 596, 597, 599, 601, 604, 607, 608, 609, 610, 611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647 , 653, 662, 664, 670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688, 689, 690, 695, 700, 701, 702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739, 7 41, 742, 743, 744, 745, 746, 748, 749, 750, 751, 752, 754, 755, 756, 757, 758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774, 777, 779, 786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794, 798, 802, 803, 804, 806,In some embodiments, the AR comprises one or more mutations at positions corresponding to 807, 812, 813, 814, 819, 820, 821, 824, 827, 828, 830, 831, 834, 840, 841, 842, 846, 854, 855, 856, 863, 864, 866, 869, 870, 871, 874, 875, 877, 879, 880, 881, 886, 888, 889, 891, 892, 895, 896, 897, 898, 902, 903, 904, 907, 909, 910, 911, 913, 916, 919, or a combination thereof. The DNA or RNA may be selected from the group consisting of E2K, P14Q, K16N, V29M, S45T, L54S, L57Q, Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S, G214R, K221N, N222D, D233K, S243L, A252V, L255P, M266T, P269S, A287D, E288K, S334P, S335T, P340L, Y363N, L368V, A369P, P390R, P390S, P390L, A403V, ​​Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P53 3S, L547F, P548S, D564Y, S567F, G568W, L574P, L547F, C559Y, G568W, G568V, Y571C, Y571H, A573D, T575A, C576R, C576F, G577R, S578T, C579Y, C579F, K580R, V581F, F582Y, F582S, R585K, A586V, A587S, A596T, A596S, S597G, S59 7I, N599Y, C601F, D604Y, R607Q, R608K, K609N, D610T, C611Y, R615H, R615P, R615G, R616C, L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T, L638M, A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H, I672T, L674P, L677P, E681L, P682T, G683A, V684I, V684A, A687V, G688Q, H689P, D690V, D695N, D695V, D695H, L700M,L701P, L701I, H701H, S702A, S703G, N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E, L712F, V715M, K717Q, K720E, A721T, L722F, P723S, G724S, G724D, G724N, F725L, R726L, N727K, L728S, L728I, V730M, D732N, D732Y, D732E, Q733H, I737T, Y739D, W741R, M742V, M742I, G743R, G743V, L744F, M745T, V746M, A748D, A748V, A748T, M749V, M749I, G750S, G750D, W751R, R 752Q, F754V, F754L, T755A, N756S, N756D, V757A, N758T, S759F, S759P, L76 2F, Y763H, Y763C, F764L, A765T, A765V, P766A, P766S, D767E, L768P, L768 M, N771H, E772G, E772A, R774H, R774C, K777T, R779W, R786Q, G795V, M780I, S782N, C784Y, M787V, R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G 803L, F804L, C806Y, M807V, M807R, M807I, L812P, F813V, S814N, N819Q, G82 0A, L821V, Q824L, Q824R, F827L, F827V, D828H, L830V, L830P, R831Q, R831 L, Y834C, R840C, R840H, I841S, I842T, R846G, R854K, R855C, R855H, F856L, L863R, D864N, D864E, D864G, V866L, V866M, V866E, I869M, A870G, A870V, R 871G, H874Y, H874R, Q875K, T877S, T877A, D879T, D879G, L880Q, L881V, M88 6V, S888L, V889M, F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903 M, P904S, P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R,or a combination thereof.

[0108] In some embodiments, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid hybridizes to one or more AR splice variants. In some embodiments, the polynucleic acid hybridizes to a target region of AR DNA or RNA that contains one or more splice variants, including but not limited to AR1 / 2 / 2b, ARV2, ARV3, ARV4, AR1 / 2 / 3 / 2b, ARV5, ARV6, ARV7, ARV9, ARV10, ARV11, ARV12, ARV13, ARV14, ARV15, ARV16, and ARV(v567es). In some embodiments, the polynucleic acid molecule hybridizes to a target region of AR DNA or RNA that contains one or more mutations in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, or a combination thereof. In some embodiments, the polynucleic acid molecule is selected from the group consisting of amino acid residues 2, 14, 16, 29, 45, 54, 57, 64, 106, 112, 176, 180, 184, 194, 198, 204, 214, 221, 222, 233, 243, 252, 255, 266, 269, 287, 288, 334, 335, 340, 363, 370, 372, 374, 376, 378, 379, 380, 382, ​​384, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 44 68, 369, 390, 403, 443, 491, 505, 513, 524, 524, 528, 533, 547, 548, 564, 567, 568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579, 580, 581, 582, 585, 586, 587, 596, 597, 59 9, 601, 604, 607, 608, 609, 610, 611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647, 653, 662, 664, 670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688, 689, 690, 695, 700, 701 , 702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717, 720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739, 741, 742, 743, 744, 745, 746, 748, 749, 750, 751, 752,754, 755, 756, 757, 758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774, 777, 779, 786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794, 798, 802, 803, 804, 806, 807, 812, 813, 814, 819, 820, 821, 824, 827, 828, 830, 831, 834 , 840, 841, 842, 846, 854, 855, 856, 863, 864, 866, 869, 870, 871, 874, 875, 877, 879, 880, 881, 886, 888, 889, 891, 892, 895, 896, 897, 898, 902, 903, 904, 907, 909, 910, 911, 913, 916, 919, or combinations thereof. In some embodiments, the polynucleic acid molecule comprises one or more of the following amino acids: E2K, P14Q, K16N, V29M, S45T, L54S, L57Q, Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S, G214R, K221N, N222D, D233K, S243L, A222R, B222R, C222R, D233K, C222R, D233R, D24 ... 252V, L255P, M266T, P269S, A287D, E288K, S334P, S335T, P340L, Y363N, L368V, A369P, P390 R, P390S, P390L, A403V, ​​Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P533S, L547F, P 548S, D564Y, S567F, G568W, L574P, L547F, C559Y, G568W, G568V, Y571C, Y571H, A573D, T57 5A, C576R, C576F, G577R, S578T, C579Y, C579F, K580R, V581F, F582Y, F582S, R585K, A586V, A587S, A596T, A596S, S597G, S597I, N599Y, C601F, D604Y, R607Q, R608K, K609N, D610T, C61 1Y, R615H, R615P, R615G, R616C, L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T,L638M, A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H, I672T, L674P, L677P, E681L, P682T, G683A, V684I, V684A, A687V, G688Q, H689P, D690V, D695N, D695V, D695H, L700M, L701P, L701I, H701H, S702A, S703G, N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E, L712F, V715M, K71 7Q, K720E, A721T, L722F, P723S, G724S, G724D, G724N, F725L, R726L, N727K , L728S, L728I, V730M, D732N, D732Y, D732E, Q733H, I737T, Y739D, W741R, M 742V, M742I, G743R, G743V, L744F, M745T, V746M, A748D, A748V, A748T, M74 9V, M749I, G750S, G750D, W751R, R752Q, F754V, F754L, T755A, N756S, N756D, V757A, N758T, S759F, S759P, L762F, Y763H, Y763C, F764L, A765T, A765V, P7 66A, P766S, D767E, L768P, L768M, N771H, E772G, E772A, R774H, R774C, K777 T, R779W, R786Q, G795V, M780I, S782N, C784Y, M787V, R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G803L, F804L, C806Y, M807V, M807R, M807I, L81 2P, F813V, S814N, N819Q, G820A, L821V, Q824L, Q824R, F827L, F827V, D828H , L830V, L830P, R831Q, R831L, Y834C, R840C, R840H, I841S, I842T, R846G, R 854K, R855C, R855H, F856L, L863R, D864N, D864E, D864G, V866L, V866M, V86 6E, I869M, A870G, A870V, R871G, H874Y, H874R, Q875K, T877S, T877A, D879T,Hybridizes to a target region of AR DNA or RNA containing one or more mutations selected from D879G, L880Q, L881V, M886V, S888L, V889M, F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903M, P904S, P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R, or a combination thereof.

[0109] Polynucleic acid molecules targeting β-catenin and β-catenin-related genes Catenin beta-1 (CTNNB1, also known as β-catenin or beta-catenin) is a member of the catenin protein family. In humans, it is encoded by the CTNNB1 gene and is known for its dual functions: cell-cell adhesion and gene transcription. Beta-catenin is an essential structural component of cadherin-based adherens junctions, which regulate cell growth and adhesion between cells and anchor the actin cytoskeleton. In some instances, beta-catenin is involved in sending contact inhibition signals that cause cells to stop dividing once epithelial sheets are completed. Beta-catenin is also a key nuclear effector of the Wnt signaling pathway. In some instances, imbalances in the structural signaling properties of beta-catenin lead to unregulated growth that is associated with various diseases and malignancies such as cancer. For example, overexpression of beta-catenin is associated with cancers such as gastric cancer (Suriano, et al., "Beta-catenin (CTNNB1) gene amplification: a new mechanism of protein overexpression in cancer," Genes Chromosomes Cancer 42(3): 238-246 (2005)). In some cases, mutations in the CTNNB1 gene are involved in the development of cancer (e.g., colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricoma, or prostate cancer) and, in some cases, in the progression of metastases. In further cases, mutations in the CTNNB1 gene translocate beta-catenin to the nucleus without external stimuli, continuously driving the transcription of its target genes. In some cases, the ability of beta-catenin to change the previous epithelial phenotype of affected cells to an invasive mesenchymal-like type contributes to metastasis formation.

[0110] In some embodiments, the CTNNB1 gene is wild-type CTNNB1 or CTNNB1 containing one or more mutations. In some examples, the CTNNB1 is wild-type CTNNB1. In some examples, the CTNNB1 is CTNNB1 containing one or more mutations. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of wild-type CTNNB1. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of CTNNB1 that contains a mutation (e.g., a substitution, deletion, or addition).

[0111] In some embodiments, the CTNNB1 DNA or RNA comprises one or more mutations. In some embodiments, the CTNNB1 DNA or RNA comprises one or more mutations in one or more exons. In some examples, the one or more exons include exon 3. In some examples, the CTNNB1 DNA or RNA comprises one or more mutations at codons 32, 33, 34, 37, 41, 45, 183, 245, 287, or combinations thereof. In some examples, the CTNNB1 DNA or RNA contains one or more mutations at positions corresponding to amino acid residues 25, 31, 32, 33, 34, 35, 36, 37, 41, 45, 140, 162, 170, 199, 213, 215, 257, 303, 322, 334, 354, 367, 373, 383, 387, 402, 426, 453, 474, 486, 515, 517, 535, 553, 555, 582, 587, 619, 623, 641, 646, 688, 703, 710, 712, 714, 724, 738, 777, or combinations thereof, of a CTNNB1 polypeptide. DNA or RNA may be used to identify the following mutations in the CTNNB1 polypeptide: W25 (nonsense mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y, S33F, S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N, T41A, T41I, S45Y, S45F, S45C, I140T, D162E, K170M, V199I, C213F, A215T, T257I, I303M, Q322K, E334K, K354T, G367V, P373S, , W383G, N387K, L402F, N426D, R453L, R453Q, R474 (nonsense mutation), R486C, R515Q, L517F, R535 (nonsense mutation), R535Q, M553V, G555A, R582Q, R587Q, C619Y, Q623E, T641 (frameshift), S646F, M688T, Q703H, R710H, D712N, P714R, Y724H, E738K, F777S, or a combination thereof.

[0112] In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA that contains one or more mutations in exon 3. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA that contains one or more mutations in codons 32, 33, 34, 37, 41, 45, 183, 245, 287, or combinations thereof. In some embodiments, the polynucleic acid molecule hybridizes to a target region of CTNNB1 DNA or RNA that contains one or more mutations at positions corresponding to amino acid residues 25, 31, 32, 33, 34, 35, 36, 37, 41, 45, 140, 162, 170, 199, 213, 215, 257, 303, 322, 334, 354, 367, 373, 383, 387, 402, 426, 453, 474, 486, 515, 517, 535, 553, 555, 582, 587, 619, 623, 641, 646, 688, 703, 710, 712, 714, 724, 738, 777, or combinations thereof, of a CTNNB1 polypeptide.In some embodiments, the polynucleic acid molecule is selected from the group consisting of W25 (nonsense mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y, S33F, S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N, T41A, T41I, S45Y, S45F, S45C, I140T, D162E, K170M, V199I, C213F, A215T, T257I, I303M, Q322K, E334K, K354T, G ... The hybridization target hybridizes to a target region of CTNNB1 DNA or RNA containing one or more mutations selected from 67V, P373S, W383G, N387K, L402F, N426D, R453L, R453Q, R474 (nonsense mutation), R486C, R515Q, L517F, R535 (nonsense mutation), R535Q, M553V, G555A, R582Q, R587Q, C619Y, Q623E, T641 (frameshift), S646F, M688T, Q703H, R710H, D712N, P714R, Y724H, E738K, F777S, or a combination thereof.

[0113] In some embodiments, the beta-catenin-related genes further include PIK3CA, PIK3CB, and MYC. In some embodiments, the beta-catenin-related genes further include PIK3CA DNA or RNA. PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha or p110α protein) is a class i PI3 kinase catalytic subunit that uses ATP to phosphorylate phosphatidylinositol. In some embodiments, the PIK3CA gene is wild-type PIK3CA or PIK3CA containing one or more mutations. In some examples, the PIK3CA DNA or RNA is wild-type PIK3CA. In some examples, the PIK3CA DNA or RNA contains one or more mutations. In some examples, the polynucleic acid molecule hybridizes to a target region of wild-type PIK3CA DNA or RNA. In some examples, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA containing a mutation (e.g., a substitution, deletion, or addition).

[0114] In some embodiments, the PIK3CA DNA or RNA comprises one or more mutations. In some embodiments, the PIK3CA DNA or RNA comprises one or more mutations in one or more exons. In some examples, the PIK3CA DNA or RNA comprises one or more mutations in exons 9 and / or 20. In some examples, the PIK3CA DNA or RNA comprises one or more mutations at amino acid residues 1, 4, 10-16, 11-18, 11, 12, 38, 39, 65, 72, 75, 79, 81, 83, 88, 90, 93, 102, 103, 103-104, 103-106, 104, 105-108, 106, 106-107, 106-108, 107, 108, 109-112, 110, 111, 113, 115, 137, 170, 258, 272, 279, 320, 328, 335, 342, 344, 345, 350, 357, 359, 363, 364, 365, 366, 378, 398, 401, 417, 420, 447-455, 449, 449-457 , 451, 453, 454, 455, 455-460, 463-465, 471, 495, 522, 538, 539, 542, 545, 546, 547, 576, 604, 614, 617, 629, 643, 663, 682, 725, 726, 777, 791, 818, 866, 901, 909, 939, 951, 9 In some embodiments, the PIK3CA polypeptide comprises one or more mutations at positions corresponding to 58, 970, 971, 975, 992, 1004, 1007, 1016, 1017, 1021, 1025, 1029, 1037, 1040, 1043, 1044, 1045, 1047, 1048, 1049, 1052, 1065, 1069, or a combination thereof.The DNA or RNA may be modified to express the following mutations in the PIK3CA polypeptide: M1V, R4 (nonsense mutation), L10-M16 (deletion), W11-P18 (deletion), W11L, G12D, R38L, R38H, R38C, R38S, E39K, E39G, E65K, S72G, Q75E, R79M, E81K, E81 (deletion), F83Y, R88Q, C90Y ... G, R93Q, R93W, I102 (deletion), E103G, E103-P104 (deletion), E103-G106 (deletion), P104L, V105-R108 (deletion), G106V, G106-N107 (deletion), G106-R108 (deletion), G106R, N107S, R108L, R108H, E109-I112 (deletion), E110 (deletion), K1 11E, K111R, K111N, K111 (deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272 (nonsense mutation), L279I, G320V, W328S, R335G, T342S, V344G, V344M, V344A, N345K, N345I, N345T, D350N, D350G, R357Q, G359R, G363A, G364R, E365K, E365V, P366R, C378R, C378Y, R398H, R401Q, E417K, C420R, C420G, P447-L455(deletion), P449L, P449-N457(deletion), G451R, G451V, E453K, E453Q, E453D, D454Y, L455(frame shiftinsertion), L455-G460 (deletion), G463-N465 (deletion), P471L, P471A, H495L, H495Y, E522A, D538N, P539R, E542K, E542V, E542G, E542Q, E542A, E545K, E545A, E545G, E545Q , E545D, Q546K, Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H, I663S, Q682(deletion), D725N, W726K, R777M, E791Q, R818C, L866W, C901F, F909L, D939 and comprising one or more mutations at positions corresponding to amino acid residues selected from G, R951C, Q958R, E970K, C971R, R975S, R992P, M1004I, G1007R, F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S, D1029H, E1037K, M1040V, M1043V, M1043I, N1044K, N1044Y, D1045V, H1047R, H1047L, H1047Y, H1047Q, H1048R, G1049R, T1052K, H1065L, 1069W (non-stop mutation), or combinations thereof.

[0115] In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA that contains one or more mutations in an exon. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA that contains one or more mutations in exon 9 or exon 20. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA that contains one or more mutations in exon 9 or exon 20. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA that contains one or more mutations in exon 9 or exon 20. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CA DNA or RNA that contains one or more mutations in exon 9 or exon 20. -108, 107, 108, 109-112, 110, 111, 113, 115, 137, 170, 258, 272, 279, 320, 328, 335, 342, 344, 345, 350, 357, 359, 363, 364, 365, 366, 378, 398, 401, 417, 420, 447-455, 449, 449-4 57, 451, 453, 454, 455, 455-460, 463-465, 471, 495, 522, 538, 539, 542, 545, 546, 547, 576, 604, 614, 617, 629, 643, 663, 682, 725, 726, 777, 791, 818, 866, 901, 909, 939, 951, 958 , 970, 971, 975, 992, 1004, 1007, 1016, 1017, 1021, 1025, 1029, 1037, 1040, 1043, 1044, 1045, 1047, 1048, 1049, 1052, 1065, 1069, or a combination thereof.In some embodiments, the polynucleic acid molecule hybridizes to a target region of DNA or RNA. In some embodiments, the polynucleic acid molecule hybridizes to a target region of the PIK3CA polypeptide, such as M1V, R4 (nonsense mutation), L10-M16 (deletion), W11-P18 (deletion), W11L, G12D, R38L, R38H, R38C, R38S, E39K, E39G, E65K, S72G, Q75E, R79M, E81K, E81 (deletion), F83Y, R88Q, C90Y, C90G, R93Q, R93W, I102 (deletion), E103G, E103-P104 (deletion), E103-G106 (deletion), P104L, V105-R108 (deletion), G106V, G106-N107 (deletion), G106-R108 (deletion), G106R, N107S, R108L, R108H, E109-I112 (deletion), E110 (deletion) deletion), K111E, K111R, K111N, K111 (deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272 (nonsense mutation), L279I, G320V, W328S, R335G, T342S, V344G, V344M, V344A, N345K, N345I, N345T, D350N, D350G, R35 7Q, G359R, G363A, G364R, E365K, E365V, P366R, C378R, C378Y, R398H, R401Q, E417K, C420R, C420G, P447-L455(deletion), P449L, P449-N457(deletion), G451R, G451V, E453K, E453Q, E453D, D454Y, L455(frame shiftinsertion), L455-G460 (deletion), G463-N465 (deletion), P471L, P471A, H495L, H495Y, E522A, D538N, P539R, E542K, E542V, E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D, Q546K, Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H, I663S, Q682(deletion), D725N, W726K, R777M, E791Q, R818C, L866W, C901F, F909L, D939G, R PIK3CA comprising one or more mutations at positions corresponding to amino acid residues selected from 951C, Q958R, E970K, C971R, R975S, R992P, M1004I, G1007R, F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S, D1029H, E1037K, M1040V, M1043V, M1043I, N1044K, N1044Y, D1045V, H1047R, H1047L, H1047Y, H1047Q, H1048R, G1049R, T1052K, H1065L, 1069W (non-stop mutation), or a combination thereof. A polynucleic acid molecule that hybridizes to a target region of DNA or RNA.

[0116] In some embodiments, the beta-catenin related gene further comprises PIK3CB. In some embodiments, the PIK3CB gene is wild type or comprises one or more mutations. In some examples, the PIK3CB DNA or RNA is wild type PIK3CB DNA or RNA. In some examples, the PIK3CB DNA or RNA comprises one or more mutations. In some examples, the polynucleic acid molecule hybridizes to a target region of wild type PIK3CB DNA or RNA. In some examples, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA that contains a mutation (e.g., a substitution, deletion, or addition).

[0117] In some embodiments, the PIK3CB DNA or RNA comprises one or more mutations.In some embodiments, the PIK3CB DNA or RNA comprises one or more mutations in one or more exons. In some examples, the PIK3CB DNA or RNA contains one or more mutations at positions corresponding to amino acid residues 18, 19, 21, 28, 50, 61, 68, 103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417, 442, 470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595, 619, 628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036, 1046, 1047, 1048, 1049, 1051, 1055, 1067, or combinations thereof, of a PIK3CB polypeptide. In some embodiments, the PIK3CB DNA or RNA comprises one or more of the following mutations in the PIK3CB polypeptide: W18 (nonsense mutation), A19V, D21H, G28S, A50P, K61T, M68I, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V, I304R, R321Q, V369I, T417M, N442K, E470K, E497D, P507S, I512M, E540 (nonsense mutation), C551R, E552K, E554K, R562 (nonsense mutation), The nucleic acid sequence includes one or more mutations at positions corresponding to amino acid residues selected from E567D, A593V, L595P, V619A, R628 (nonsense mutation), R668W, L768F, K805E, D824E, A830T, E887 (nonsense mutation), V967A, I992T, A1005V, D1020H, E1036K, D1046N, E1047K, A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or combinations thereof.

[0118] In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of PIK3CB DNA or RNA that contains one or more mutations within an exon. In some embodiments, the polynucleic acid molecule comprises a PIK3CB polypeptide comprising one or more mutations at positions corresponding to amino acid residues 18, 19, 21, 28, 50, 61, 68, 103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417, 442, 470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595, 619, 628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036, 1046, 1047, 1048, 1049, 1051, 1055, 1067, or a combination thereof. In some embodiments, the polynucleic acid molecule hybridizes to a target region of DNA or RNA. In some embodiments, the polynucleic acid molecule hybridizes to a target region of a PIK3CB polypeptide, such as W18 (nonsense mutation), A19V, D21H, G28S, A50P, K61T, M68I, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V, I304R, R321Q, V369I, T417M, N442K, E470K, E497D, P507S, I512M, E540 (nonsense mutation), C551R, E552K, E554K, R562 (nonsense mutation), The present invention hybridizes to a target region of PIK3CB DNA or RNA containing one or more mutations at positions corresponding to amino acid residues selected from among the following: E567D, A593V, L595P, V619A, R628 (nonsense mutation), R668W, L768F, K805E, D824E, A830T, E887 (nonsense mutation), V967A, I992T, A1005V, D1020H, E1036K, D1046N, E1047K, A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or combinations thereof.

[0119] In some embodiments, the beta-catenin related gene further comprises MYC. In some embodiments, the MYC gene is wild-type MYC or MYC containing one or more mutations. In some examples, the MYC is wild-type MYC DNA or RNA. In some examples, the MYC DNA or RNA contains one or more mutations. In some examples, the polynucleic acid molecule hybridizes to a target region of wild-type MYC DNA or RNA. In some examples, the polynucleic acid molecule is a polynucleic acid molecule that hybridizes to a target region of MYC DNA or RNA that contains a mutation (e.g., a substitution, deletion, or addition).

[0120] In some embodiments, the MYC DNA or RNA contains one or more mutations. In some embodiments, the MYC DNA or RNA contains one or more mutations in one or more exons. In some examples, the MYC DNA or RNA contains one or more mutations in exon 2 or exon 3. In some examples, the MYC DNA or RNA contains one or more mutations at positions corresponding to amino acid residues 2, 7, 17, 20, 32, 44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200, 202, 203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419, 431, 439, or combinations thereof, of a MYC polypeptide. In some embodiments, the MYC DNA or RNA comprises one or more mutations at positions corresponding to amino acid residues selected from P2L, F7L, D17N, Q20E, Y32N, A44V, A44T, T58I, P59L, A76V, F115L, F138S, A141S, V145I, S146L, S169C, S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E, V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K, R439Q, or a combination thereof.

[0121] In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA that contains one or more mutations in an exon. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA that contains one or more mutations in exon 2 or exon 3. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA containing one or more mutations at positions corresponding to amino acid residues 2, 7, 17, 20, 32, 44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200, 202, 203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419, 431, 439, or combinations thereof of a MYC polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of MYC DNA or RNA that contains one or more mutations at a position corresponding to an amino acid residue selected from P2L, F7L, D17N, Q20E, Y32N, A44V, A44T, T58I, P59L, A76V, F115L, F138S, A141S, V145I, S146L, S169C, S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E, V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K, R439Q, or a combination thereof of a MYC polypeptide.

[0122] Polynucleic acid molecules targeting hypoxanthine phosphoribosyltransferase 1 (HPRT1) Hypoxanthine guanine phosphoribosyltransferase (HGPRT) is a transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. HGPRT is encoded by the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene.

[0123] In some embodiments, the HPRT1 DNA or RNA is wild-type or contains one or more mutations. In some examples, the HPRT1 DNA or RNA contains one or more mutations in one or more exons. In some examples, the one or more exons include exon 2, exon 3, exon 4, exon 6, exon 8, or exon 9. In some examples, the HPRT1 DNA or RNA contains one or more mutations at positions corresponding to amino acid residues 35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or combinations thereof, of an HPRT1 polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA that contains one or more mutations selected from V35M, R48H, E56D, F74L, R87I, N129 (splice site mutation), N154H, S162 (splice site mutation), Y195C, Y195N, R200M, E210K, or combinations thereof in the HPRT1 polypeptide.

[0124] In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA that contains one or more mutations. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA that contains one or more mutations in exon 2, exon 3, exon 4, exon 6, exon 8, or exon 9. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA that contains one or more mutations at positions corresponding to amino acid residues 35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or combinations thereof, of an HPRT1 polypeptide. In some embodiments, the polynucleic acid molecule hybridizes to a target region of HPRT1 DNA or RNA that contains one or more mutations selected from V35M, R48H, E56D, F74L, R87I, N129 (splice site mutation), N154H, S162 (splice site mutation), Y195C, Y195N, R200M, E210K, or combinations thereof in the HPRT1 polypeptide.

[0125] Polynucleic acid molecule sequence In some embodiments, the polynucleic acid molecule comprises a sequence that hybridizes to a target sequence illustrated in Tables 1, 3, 5, 6, or 7. In some examples, the polynucleic acid molecule is B. In some examples, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 1 (KRAS target sequence). In some examples, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 3 (EGFR target sequence). In some examples, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 5 (AR target sequence). In some examples, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 6 (β-catenin target sequence). In additional examples, the polynucleic acid molecule B comprises a sequence that hybridizes to a target sequence illustrated in Table 7 (PIK3CA and PIK3CB target sequences).

[0126] In some embodiments, polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 2. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:165-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO:16-45.In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO:16-45. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO:16-45.

[0127] In some embodiments, polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16-45. In some examples, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16-45. In some examples, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16-45 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16-45.

[0128] In some embodiments, polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 4. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO: 422-1173. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO: 422-1173.In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO:422-1173. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO:422-1173.

[0129] In some embodiments, polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:422-1173. In some examples, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:422-1173. In some examples, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:422-1173 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:422-1173.

[0130] In some embodiments, polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 8. In some embodiments, polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1195-1214. In some embodiments, polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 1195-1214.In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO:1195-1214. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO:1195-1214.

[0131] In some embodiments, polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1195-1214. In some examples, the second polynucleotide comprises a sequence that is complementary to a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1195-1214. In some examples, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1195-1214 and a second polynucleotide that is complementary to a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1195-1214.

[0132] In some embodiments, polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 9. In some embodiments, polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1215-1242. In some embodiments, polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs: 1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs: 1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs: 1215-1242.In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO:1215-1242. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO:1215-1242.

[0133] In some embodiments, polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1215-1242. In some examples, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1215-1242. In some examples, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1215-1242 and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1215-1242.

[0134] Polynucleic acid molecule In some embodiments, the polynucleic acid molecules described herein comprise RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some cases, the RNA comprises small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some cases, the RNA comprises shRNA. In some cases, the RNA comprises miRNA. In some cases, the RNA comprises dsRNA. In some cases, the RNA comprises tRNA. In some cases, the RNA comprises rRNA. In some cases, the RNA comprises hnRNA. In some cases, the RNA comprises siRNA. In some cases, the polynucleic acid molecule comprises siRNA. In some cases, B comprises siRNA.

[0135] In some embodiments, the nucleic acid polymer is about 10 to about 50 nucleotides in length. In some embodiments, the polynucleic acid molecule is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length.

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

[0137] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide. In some examples, the polynucleic acid molecule comprises a second polynucleotide. In some examples, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide is a sense strand or a passenger strand. In some examples, the second polynucleotide is an antisense strand or a guide strand.

[0138] In some embodiments, the polynucleic acid molecule is a first polynucleotide. In some embodiments, the first polynucleotide is about 10 to about 50 nucleotides in length. In some embodiments, the first polynucleotide 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.

[0139] In some examples, the first polynucleotide is about 50 nucleotides in length. In some examples, the first polynucleotide is about 45 nucleotides in length. In some examples, the first polynucleotide is about 40 nucleotides in length. In some examples, the first polynucleotide is about 35 nucleotides in length. In some examples, the first polynucleotide is about 30 nucleotides in length. In some examples, the first polynucleotide is about 25 nucleotides in length. In some examples, the first polynucleotide is about 20 nucleotides in length. In some examples, the first polynucleotide is about 19 nucleotides in length. In some examples, the first polynucleotide is about 18 nucleotides in length. In some examples, the first polynucleotide is about 17 nucleotides in length. In some examples, the first polynucleotide is about 16 nucleotides in length. In some examples, the first polynucleotide is about 15 nucleotides in length. In some examples, the first polynucleotide is about 14 nucleotides in length. In some examples, the first polynucleotide is about 13 nucleotides in length. In some examples, the first polynucleotide is about 12 nucleotides in length. In some examples, the first polynucleotide is about 11 nucleotides in length. In some examples, the first polynucleotide is about 10 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 50 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 45 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 40 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 35 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 30 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 25 nucleotides in length. In some examples, the first polynucleotide is about 10 to about 20 nucleotides in length. In some examples, the first polynucleotide is about 15 to about 25 nucleotides in length.In some examples, the first polynucleotide is about 15 to about 30 nucleotides in length. In some examples, the first polynucleotide is about 12 to about 30 nucleotides in length.

[0140] In some embodiments, the polynucleic acid molecule is a second polynucleotide. In some embodiments, the second polynucleotide is about 10 to about 50 nucleotides in length. In some embodiments, the second polynucleotide 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.

[0141] In some examples, the second polynucleotide is about 50 nucleotides in length. In some examples, the second polynucleotide is about 45 nucleotides in length. In some examples, the second polynucleotide is about 40 nucleotides in length. In some examples, the second polynucleotide is about 35 nucleotides in length. In some examples, the second polynucleotide is about 30 nucleotides in length. In some examples, the second polynucleotide is about 25 nucleotides in length. In some examples, the second polynucleotide is about 20 nucleotides in length. In some examples, the second polynucleotide is about 19 nucleotides in length. In some examples, the second polynucleotide is about 18 nucleotides in length. In some examples, the second polynucleotide is about 17 nucleotides in length. In some examples, the second polynucleotide is about 16 nucleotides in length. In some examples, the second polynucleotide is about 15 nucleotides in length. In some examples, the second polynucleotide is about 14 nucleotides in length. In some examples, the second polynucleotide is about 13 nucleotides in length. In some examples, the second polynucleotide is about 12 nucleotides in length. In some examples, the second polynucleotide is about 11 nucleotides in length. In some examples, the second polynucleotide is about 10 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 50 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 45 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 40 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 35 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 30 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 25 nucleotides in length. In some examples, the second polynucleotide is about 10 to about 20 nucleotides in length. In some examples, the first polynucleotide is about 15 to about 25 nucleotides in length.In some examples, the first polynucleotide is about 15 to about 30 nucleotides in length. In some examples, the first polynucleotide is about 12 to about 30 nucleotides in length.

[0142] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. 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.

[0143] 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 examples, the sequence of the polynucleic acid molecule is 100% complementary to the target sequence described herein.

[0144] In some embodiments, the sequence of the polynucleic acid molecule has 5 or less mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has 4 or less mismatches to the target sequences described herein. In some cases, the sequence of the polynucleic acid molecule may have 3 or less mismatches to the target sequences described herein. In some cases, the sequence of the polynucleic acid molecule may have 2 or less mismatches to the target sequences described herein. In some cases, the sequence of the polynucleic acid molecule may have 1 or less mismatch to the target sequences described herein.

[0145] In some embodiments, the specificity of a polynucleic acid molecule hybridizing to a target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence, in some instances, hybridization is under high stringency hybridization conditions.

[0146] 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. 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 the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 15 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 16 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 17 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 18 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 19 contiguous bases of the target sequence described herein. In some embodiments, the polynucleic acid molecule hybridizes to at least 20 contiguous bases of the target sequence described herein.

[0147] In some embodiments, the polynucleic acid molecule has reduced off-target effects. In some instances, "off-target" or "off-target effect" refers to any instance where a polynucleic acid polymer for a given 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.

[0148] In some embodiments, the polynucleic acid molecule comprises natural, synthetic, or artificial nucleotide analogs or bases. In some cases, the polynucleic acid molecule comprises a combination of DNA, RNA, and / or nucleotide analogs. In some examples, the synthetic or artificial nucleotide analogs or bases comprise modifications at one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or a combination thereof.

[0149] 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. In some embodiments, the 5'-vinylphosphonate modified nucleotide is selected from the nucleotides provided below:

[0150] [ka]

[0151] In some embodiments, the modification of 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.

[0152] In some instances, the 5'-vinylphosphonate is further modified with a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which an 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'-vinylphosphonate modified LNA is illustrated below, where J is an internucleotide bond.

[0153] [ka]

[0154] In some embodiments, additional modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0155] In some embodiments, the nucleotide analogs include modified bases, such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides (7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azocytidine). thymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, O- and N-alkylated purines and pyrimidines, such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine, 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracil and thymine, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. 5'-vinylphosphonate modified nucleotides further include those nucleotides that are modified on the sugar moiety, as well as 5'-vinylphosphonate modified nucleotides having non-ribosyl sugars or analogs thereof, for example, in some cases the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles.The term nucleotide further includes those known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0156] In some embodiments, the 5'-vinylphosphonate modified nucleotide analogs further include morpholinos, peptide nucleic acids (PNAs), methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5' phosphoramidites, or 1',5'-anhydrohexitol nucleic acids (HNAs). Morpholinos or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics the natural nucleic acid structure but deviates from the normal sugar and phosphate 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, the ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some cases, backbone alterations remove all positive and negative charges making the morpholino neutral molecule capable of crossing cell membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides. Non-limiting examples of 5'-vinylphosphonate modified morpholino oligonucleotides are illustrated below.

[0157] [ka]

[0158] In some embodiments, the 5'-vinylphosphonate modified morpholino or PMO described above is a PMO that contains a positive or cationic charge. In some examples, the PMO is PMOplus (Sarepta). PMOplus refers to phosphorodiamidate morpholino oligomers that contain any number of (1-piperazino)phosphinylideneoxy, (1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy bonds (such as those described in PCT International Publication No. WO2008 / 036127). In some examples, the PMO is a PMO described in U.S. Patent No. 7,943,762.

[0159] In some embodiments, the morpholino or PMO described above is PMO-X (Sarepta). In some cases, PMO-X refers to a phosphorodiamidate morpholino oligomer that includes at least one bond or at least one of the disclosed terminal modifications, such as those described in PCT International Publication No. WO2011 / 150408 and U.S. Publication No. 2012 / 0065169.

[0160] In some embodiments, the morpholino or PMO described above is a PMO as described in Table 5 of U.S. Publication No. 2014 / 0296321.

[0161] An exemplary representation of the chemical structure of a 5'-vinylphosphonate modified nucleic acid is illustrated below, where J is an internucleotide linkage.

[0162] [ka]

[0163] In some embodiments, peptide nucleic acids (PNAs) contain no sugar backbone rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating the backbone charge.

[0164] [ka]

[0165] In some embodiments, one or more modifications of the 5'-vinylphosphonate modified oligonucleotides are optionally made at the internucleotide bond. In some examples, the modified internucleotide bond may be, but is not limited to, phosphorothioate; phosphorodithioate; methyl phosphonate; 5'-alkylene phosphonate; 5'-methyl phosphonate; 3'-alkylene phosphonate; boron trifluoride; 3'-5' or 2'-5' boranophosphate and selenophosphate; phosphotriester; thionoalkyl phosphotriester; hydrogen phosphonate bond; alkyl phosphonate; alkyl phosphonothioate; aryl phosphonothioate; phosphoroselenoate; phosphorodiselenoate; phosphinate; phosphoramidate; 3'-alkyl phosphoramidate; aminoalkyl phosphoramidate; thionophosphoramidate; phospho. Examples of such linkers include lopiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazo; methylenedimethylhydrazo; formacetals; thioformacetals; oximes; methyleneimino; methylenemethylimino; thioamidates; linkages with riboacetyl groups; aminoethylglycines; silyl or siloxane linkages; alkyl or cycloalkyl linkages, e.g., saturated or unsaturated and / or containing heteroatoms, with or without heteroatoms of 1 to 10 carbons; linkages with morpholino structures, amides, or polyamides in which the base is attached directly or indirectly to the aza nitrogen of the backbone; and combinations thereof.

[0166] In some examples, the modification is a methyl or thiol modification, such as a methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left), phosphorodithioate (middle), and methylphosphonate nucleotides (right) are illustrated below.

[0167] [ka]

[0168] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, phosphoramidites exemplified as follows:

[0169] [ka]

[0170] In some examples, the modified internucleotide linkage is a phosphorodiamidate linkage. Non-limiting examples of phosphorodiamidate linkages using the morpholino system are shown below.

[0171] [ka]

[0172] In some examples, the modified internucleotide linkage is a methylphosphonate linkage. Non-limiting examples of methylphosphonate linkages are shown below.

[0173] [ka]

[0174] In some instances, the modified internucleotide bond is an amide bond. Non-limiting examples of amide bonds are shown below.

[0175] [ka]

[0176] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, the modified nucleic acids exemplified below.

[0177] In some embodiments, the one or more modifications include a modified phosphate backbone, where the modification produces a neutral or uncharged backbone. In some examples, the phosphate backbone is modified by alkylation, which produces an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, an alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing 1-6 carbon atoms. In some examples, 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 cases, the modified phosphate is a phosphate group described in U.S. Pat. No. 9,481,905.

[0178] In some embodiments, the additional modified phosphate backbone comprises a methylphosphonate, an ethylphosphonate, a methylthiophosphonate, or a methoxyphosphonate. In some embodiments, the modified phosphate is a methylphosphonate. In some embodiments, the modified phosphate is an ethylphosphonate. In some embodiments, the modified phosphate is a methylthiophosphonate. In some embodiments, the modified phosphate is a methoxyphosphonate.

[0179] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or the nucleotide analog at the 3' or 5' end. For example, the 3' end optionally includes a 3' cationic group, or the nucleoside is inverted at the 3' end including a 3'-3' bond. In another alternative, the 3' end is optionally linked to an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In an additional alternative, the 3' end is optionally linked to an abasic site, such as an apurinic or apyrimidinic site.

[0180] In some embodiments, the polynucleic acid molecule comprises one or more artificial nucleotide analogs described herein. In some examples, the 5'-vinylphosphonate modified polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 5'-vinylphosphonate modified nucleotide analogs described herein. In some embodiments, the artificial 5'-vinylphosphonate modified nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5' phosphoramidites, or combinations thereof. In some examples, the 5'-vinyl phosphonate is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or ...methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'-O-methyl, 2'- or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 20, 25 or more artificial nucleotide analogs selected from 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5' phosphoramidites, or combinations thereof.In some embodiments, a 5'-vinylphosphonate modified polynucleic acid molecule 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 embodiments, a 5'-vinylphosphonate modified polynucleic acid molecule 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, the 5'-vinylphosphonate modified polynucleic acid molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiolphosphonate nucleotides.

[0181] In some embodiments, the 5'-vinylphosphonate modified polynucleic acid molecule contains 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 or more modifications. In some examples, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242.

[0182] In some examples, the 5'-vinylphosphonate modified polynucleic acid molecule 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 or more modified nucleotides. In some examples, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242.

[0183] In some examples, the 5'-vinylphosphonate modified polynucleic acid molecule comprises at least one of the following: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification. In some examples, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242.

[0184] In some examples, about 5 to about 100% of the 5'-vinylphosphonate modified polynucleic acid molecules comprise 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 comprise 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 of SEQ ID NOs:16-45, 422-1173, 1195-1214, or 1215-1242 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 of SEQ ID NO: 16-45 contain artificial nucleotide analogs as described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules of SEQ ID NO: 422-1173 contain artificial nucleotide analogs as described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules of SEQ ID NOs: 1195-1214 contain artificial nucleotide analogs as described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules of SEQ ID NOs: 1215-1242 contain artificial nucleotide analogs as described herein.In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, -methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0185] In some cases, one or more of the artificial 5'-vinylphosphonate modified nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases, as compared to naturally occurring polynucleic acid molecules. In some examples, the 5'-vinylphosphonate modified nucleotide analogs are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some examples, the 5'-vinylphosphonate modified nucleotide analogs are resistant to nucleases, e.g., 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). Artificial nucleotide analogs, including modified LNA, ENA, PNA, HNA, morpholino, -methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, 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. In some examples, 2'-O-methyl modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, 2'-deoxy modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, T-deoxy-2'-O-fluoro modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, LNA-modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, ENA-modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, HNA-modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). Morpholinos are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, PNA-modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, methylphosphonate nucleotide modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the thiophosphonate nucleotide modified polynucleic acid molecules are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some examples, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0186] In some embodiments, one or more of the artificial 5'-vinylphosphonate modified nucleotide analogs described herein have increased binding affinity for their mRNA targets compared to a comparable naturally occurring polynucleic acid molecule. One or more of the artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5' phosphoramidites, or combinations thereof, may have increased binding affinity to the mRNA compared to a comparable naturally occurring polynucleic acid molecule. In some examples, a 2'-O-methyl modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule. In some examples, a 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule. In some examples, a 2'-O-aminopropyl modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule. In some examples, a 2'-deoxy modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule. In some examples, a T-deoxy-2'-fluoro modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule. In some examples, a 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule.In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, LNA-modified polynucleic acid molecules have increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, an ENA-modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some examples, a PNA-modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some examples, an HNA-modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some examples, a morpholino-modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some examples, a methylphosphonate nucleotide-modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some examples, a thiolphosphonate nucleotide-modified polynucleic acid molecule has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some examples, a polynucleic acid molecule comprising a 2'-fluoro N3-P5'-phosphoramidite has increased binding affinity to its mRNA target compared to an equivalent natural polynucleic acid molecule. In some cases, the increased affinity is demonstrated by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0187] In some embodiments, the 5'-vinylphosphonate modified polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules that include a single enantiomer. In some examples, the polynucleic acid molecules include L-nucleotides. In some examples, the polynucleic acid molecules include D-nucleotides. In some examples, the polynucleic acid molecule composition includes 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some cases, the polynucleic acid molecule composition includes 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; WO2015107425.

[0188] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer binding moiety. In some examples, the aptamer binding moiety is a DNA aptamer binding moiety. In some examples, the aptamer binding moiety is Alphamer (Centauri Therapeutics), which includes an aptamer portion that recognizes a specific cell surface target and a portion 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 binding moiety as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0189] In additional 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 two hydroxyl positions, such as with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, or with locked or bridged ribose structures (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'-fluoro N3-P5'-phosphoramidite to increase its stability. In some examples, the polynucleic acid molecule is a chirally pure (or stereopure) polynucleic acid molecule. In some examples, the chirally pure (or stereopure) polynucleic acid molecule is modified to increase its stability. The appropriate modification of RNA to increase the stability of delivery is clear to those skilled in the art.

[0190] In some embodiments, the polynucleic acid molecules described herein have RNAi activity that regulates the expression of the RNA encoded by the gene. In some examples, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of a gene, where one strand of the double-stranded siRNA molecule comprises a nucleotide sequence that is complementary to the nucleotide sequence of the gene or the RNA encoded by the gene or a portion thereof, and the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence that is substantially similar to the nucleotide sequence of the gene or the RNA encoded by the gene or a portion thereof. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of a gene, where each strand of the siRNA molecule comprises about 15-25, 18-24, or 19-about 23 nucleotides, and each strand comprises at least about 14, 17, or 19 nucleotides that are complementary to the nucleotides of the other strand. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate expression of a gene, wherein each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and each strand comprises at least about 19 nucleotides that are complementary to nucleotides of the other strand. In some examples, the gene is KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or a β-catenin-related gene.

[0191] 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 those described in: 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., the transcribed RNA of the inserted polynucleic acid molecule will be in an antisense orientation to the desired target polynucleic acid molecule).

[0192] One embodiment is a compound of formula (I): AXBYC Formula I providing a molecule of During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first non-polymeric linker; and Y is a bond or a second linker; wherein the polynucleotide comprises at least one 5'-vinylphosphonate modified non-natural nucleotide; and wherein A and C are not linked to B at the same end.

[0193] Another embodiment provides a molecule of formula (I): wherein the polynucleotide optionally comprises at least one modified internucleotide linkage or at least one inverted abasic moiety.

[0194] Another embodiment provides a molecule of formula (I): at least one 5'-vinylphosphonate modified non-natural nucleotide is located at the 5'-terminus of the polynucleotide.

[0195] Another embodiment provides a molecule of formula (I): at least one 5'-vinylphosphonate modified non-natural nucleotide is positioned at an internucleotide linkage of the polynucleotide.

[0196] Another embodiment provides a molecule of formula (I): at least one 5'-vinylphosphonate modified non-natural nucleotide is further modified at the 2'-position.

[0197] Another embodiment provides a molecule of formula (I): the 2'-modification is selected from a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2-O-NMA) modified nucleotide.

[0198] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0199] [ka] B is a heterocyclic base moiety.

[0200] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0201] [ka] B is a heterocyclic base moiety; R1, R2, and R3 are independently selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0202] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0203] [ka] B is a heterocyclic base moiety; R4 and R5 are independently selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that links adjacent nucleotides of the polynucleotide.

[0204] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0205] [ka] B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or acyl; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0206] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified unnatural nucleotide is selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

[0207] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0208] [ka] B is a heterocyclic base moiety; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0209] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0210] [ka] B is a heterocyclic base moiety; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0211] Another embodiment provides a molecule of formula (I): wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is:

[0212] [ka] B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or acyl; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0213] Another embodiment provides a molecule of formula (I): at least one modified internucleotide linkage comprises a phosphorothioate linkage, a phosphorodithioate linkage, a phosphorodiamidate linkage, a methylphosphonate linkage, or an amide linkage.

[0214] Another embodiment provides a molecule of formula (I): wherein at least one inverted abasic moiety is at least one terminal.

[0215] One embodiment provides an oligonucleotide of Formula (II), wherein the oligonucleotide comprises at least one 5'-vinylphosphonate modified non-natural nucleotide.

[0216] Another embodiment provides an oligonucleotide of Formula (II), wherein the oligonucleotide further comprises at least one modified internucleotide linkage or at least one inverted abasic moiety.

[0217] Another embodiment provides an oligonucleotide of Formula (II), wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is located at the 5'-terminus of the polynucleotide.

[0218] Another embodiment provides an oligonucleotide of formula (II), wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is positioned at an internucleotide linkage of the polynucleotide.

[0219] Another embodiment provides an oligonucleotide of formula (II), wherein at least one 5'-vinylphosphonate modified non-natural nucleotide is further modified at the 2'-position.Another embodiment provides an oligonucleotide of formula (II), wherein the 2'-modification is selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2-O-NMA) modified nucleotides.

[0220] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0221] [ka] B is a heterocyclic base moiety.

[0222] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0223] [ka] B is a heterocyclic base moiety; R1, R2, and R3 are independently selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0224] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0225] [ka] B is a heterocyclic base moiety; R4 and R5 are independently selected from hydrogen, halogen, alkyl, or alkoxy; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0226] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0227] [ka] B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or acyl; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0228] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified unnatural nucleotide is selected from a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA).

[0229] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0230] [ka] B is a heterocyclic base moiety; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0231] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from:

[0232] [ka] B is a heterocyclic base moiety; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0233] Another embodiment provides an oligonucleotide of formula (II), wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from the following:

[0234] [ka] B is a heterocyclic base moiety; R6 is selected from hydrogen, halogen, alkyl, or acyl; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0235] Another embodiment provides an oligonucleotide of formula (II), wherein at least one modified internucleotide linkage comprises a phosphorothioate linkage, a phosphorodithioate linkage, a phosphorodiamidate linkage, a methylphosphonate linkage, or an amide linkage.

[0236] Another embodiment provides an oligonucleotide of formula (II), wherein at least one inverted abasic moiety is at least one terminus.

[0237] Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is single-stranded.Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is double-stranded.

[0238] Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 100 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 90 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 80 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 70 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 60 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 50 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 40 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 30 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 20 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 2 to about 10 residues long. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 8 to about 30 residues in length. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 10 to about 30 residues in length. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 14 to about 30 residues in length. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 18 to about 30 residues in length. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 22 to about 30 residues in length. Another embodiment provides an oligonucleotide of formula (II), wherein the oligonucleotide is 26 to about 30 residues in length.

[0239] One embodiment provides a compound suitable for the synthesis of an oligonucleotide selected from the group consisting of:

[0240] [ka] B is a heterocyclic base moiety.

[0241] bond 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, as well as all analogs or derivatives of these classes of substances. Additional examples of binding moieties further include cholesterol, phospholipids, diacylglycerol and triacylglycerol, fatty acids, carbohydrates (e.g., saturated, unsaturated, or substituted), enzyme substrates, steroids such as biotin, digoxigenin, and polysaccharides. 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.

[0242] In some embodiments, the polynucleic acid molecule is attached to the binding moiety by a chemical ligation process. In some examples, the polynucleic acid molecule is attached to the binding moiety by native ligation. In some instances, the conjugation may be performed using methods such as those described in Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, No. 4,116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleic acid molecule is site-specifically or non-specifically conjugated to a binding moiety via native ligation chemistry.

[0243] In some instances, polynucleic acid molecules are coupled 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 then coupled to a polynucleic acid molecule that contains 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)).

[0244] In some examples, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an unnatural amino acid introduced into the binding moiety. In some examples, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some examples, the keto group of pAcPhe is selectively conjugated to an alkoxy-amine derived binding 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)).

[0245] In some examples, the polynucleic acid molecule is attached to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process. In some examples, the site-directed method utilizes SMARTag™ technology (Redwood). In some examples, SMARTag™ technology involves the generation of formylglycine (FGly) residues from cysteine ​​by formylglycine generating enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent attachment of FGly to an alkylhydrazine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (See 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))

[0246] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some examples, the polynucleic acid molecule is conjugated to the binding moiety using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some examples, mTG is produced from Streptomyces 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))

[0247] 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.

[0248] In some examples, the polynucleic acid molecule is attached to the binding moiety by methods such as those described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0249] joining part In some embodiments, binding moiety A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , F(ab)' 3 Fragments, single chain variable fragments (scFv), bis-scFv (scFv) 2 , diabodies, minibodies, nanobodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv), single domain antibodies (sdAbs), Ig NARs, camelid antibodies or binding fragments thereof, bispecific antibodies or binding fragments thereof, or chemically modified derivatives thereof.

[0250] In some examples, A is an antibody or binding fragment thereof. In some examples, A is a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , F(ab)' 3 Fragments, single chain variable fragments (scFv), bis-scFv (scFv) 2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some examples, A is a humanized antibody or binding fragment thereof. In some examples, A is a murine antibody or binding fragment thereof. In some examples, A is a chimeric antibody or binding fragment thereof. In some examples, A is a monoclonal antibody or binding fragment thereof. In some examples, A is a monovalent Fab'. In some examples, A is a bivalent Fab. 2 In some examples, A is a single chain variable fragment (scFv).

[0251] In some embodiments, binding moiety A is a bispecific antibody or a binding fragment thereof. In some examples, the bispecific antibody is a trifunctional antibody or a bispecific miniantibody. In some cases, the bispecific antibody is a trifunctional antibody. In some examples, the trifunctional antibody is a full-length monoclonal antibody that contains binding sites for two different antigens. Exemplary trifunctional antibodies include catumaxomab (targeting EpCAM and CD3; Fresenius Biotech / Trion Pharma), ertumaxomab (targeting HER2 / neu / CD3; Fresenius Biotech / Trion Pharm), lymphomun FBTA05 (targeting CD20 / CD3; Fresenius Biotech / Trion Pharm), and erythromycin (targeting HER2 / neu / CD3; Fresenius Biotech / Trion Pharm). Pharm), RG7221 (RO5520985; targets Angiopoietin 2 / VEGF; Roche), RG7597 (targets Her1 / Her3; Genentech / Roche), MM141 (targets IGF1R / Her3; Merrimack), ABT122 (targets TNFα / IL17; Abbvie), ABT981 (targets IL1α / IL1β; Abbott), LY3164530 (targets Her1 / cMET; Eli Lilly), and TRBS07 (Ektomab; targets GD2 / CD3; Trion Research Gmbh). Further exemplary trifunctional antibodies include the mAb from F-star Biotechnology Ltd. 2In some examples, A is a bispecific trifunctional antibody. In some embodiments, A is a bispecific trifunctional antibody selected from catumaxomab (targets EpCAM and CD3; Fresenius Biotech / Trion Pharma), ertumaxomab (targets HER2 / neu / CD3; Fresenius Biotech / Trion Pharm), lymphomun FBTA05 (targets CD20 / CD3; Fresenius Biotech / Trion Pharm), RG7221 (RO5520985; angiopoietin 2 / VEGF; Roche), RG7597 (targeting Her1 / Her3; Genentech / Roche), MM141 (targeting IGF1R / Her3; Merrimack), ABT122 (targeting TNFα / IL17; Abbvie), ABT981 (targeting IL1α / IL1β; Abbott), LY3164530 (targeting Her1 / cMET; Eli Lilly), TRBS07 (Ektomab; targeting GD2 / CD3; Trion Research Gmbh), and mAbs from F-star Biotechnology Ltd. 2 .

[0252] In some cases, the bispecific antibody is a bispecific miniantibody. In some examples, the bispecific miniantibody is a bivalent Fab 2 , F(ab)' 3 Fragment, bis-scFv (scFv) 2, diabodies, minibodies, triabodies, tetrabodies, or bispecific T cell engagers (BiTEs). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single chain variable fragments (scFvs), where the two scFvs target epitopes of two different antigens. Exemplary bispecific miniantibodies include, but are not limited to: DART (Dual Affinity Retargeting Platform; MacroGenics), blinatumomab (MT103 or AMG103; targets CD19 / CD3; Micromet), MT111 (targets CEA / CD3; Micromet / Amegen), MT112 (BAY2010112; targets PSMA / CD3; Micromet / Bayer), MT110 (AMG 110; targets EPCAM / CD3; Amgen / Micromet), MGD006 (targets CD123 / CD3; MacroGenics), MGD007 (targets GPA33 / CD3; MacroGenics), BI1034020 (targets two different epitopes on β-amyloid; Ablynx), ALX0761 (targets IL17A / IL17F; Ablynx), TF2 (targets CEA / Heptene; Immunomedics), IL-17 / IL-34 biAb (BMS), AFM13 (targets CD30 / CD16; Affimed), AFM11 (targets CD19 / CD3; Affimed), and domain antibodies (dAbs from Domantis / GSK).

[0253] 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)' 3In some embodiments, A is a bispecific bis-scFv. In some embodiments, A is a bispecific (scFv). 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 miniantibody selected from: 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 110; targets EPCAM / CD3; Amgen / Micromet), MGD006 (targets CD123 / CD3; MacroGenics), MGD007 (targets GPA33 / CD3; MacroGenics), BI1034020 (targets two different epitopes on β-amyloid; Ablynx), ALX0761 (targets IL17A / IL17F; Ablynx), TF2 (targets CEA / Heptene; Immunomedics), IL-17 / IL-34 biAb (BMS), AFM13 (targets CD30 / CD16; Affimed), AFM11 (targets CD19 / CD3; Affimed), and domain antibodies (dAbs from Domantis / GSK).

[0254] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody is F(ab)' 3 In some embodiments, A is a trispecific F(ab)' 3In 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).

[0255] In some embodiments, binding moiety A is an antibody or binding fragment thereof that recognizes a cell surface protein. In some examples, the cell surface protein is an antigen expressed by a cancer cell. Exemplary cancer antigens include, but are not limited to, alpha fetoprotein, ASLG659, B7-H3, BAFF-R, brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, S PAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein (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 migration 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 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), and others.

[0256] 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, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw6 0, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, These include 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), and the like.

[0257] 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.

[0258] 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, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, C an antibody or binding fragment thereof that recognizes D49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, 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), or a combination thereof;

[0259] 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, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblitutumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek Inc.), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), framvotumab (ImClone Systems), frezolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), direntuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImClone Systems Inc.), imgatuzumab (Genentech / Roche), intetumumab (Centocor), ipilimumab (Yervoy®, Bristol-Myers Squibb), Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), lumiliximab, mapatuzumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek Inc.), oregovomab (OvaRex® (AltaRex, Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotopex GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech / Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumumab (Schering-Plough), seribantomab (Sanofi / Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab peptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab-cell moleukin (EMD These include ublituximab (BMS-663513, Bristol-Myers Squibb), volociximab (M200, Biogen Idec), and zatuximab.

[0260] 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, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblitutumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek Inc.), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), framvotumab (ImClone Systems), frezolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), direntuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImClone Systems Inc.), imgatuzumab (Genentech / Roche), intetumumab (Centocor), ipilimumab (Yervoy®, Bristol-Myers Squibb), Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), lumiliximab, mapatuzumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek Inc.), oregovomab (OvaRex® (AltaRex, Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotopex GMBH), parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech / Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumumab (Schering-Plough), seribantomab (Sanofi / Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab peptox, tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab-cell moleukin (EMD In some embodiments, binding moiety A is zalutumumab (HuMax-EFGr by Genmab).

[0261] In some embodiments, the binding moiety A is linked to a polynucleic acid molecule (B) according to formula (I) and to a polymer (C), and optionally to an endosomolytic moiety (D) according to formula (II) described herein. In some examples, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listed in Table 2, 4, 8, or 9. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242. In some examples, the polynucleic acid molecule has a sequence selected from SEQ ID NO: 16-45, 422-1173, 1195-1214, or 1215-1242. In some examples, the polymer C comprises a polyalkylene oxide (e.g., polyethylene glycol). In some embodiments, the endosomolytic moiety D comprises INF7 or melittin, or a derivative of each.

[0262] In some embodiments, binding moiety A binds to a polynucleic acid molecule (B) and a polymer (C), and optionally to an endosomolytic moiety (D). In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0263] In some embodiments, binding moiety A binds non-specifically to polynucleic acid molecule (B). In some examples, binding moiety A binds to polynucleic acid molecule (B) in a non-site specific manner via a lysine or cysteine ​​residue. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a non-site specific manner via a lysine residue. In some cases, binding moiety A binds to polynucleic acid molecule (B) in a non-site specific manner via a cysteine ​​residue. In some examples, binding moiety A is an antibody or binding fragment thereof.

[0264] In some embodiments, the binding moiety A binds to the polynucleic acid molecule (B) in a non-site specific manner. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner via a lysine residue, a cysteine ​​residue, at the 5'-end, at the 3'-end, at an unnatural amino acid, or at an enzyme-modified or enzyme-catalyzed residue. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner via a lysine residue. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner via a cysteine ​​residue. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner at the 5'-end. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner at the 3'-end. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner via an unnatural amino acid. In some examples, the binding moiety A binds to the polynucleic acid molecule (B) in a site specific manner via an enzyme-modified or enzyme-catalyzed residue. In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0265] In some embodiments, one or more regions of binding moiety A (e.g., an antibody or binding fragment thereof) bind to polynucleic acid molecule (B). In some examples, one or more regions of binding moiety A include N-terminus, C-terminus, within the constant region, hinge region, or Fc region of binding moiety A. In some examples, polynucleic acid molecule (B) binds 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) binds 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) binds 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) binds 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) binds to the Fc region of binding moiety A (e.g., the constant region of an antibody or binding fragment thereof).

[0266] In some embodiments, one or more polynucleic acid molecules (B) are bound to 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 bound to one binding moiety A. In some examples, about 1 polynucleic acid molecule is bound to one binding moiety A. In some examples, about 2 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 3 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 4 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 5 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 6 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 7 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 8 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 9 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 10 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 11 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 12 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 13 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 14 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 15 polynucleic acid molecules are bound to one binding moiety A. In some examples, about 16 polynucleic acid molecules are bound to one binding moiety A. In some cases, one or more of the polynucleic acid molecules are the same. In other examples, one or more of the polynucleic acid molecules are different. In some examples, binding moiety A is an antibody or a binding fragment thereof.

[0267] In some embodiments, the number of polynucleic acid molecules (B) bound to binding moiety A (e.g., an antibody or binding fragment thereof) forms a ratio. In some examples, the ratio is referred to as a DAR (drug to antibody) ratio, and the drug as referred to herein is the polynucleic acid molecule (B). In some examples, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 2 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 3 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 4 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 5 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 6 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 7 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 8 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 9 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 10 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 11 or greater. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 12 or greater.

[0268] In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A (e.g., an antibody or binding fragment thereof) is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 2. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 3. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 4. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 5. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 6. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 7. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 8. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 9. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 10. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 11. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 12. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 13. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 14. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 15. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 16.

[0269] In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 1. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 2. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 4. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 6. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 8. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is 12.

[0270] In some embodiments, the antibody or binding fragment thereof is further modified, alone or in combination, using conventional techniques known in the art, for example, by using amino acid deletion, insertion, substitution, addition, and / or recombination, and / or other modifications known in the art (e.g., post-translational and chemical modifications such as glycosylation and phosphorylation). In some examples, the modifications further include modifications to modulate interaction with Fc receptors. In some examples, one or more modifications include, for example, those described in International Publication No. WO97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence underlying the amino acid sequence of the antibody or binding fragment thereof are well known to those skilled in the art.

[0271] In some instances, an antibody binding fragment further includes derivatives thereof and comprises a polypeptide sequence comprising at least one CDR.

[0272] In some instances, the term "single-chain" as described 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.

[0273] In some examples, the bispecific single chain antibody construct relates to a construct comprising binding domains from two antibodies. In such embodiments, the bispecific single chain antibody construct is a tandem bi-scFv or diabody. In some examples, the scFv comprises a VH and a VL domain connected by a linker peptide. In some examples, the linker is of sufficient length and sequence to allow each of the first and second domains to retain their differential binding specificity independently of each other.

[0274] In some embodiments, as used herein, binding with or interaction by defines the binding / interaction of at least two antigen interaction sites with each other. In some examples, the antigen interaction site defines a motif of a polypeptide that exhibits a capacity for a specific interaction with a specific antigen or a specific group of antigens. In some cases, the binding / interaction is also understood to define a specific recognition. In such cases, the specific recognition refers to the ability of an antibody or a binding fragment thereof to specifically interact and / or bind to at least two amino acids of each of the target molecules. For example, the specific recognition relates to the specificity of the antibody molecule or its ability to distinguish a specific range of target molecules. In additional examples, the specific interaction of the antigen interaction site with its specific antigen results in the initiation of a signal, for example, by inducing a conformational change of the antigen, oligomerization of the antigen, and the like. In further embodiments, the binding is exemplified by the specificity of the "key-lock-principle". Thus, in some examples, the antigen interaction site and the specific motif in the amino acid sequence of the antigen bind to each other as a result of their primary, secondary, or tertiary structure, as well as as a result of a second modification of said structure. In such cases, the specific interaction of the antigen interaction site with its specific antigen results in the simple binding of the site to the antigen.

[0275] In some instances, specific interaction further refers to reduced cross-reactivity of an antibody or its binding fragment, or reduced off-target effects. For example, an antibody or its binding fragment that binds to a desired polypeptide / protein but does not essentially bind to any other polypeptide is considered to be specific for the desired polypeptide / protein. Specificity of antigen interaction site Examples of specific interactions with an antigen include the specificity of a ligand with its receptor, for example, the interaction of a group of antigenic determinants (epitopes) with the antigen binding site of an antibody.

[0276] Additional binding moieties In some embodiments, binding moiety A is a plasma protein. In some examples, the plasma protein includes albumin. In some examples, binding moiety A is albumin. In some examples, albumin is modified to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some examples, albumin is conjugated to the polynucleic acid molecule by native ligation chemistry. In some examples, albumin is conjugated to the polynucleic acid molecule by lysine linkage.

[0277] In some examples, the binding moiety A is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, saturated, unsaturated, substituted hydrocarbons, or combinations thereof. In some examples, the steroid is cholesterol. In some examples, the binding moiety is cholesterol. In some examples, the cholesterol is bound to the polynucleic acid molecule by one or more of the binding chemistries described herein. In some examples, the cholesterol is bound to the polynucleic acid molecule by native ligation chemistry. In some examples, the cholesterol is bound to the polynucleic acid molecule by a lysine bond.

[0278] In some examples, the binding moiety is a polymer, including but not limited to a polynucleic acid molecule aptamer, that binds to a specific surface marker on a cell. In this example, the binding moiety is a polynucleic acid that does not hybridize to a target gene or mRNA, but instead can selectively bind to the cell surface marker similar to an antibody that binds to that specific epitope of the cell surface marker.

[0279] In some examples, the binding moiety A is a peptide. In some cases, the peptide comprises about 1 to about 3 kDa. In some cases, the peptide comprises about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some examples, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some examples, the binding moiety is a bicyclic peptide (e.g., Bicycle from Bicycle Therapeutics).

[0280] In further cases, 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, where the target-binding end can recognize and interact with a cell surface receptor. For example, in some instances, the target-binding end that comprises a glutamate urea compound allows for interaction with PSMA, thereby enhancing antibody interaction with cells that express PSMA (e.g., cancer cells). In some instances, 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).

[0281] Production of antibodies or binding fragments thereof In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced using any method known in the art to aid in the synthesis of polypeptides (e.g., antibodies), inter alia, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0282] In some examples, antibodies or binding fragments thereof are recombinantly expressed and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesis of overlapping oligonucleotides containing portions of the antibody encoding sequence, annealing and ligation of the oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0283] Alternatively, nucleic acid molecules encoding antibodies are optionally produced from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing 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.

[0284] In some examples, the antibody or binding thereof is optionally produced by immunizing an animal such as a rabbit to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72) or 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 Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of FAb fragments that bind specific antigens, or by screening antibody libraries (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0285] In some embodiments, techniques developed for the production of "chimeric antibodies" (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) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity 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 mouse monoclonal antibody and a human immunoglobulin constant region (e.g., humanized antibodies).

[0286] In some embodiments, techniques described for the production of single chain antibodies (US Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad.Sci.USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are suitable for producing single chain antibodies. Single chain antibodies are formed by linking heavy or light chain fragments of the Fv region by an amino acid bridge to produce 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).

[0287] In some embodiments, an expression vector containing an antibody nucleotide sequence or the antibody nucleotide sequence is introduced into a host cell 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, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0288] In some embodiments, a variety of host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent vehicles in which antibody coding sequences are produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or binding fragments thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment coding sequence; insect cell systems (e.g., baculovirus) infected with recombinant virus expression vectors containing the antibody or binding fragment coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., Cauliflower Mosaic Virus (CaMV) and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or binding fragment coding sequence; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0289] For long-term and 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., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, the cells are engineered to grow in enriched medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cells to stably integrate the plasmid into their chromosomes and grow to form foci that are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that advantageously express antibodies or binding fragments thereof.

[0290] In some instances, 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 employed in tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance is used as the selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods known in the art of recombinant DNA technology that can be used are generally 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).

[0291] In some instances, antibody expression levels are increased by vector amplification (for review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture increases the number of copies of the marker gene. Because the amplified region is related to the antibody nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0292] In some examples, any method known in the art for purification of antibodies is used, for example, by chromatography (e.g., ion exchange, affinity, especially affinity to specific antigens followed by Protein A and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purification of proteins.

[0293] Polymer Binding Moiety In some embodiments, the polymer moiety C is further linked to a polynucleic acid molecule as described herein, a binding moiety as described herein, or a combination thereof. In some examples, the polymer moiety C is linked to a polynucleic acid molecule. In some cases, the polymer moiety C is linked to a binding moiety. In other cases, the polymer moiety C is linked to a polynucleic acid molecule binding moiety molecule. In further cases, the polymer moiety C is linked and is as discussed in the therapeutic molecular platform section.

[0294] In some examples, the polymer moiety C is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of two- or three-dimensional monomers. In some examples, the polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C includes, but is not limited to, alpha, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, a mixture refers to the use of various polymers within the same compound, as in the context of block copolymers. In some cases, a block copolymer is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some examples, polymer portion C includes a polyalkylene oxide. In some examples, polymer portion C includes PEG. In some examples, polymer portion C includes polyethylene imide (PEI) or hydroxyethyl starch (HES).

[0295] In some examples, C is a PEG moiety. In some examples, the PEG moiety is attached at the 5' end of the polynucleic acid molecule, while the conjugation moiety is attached at the 3' end of the polynucleic acid molecule. In some examples, the PEG moiety is attached at the 3' end of the polynucleic acid molecule, while the conjugation moiety is attached at the 5' end of the polynucleic acid molecule. In some examples, the PEG moiety is attached to an internal site of the polynucleic acid molecule. In some examples, the PEG moiety, the conjugation moiety, or a combination thereof, is attached to an internal site of the polynucleic acid molecule. In some examples, the conjugate is a direct conjugate. In some examples, the conjugation is via native ligation.

[0296] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some examples, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some examples, 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 given represents the average molecular weight of the polyalkylene oxide (e.g., PEG) molecules.

[0297] 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, 83 700, 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.

[0298] In some embodiments, C is a polyalkylene oxide (e.g., 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, 270 having a molecular weight of 0, 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 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, 8500 In some examples, the molecular weight of C is about 200 Da. In some examples, the molecular weight of C is about 300 Da. In some examples, the molecular weight of C is about 400 Da. In some examples, the molecular weight of C is about 500 Da. In some examples, the molecular weight of C is about 600 Da. In some examples, the molecular weight of C is about 700 Da. In some examples, the molecular weight of C is about 800 Da. In some examples, the molecular weight of C is about 900 Da. In some examples, the molecular weight of C is about 1000 Da. In some examples, the molecular weight of C is about 1100 Da. In some examples, the molecular weight of C is about 1200 Da. In some examples, the molecular weight of C is about 1300 Da. In some examples, the molecular weight of C is about 1400 Da.In some examples, the molecular weight of C is about 1450 Da. In some examples, the molecular weight of C is about 1500 Da. In some examples, the molecular weight of C is about 1600 Da. In some examples, the molecular weight of C is about 1700 Da. In some examples, the molecular weight of C is about 1800 Da. In some examples, the molecular weight of C is about 1900 Da. In some examples, the molecular weight of C is about 2000 Da. In some examples, the molecular weight of C is about 2100 Da. In some examples, the molecular weight of C is about 2200 Da. In some examples, the molecular weight of C is about 2300 Da. In some examples, the molecular weight of C is about 2400 Da. In some examples, the molecular weight of C is about 2500 Da. In some examples, the molecular weight of C is about 2600 Da. In some examples, the molecular weight of C is about 2700 Da. In some examples, the molecular weight of C is about 2800 Da. In some examples, the molecular weight of C is about 2900 Da. In some examples, the molecular weight of C is about 3000 Da. In some examples, the molecular weight of C is about 3250 Da. In some examples, the molecular weight of C is about 3350 Da. In some examples, the molecular weight of C is about 3500 Da. In some examples, the molecular weight of C is about 3750 Da. In some examples, the molecular weight of C is about 4000 Da. In some examples, the molecular weight of C is about 4250 Da. In some examples, the molecular weight of C is about 4500 Da. In some examples, the molecular weight of C is about 4600 Da. In some examples, the molecular weight of C is about 4750 Da. In some examples, the molecular weight of C is about 5000 Da. In some examples, the molecular weight of C is about 5500 Da. In some examples, the molecular weight of C is about 6000 Da. In some examples, the molecular weight of C is about 6500 Da. In some examples, the molecular weight of C is about 7000 Da. In some examples, the molecular weight of C is about 7500 Da. In some examples, the molecular weight of C is about 8,000 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.

[0299] 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 2-60, 2-50, or 2-48 repeating ethylene oxide units. In some examples, the dPEG contains about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50, or more repeating ethylene oxide units. In some examples, the dPEG contains about 2 or more repeating ethylene oxide units. In some examples, the dPEG contains about 3 or more repeating ethylene oxide units. In some examples, the dPEG contains about 4 or more repeating ethylene oxide units. In some examples, the dPEG contains about 5 or more repeating ethylene oxide units. In some examples, the dPEG contains about 6 or more repeating ethylene oxide units. In some examples, dPEG contains about 7 or more repeating ethylene oxide units. In some examples, dPEG contains about 8 or more repeating ethylene oxide units. In some examples, dPEG contains about 9 or more repeating ethylene oxide units. In some examples, dPEG contains about 10 or more repeating ethylene oxide units. In some examples, dPEG contains about 11 or more repeating ethylene oxide units. In some examples, dPEG contains about 12 or more repeating ethylene oxide units. In some examples, dPEG contains about 13 or more repeating ethylene oxide units. In some examples, dPEG contains about 14 or more repeating ethylene oxide units. In some examples, dPEG contains about 15 or more repeating ethylene oxide units. In some examples, dPEG contains about 16 or more repeating ethylene oxide units. In some examples, dPEG contains about 17 or more repeating ethylene oxide units. In some examples, dPEG contains about 18 or more repeating ethylene oxide units. In some examples, dPEG contains about 19 or more repeating ethylene oxide units. In some examples, dPEG contains about 20 or more repeating ethylene oxide units.In some examples, dPEG contains about 22 or more repeating ethylene oxide units. In some examples, dPEG 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 as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials in a stepwise manner. 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.

[0300] In some embodiments, the polymer portion C comprises a cationic mucic acid-based polymer (cMAP). In some examples, the cMPA comprises one or more subunits of at least one repeating subunit, the subunit structure being represented as formula (III):

[0301] [ka]

[0302] wherein m at each occurrence is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4-6, or 5; and n at each occurrence is independently 0, 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0303] 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 is about 500 Da to about 1000 Da, from 1000 Da to about 5000 Da, from 5000 Da to about 10,000 Da, from 10,000 Da to about 25,000 Da, from 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

[0304] In some examples, the polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the polymer moiety C is a cMAP-PEG copolymer. In other cases, the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In further cases, the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0305] In some embodiments, the polymer moiety C is linked to the polynucleic acid molecule, the binding moiety, and optionally the endosomolytic moiety.

[0306] Endosomolytic moiety In some embodiments, the molecule of formula (I), AXBYC, further comprises an additional binding moiety. In some examples, the additional binding moiety is an endosomolytic moiety. In some cases, the endosomolytic moiety is a cell compartment releasing component, such as a compound that can be released from any of the cell compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum with cells. 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.

[0307] Endosomolytic Polypeptides In some embodiments, the molecule of formula (I):AXBYC is further linked to an endosomolytic polypeptide. In some cases, the endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the endosomolytic polypeptide is an amphipathic polypeptide. In additional cases, the endosomolytic polypeptide is a peptidomimetic. In some examples, the endosomolytic polypeptide comprises INF, melittin, mucin, or their respective derivatives. In some examples, the endosomolytic polypeptide comprises INF or their respective derivatives. In other cases, the endosomolytic polypeptide comprises melittin or their respective derivatives. In further cases, the endosomolytic polypeptide comprises mucin or their respective derivatives.

[0308] In some examples, INF7 is a 24 residue polypeptide and these sequences include CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 1243), or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 1244). In some examples, INF7 or a derivative thereof includes the following sequences: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 1245), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 1246), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 1247).

[0309] In some instances, melittin is a 26 residue polypeptide and the sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 1248), or alternatively, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 1249). In some instances, melittin comprises a polypeptide sequence described in U.S. Patent No. 8,501,930.

[0310] In some instances, the mucin is an antimicrobial peptide (AMP) derived from the venom gland of the scorpion Mesobuthus eupeus. In some instances, the mucin is comprised of mucin-13, the sequence of which includes IFGAIAGLLKNIF-NH2 (SEQ ID NO: 1250), and mucin-18, the sequence of which includes FFGHLFKLATKIIPSLFQ (SEQ ID NO: 1251).

[0311] In some examples, the endosomolytic polypeptide comprises a polypeptide whose sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to INF7 or a derivative thereof, melittin or a derivative thereof, or a mucin or a derivative thereof, hi some examples, the endosomolytic moiety comprises INF7 or a derivative thereof, melittin or a derivative thereof, or a mucin or a derivative thereof.

[0312] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1243-1247. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1243. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1244-1247. Optionally, the endosomolytic portion comprises SEQ ID NO:1243. Optionally, the endosomolytic portion comprises SEQ ID NO:1244-1247. Optionally, the endosomolytic portion consists of SEQ ID NO:1243. Optionally, the endosomolytic portion consists of SEQ ID NO:1244-1247.

[0313] In some examples, the endosomolytic moiety is melittin or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1248 or 1249. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1248. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1249. Optionally, the endosomolytic portion comprises SEQ ID NO:1248. Optionally, the endosomolytic portion comprises SEQ ID NO:1249. Optionally, the endosomolytic portion consists of SEQ ID NO:1248. Optionally, the endosomolytic portion consists of SEQ ID NO:1249.

[0314] In some examples, the endosomolytic moiety is a mucin or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1250 or 1251. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1250. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1251. Optionally, the endosomolytic portion comprises SEQ ID NO:1250. Optionally, the endosomolytic portion comprises SEQ ID NO:1251. Optionally, the endosomolytic portion consists of SEQ ID NO:1250. Optionally, the endosomolytic portion consists of SEQ ID NO:1251.

[0315] In some examples, the endosomolytic moiety comprises a sequence as illustrated in Table 10.

[0316] [Table 1-1]

[0317] [Table 1-2]

[0318] In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide that induces apoptosis through antagonism of inhibitory targets 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).

[0319] In some examples, the endosomolytic moiety comprises a polypeptide (e.g., a cell-permeable polypeptide) as described in PCT International Publication No. WO2013 / 166155 or WO2015 / 069587.

[0320] Endosomolytic polymers In some embodiments, the molecule of formula (I):AXBYC is further conjugated to an endosomolytic polymer. As used herein, endosomolytic polymers include linear, branched network, star, comb, or ladder polymers. In some examples, the endosomolytic polymer is a homopolymer or a copolymer comprising two or more different types of monomers. In some cases, the endosomolytic polymer is a polycationic polymer. In other cases, the endosomolytic polymer is a polyanionic polymer.

[0321] In some instances, polycationic polymers are composed of monomer units that are positively charged, neutrally charged, or negatively charged, resulting in a net positive charge. In other instances, polycationic polymers include non-polymeric molecules that contain two or more positive charges. Exemplary cationic polymers include, but are not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyethyleneimine (PEI), poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or N,N-diethylaminoethyl methacrylate (DEAEMA).

[0322] In some cases, polyanionic polymers are composed of monomer units that are positively charged, neutrally charged, or negatively charged, and the net charge is negative. In other cases, polyanionic polymers include non-polymeric molecules that contain two or more negative charges. Exemplary anionic polymers include p(alkyl acrylates) (e.g., poly(propylacrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIPAM). Additional examples include PP75, L-phenylalanine-poly(L-lysine isophthalamide) polymer, described in Khormaee, et al., "Edosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications," Advanced Functional Materials 23: 565-574 (2013).

[0323] In some embodiments, the endosomolytic polymers described herein are pH-responsive endosomolytic polymers. pH-responsive polymers include polymers that increase in size (swell) or disintegrate depending on the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers.

[0324] In some instances, the endosomolytic moieties described herein are membrane disruptive polymers. In some instances, the membrane disruptive polymers include cationic polymers, neutral or hydrophobic polymers, or anionic polymers. In some instances, the membrane disruptive polymers are hydrophilic polymers.

[0325] In some examples, the endosomolytic moiety described herein is a membrane-disruptive polymer. Exemplary pH-responsive membrane-disruptive polymers include p(alkylacrylic acid), poly(N-isopropylacrylamide) (NIPAM) copolymers, succinylated p(glycidol), and p(β-malic acid) polymers.

[0326] In some examples, the p(alkylacrylic acid) includes poly(propylacrylic acid (polyPAA), poly(methacrylic acid (PMAA)), poly(ethylacrylic acid (PEAA), and poly(propylacrylic acid) (PPAA). In some examples, the p(alkylacrylic acid) includes the p(alkylacrylic acid) described in Jones, et al., Biochemistry Journal 372: 65-75 (2003).

[0327] In some embodiments, the pH-responsive membrane disruptive polymer comprises p(butyl acrylate-co-methacrylic acid). (See Bulmus, et al., Journal of Controlled Release 93: 105-120(2003); and Yessine, et al., Biochimica et Biophysica Acta 1613: 28-38(2003)).

[0328] In some embodiments, the pH-responsive membrane disruptive polymer comprises p(styrene-alt-maleic anhydride). (See, e.g., Henry, et al., Biomacromolecules 7: 2407-2414 (2006)).

[0329] In some embodiments, the pH-responsive membrane disruptive polymer comprises a pyridyl disulfide acrylate (PDSA) polymer, such as poly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-co-PDSA), poly(MAA-co-BA-co-PDSA), poly(EAA-co-BA-co-PDSA), or poly(PAA-co-BA-co-PDSA) polymer (see El-Sayed, et al., "Rational design of composition and activity correlations for pH-responsive and glutathione-reactive polymer therapeutics," Journal of Controlled Release 104: 417-427(2005); or Flanary et al., "Antigen delivery with poly(propylacrylic acid) conjugation enhanced MHC-1 presentation and T-cell activation," Bioconjugate Chem. 20: 241-248(2009)).

[0330] In some embodiments, the pH-responsive membrane disruptive polymer comprises a cytolytic polymer comprising the following base structure:

[0331] [ka]

[0332] In some examples, the endosomolytic moieties described herein are further linked to an additional conjugate, such as a polymer (e.g., PEG) or a modified polymer (e.g., a cholesterol-modified polymer).

[0333] In some examples, the additional conjugate comprises a detergent (e.g., Triton X-100). In some examples, the endosomolytic moieties described herein comprise a polymer (e.g., a poly(amidoamine)) conjugated to a detergent (e.g., Triton X-100). In some examples, the endosomolytic moieties described herein comprise poly(amidoamine)-Triton X-100 (Duncan, et al., "A polymer-Triton X-100 conjugate capable of pH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments," Journal of Drug Targeting 2: 341-347(1994)).

[0334] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the molecule of Formula (I):AXBYC 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).

[0335] In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in PCT International Publication No. WO 09 / 126,933.

[0336] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, the molecule of formula (I):AXBYC 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), amopyrroquine, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, the quinoline endosomolytic moiety may be, but is not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline; 4-(4-diethyl-amino-1-butylamino)quinoline; 7-hydroxy -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 ... 7-Fluoro-4-(1-carboxy-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;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-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline; 3-Fluoro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline 4-(4-hydroxy-alpha,alpha'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde; 1,1'-pentamethylenediquinolinium diiodide; 8-quinolinol sulfate, and amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs thereof. In some examples, the endosomolytic moiety is a small molecule described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Pat. No. 5,736,557.

[0337] Molecule of formula (I)-endosomolytic moiety conjugate In some embodiments, one or more endosomolytic moieties are conjugated to a molecule comprising at least one binding moiety, at least one polynucleotide, at least one polymer, or any combination thereof. In some examples, the endosomolytic moiety has formula (II): (AXBYC c )-LD Formula II Binds to During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer between 0 and 1; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide; and D is linked to any of A, B, or C.

[0338] In some embodiments, A and C are not attached to B at the same end.

[0339] In some embodiments, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide. In some examples, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA). In some cases, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some examples, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a single bond or a non-polymeric linker group. In some examples, A is an antibody or binding fragment thereof. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or a binding fragment thereof. In some cases, C is polyethylene glycol.

[0340] In some examples, the endosomolytic moiety comprises a polypeptide, polymer, lipid, or small molecule. In some examples, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In further cases, the endosomolytic moiety is an endosomolytic small molecule.

[0341] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1243. In some examples, the endosomolytic moiety comprises SEQ ID NO:1243. In some examples, the endosomolytic moiety consists of SEQ ID NO:1243.

[0342] In some examples, the endosomolytic moiety is melittin or a derivative thereof. Optionally, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1248. Optionally, the endosomolytic moiety comprises SEQ ID NO:1248. Optionally, the endosomolytic moiety consists of SEQ ID NO:1248.

[0343] In some examples, the endosomolytic moiety is a sequence as illustrated in Table 10.

[0344] In further cases, the endosomolytic moiety is an endosomolytic polymer, such as, for example, a pH-responsive endosomolytic polymer, a membrane disruptive polymer, a polycationic polymer, a polyanionic polymer, a pH-responsive membrane disruptive polymer, or a combination thereof. In further cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butylacrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyl disulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-surfactant conjugate, or a combination thereof.

[0345] In some embodiments, the endosomolytic moiety conjugate comprises According to formula (IIa): DLAXBYC c Formula IIa During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer equal to 1; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide.

[0346] In some embodiments, A and C are not attached to B at the same end.

[0347] In some embodiments, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide. In some examples, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA). In some cases, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some examples, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a single bond or a non-polymeric linker group. In some examples, A is an antibody or binding fragment thereof. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or a binding fragment thereof. In some cases, C is polyethylene glycol.

[0348] In some examples, the endosomolytic moiety comprises a polypeptide, polymer, lipid, or small molecule. In some examples, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In further cases, the endosomolytic moiety is an endosomolytic small molecule.

[0349] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1243. In some examples, the endosomolytic moiety comprises SEQ ID NO:1243. In some examples, the endosomolytic moiety consists of SEQ ID NO:1243.

[0350] In some examples, the endosomolytic moiety is melittin or a derivative thereof. Optionally, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1248. Optionally, the endosomolytic moiety comprises SEQ ID NO:1248. Optionally, the endosomolytic moiety consists of SEQ ID NO:1248.

[0351] In some examples, the endosomolytic moiety is a sequence as illustrated in Table 10.

[0352] In further cases, the endosomolytic moiety is an endosomolytic polymer, such as, for example, a pH-responsive endosomolytic polymer, a membrane disruptive polymer, a polycationic polymer, a polyanionic polymer, a pH-responsive membrane disruptive polymer, or a combination thereof. In further cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butylacrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyl disulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-surfactant conjugate, or a combination thereof.

[0353] In some examples, the endosomolytic moiety conjugate is according to formula (IIb): AXBLD Formula IIb During the ceremony, A is a binding moiety; B is a polynucleotide; X is a bond or a first linker; L is a bond or a third linker; and D is an endosomolytic moiety; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide.

[0354] In some embodiments, A and C are not attached to B at the same end.

[0355] In some embodiments, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide. In some examples, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA). In some cases, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some examples, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X and L are independently a single bond or a non-polymeric linker group. In some examples, A is an antibody or binding fragment thereof. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or a binding fragment thereof. In some cases, C is polyethylene glycol.

[0356] In some examples, the endosomolytic moiety comprises a polypeptide, polymer, lipid, or small molecule. In some examples, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In further cases, the endosomolytic moiety is an endosomolytic small molecule.

[0357] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1243. In some examples, the endosomolytic moiety comprises SEQ ID NO:1243. In some examples, the endosomolytic moiety consists of SEQ ID NO:1243.

[0358] In some examples, the endosomolytic moiety is melittin or a derivative thereof. Optionally, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1248. Optionally, the endosomolytic moiety comprises SEQ ID NO:1248. Optionally, the endosomolytic moiety consists of SEQ ID NO:1248.

[0359] In some examples, the endosomolytic moiety is a sequence as illustrated in Table 10.

[0360] In further cases, the endosomolytic moiety is an endosomolytic polymer, such as, for example, a pH-responsive endosomolytic polymer, a membrane disruptive polymer, a polycationic polymer, a polyanionic polymer, a pH-responsive membrane disruptive polymer, or a combination thereof. In further cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butylacrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyl disulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-surfactant conjugate, or a combination thereof.

[0361] In some examples, the endosomolytic moiety conjugate is according to formula (IIc): AXBYC c -LD Formula IIc During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer equal to 1; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide.

[0362] In some embodiments, A and C are not attached to B at the same end.

[0363] In some embodiments, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide. In some examples, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA). In some cases, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some examples, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a single bond or a non-polymeric linker group. In some examples, A is an antibody or binding fragment thereof. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or a binding fragment thereof. In some cases, C is polyethylene glycol.

[0364] In some examples, the endosomolytic moiety comprises a polypeptide, polymer, lipid, or small molecule. In some examples, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In further cases, the endosomolytic moiety is an endosomolytic small molecule.

[0365] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1243. In some examples, the endosomolytic moiety comprises SEQ ID NO:1243. In some examples, the endosomolytic moiety consists of SEQ ID NO:1243.

[0366] In some examples, the endosomolytic moiety is melittin or a derivative thereof. Optionally, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1248. Optionally, the endosomolytic moiety comprises SEQ ID NO:1248. Optionally, the endosomolytic moiety consists of SEQ ID NO:1248.

[0367] In some examples, the endosomolytic moiety is a sequence as illustrated in Table 10.

[0368] In further cases, the endosomolytic moiety is an endosomolytic polymer, such as, for example, a pH-responsive endosomolytic polymer, a membrane disruptive polymer, a polycationic polymer, a polyanionic polymer, a pH-responsive membrane disruptive polymer, or a combination thereof. In further cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butylacrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyl disulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-surfactant conjugate, or a combination thereof.

[0369] In some examples, the endosomolytic moiety conjugate is according to formula (IId): ALDXBY-Cc Formula IId During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer equal to 1; and The polynucleotide comprises at least one 5'-vinylphosphonate modified nucleotide.

[0370] In some embodiments, A and C are not attached to B at the same end.

[0371] In some embodiments, at least one 2'-modified nucleotide comprises a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide. In some examples, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA) or an ethylene nucleic acid (ENA). In some cases, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some examples, the second polynucleotide comprises at least one modification. In some cases, the first polynucleotide and the second polynucleotide are RNA molecules. In some cases, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X, Y, and L are independently a single bond or a non-polymeric linker group. In some examples, A is an antibody or binding fragment thereof. In some examples, the antibody or binding fragment thereof is a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab 2 , a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or a binding fragment thereof. In some cases, C is polyethylene glycol.

[0372] In some examples, the endosomolytic moiety comprises a polypeptide, polymer, lipid, or small molecule. In some examples, the endosomolytic moiety is an endosomolytic polypeptide. In some cases, the endosomolytic moiety is an endosomolytic polymer. In other cases, the endosomolytic moiety is an endosomolytic lipid. In further cases, the endosomolytic moiety is an endosomolytic small molecule.

[0373] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1243. In some examples, the endosomolytic moiety comprises SEQ ID NO:1243. In some examples, the endosomolytic moiety consists of SEQ ID NO:1243.

[0374] In some examples, the endosomolytic moiety is melittin or a derivative thereof. Optionally, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:1248. Optionally, the endosomolytic moiety comprises SEQ ID NO:1248. Optionally, the endosomolytic moiety consists of SEQ ID NO:1248.

[0375] In some examples, the endosomolytic moiety is a sequence as illustrated in Table 10.

[0376] In further cases, the endosomolytic moiety is an endosomolytic polymer, such as, for example, a pH-responsive endosomolytic polymer, a membrane disruptive polymer, a polycationic polymer, a polyanionic polymer, a pH-responsive membrane disruptive polymer, or a combination thereof. In further cases, the endosomolytic moiety comprises a p(alkylacrylic acid) polymer, a p(butylacrylate-co-methacrylic acid) polymer, a p(styrene-alt-maleic anhydride) polymer, a pyridyl disulfide acrylate (PDSA) polymer, a polymer-PEG conjugate, a polymer-surfactant conjugate, or a combination thereof.

[0377] Linker In some embodiments, the linkers described herein are cleavable or non-cleavable linkers. In some examples, the linker is a cleavable linker. In some examples, the linker is an acid cleavable linker. In some examples, the linker is a non-cleavable linker. In some examples, the linker is a C 1 -C 6 In some examples, the linker comprises an 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.

[0378] 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 (sulfoDS), and the like. T), 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'-pyridinyl) 1,5-difluoro-2,4-dinitrobenzene, 1,3-difluoro-4,6-dinitrobenzene and other halogenated aryl-containing compounds (DFDNB), 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BAS ED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic 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).

[0379] 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 ... Imidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacteyl) 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), succinimidylCarbonyl-reactive and -reactive hydrazides such as 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH). and sulfhydryl-reactive cross-linkers, 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-(p-azidosalicylamido)ethyl-2-(p ... ,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) ) propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl reactive and photoreactive cross-linkers, such as ρ-azidobenzoylhydrazide (ABH), carboxylate reactive and photoreactive cross-linkers, such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine reactive and photoreactive cross-linkers, such as ρ-azidophenylglyoxal (APG).

[0380] 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.

[0381] In some embodiments, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further includes caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) as described above, or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC).

[0382] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination via a retro-Michael reaction. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062(2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0383] 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:2111), 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:2112), or Gly-Phe-Leu-Gly (SEQ ID NO:2113). In some examples, the linker comprises a peptide moiety such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 2111), 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: 2112), or Gly-Phe-Leu-Gly (SEQ ID NO: 2113). In some instances, the linker comprises Val-Cit. In some instances, the linker is Val-Cit.

[0384] 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).

[0385] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises a mc-val-cit-PABA group.

[0386] 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 International Publication No. WO2015038426.

[0387] 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.

[0388] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or a linker group) to 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 silicon linker, a sulfur linker, a selenium linker, a nitrogen linker, a phosphorus linker, a boron linker, a chromium linker, or a phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15): 2493-2497 (2013). In some examples, the linker is a traceless linker as described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem. Rev. 102:2607-2024 (2002). In some examples, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0389] In some examples, the linker includes a functional group that exerts steric hinderance at the attachment site of the linker and the conjugated 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.

[0390] In some examples, the linker is an acid-cleavable linker. In some examples, the acid-cleavable linker comprises a hydrazone bond that is susceptible to hydrolytic cleavage. In some examples, the acid-cleavable linker comprises a thiomaleamic acid linker. In some examples, the acid-cleavable linker is a thiomaleamic acid linker as described in "Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation," Chem. Commun. 49: 8187-8189 (2013).

[0391] In some examples, the linker is a linker 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 / 0309256; 2015 / 037360; or 2014 / 0294851; or PCT Publication Nos. WO2015057699; WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

[0392] 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.

[0393] In some examples, X is a single bond or a linker. In some examples, X is a single bond. In some examples, X is a linker. In some examples, the linker is C 1 -C 6 In some cases, X is, for example, C 5 , C 4 , C 3 , C 2 , or C 1 C such as alkyl group 1-C 6 It is an alkyl group. In some cases, C 1 -C 6 The alkyl group is unsubstituted C 1 -C 6 It is an alkyl group. As used in the context of linkers, particularly in the context of X, alkyl means a saturated straight or branched chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X is a non-polymeric linker. In some examples, X comprises a homobifunctional linker or a heterobifunctional linker as described above. In some cases, X comprises a heterobifunctional linker. In some cases, X comprises sMCC. In other examples, X comprises C 1 -C 6 In another embodiment, X is C 1 -C 6 and optionally, sMCC linked to an alkyl group. In a further example, X does not include a homobifunctional linker or a heterobifunctional linker as described above.

[0394] In some examples, Y is a single bond or a linker. In some examples, Y is a single bond. In other cases, Y is a linker. In some embodiments, Y is C 1 -C 6 is an alkyl group. In some examples, Y is a homobifunctional or heterobifunctional linker as described above. In some examples, Y is a homobifunctional linker as described above. In some examples, Y is a heterobifunctional linker as described above. In some examples, Y comprises a maleimide group, such as maleimidocaproyl (mc), as described above, or a self-stabilizing maleimide group. In some examples, Y comprises a peptide moiety, such as Val-Cit. In some examples, Y comprises a benzoic acid group, such as PABA. In further examples, Y comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In additional examples, Y comprises a mc group. In additional examples, Y comprises a mc-val-cit group. In additional examples, Y comprises a val-cit-PABA group. In additional examples, Y comprises a mc-val-cit-PABA group.

[0395] In some examples, L is a single bond or a linker. In some cases, L is a single bond. In some cases, L is a linker. In some embodiments, L is C 1 -C 6 is an alkyl group. In some examples, L is a homobifunctional or heterobifunctional linker as described above. In some examples, L is a homobifunctional linker as described above. In some examples, L is a heterobifunctional linker as described above. In some examples, L comprises a maleimide group, such as maleimidocaproyl (mc), as described above, or a self-stabilizing maleimide group. In some examples, L comprises a peptide moiety, such as Val-Cit. In some examples, L comprises a benzoic acid group, such as PABA. In further examples, L comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In additional examples, L comprises a mc group. In additional examples, L comprises a mc-val-cit group. In additional examples, L comprises a val-cit-PABA group. In additional examples, L comprises a mc-val-cit-PABA group.

[0396] How to use In some embodiments, the compositions or pharmaceutical preparations described herein, including the polynucleic acid molecule and the binding moiety attached to the polymer, are used to treat a disease or condition. In some examples, the disease or condition is cancer. In some embodiments, the compositions or pharmaceutical preparations described herein are used as immunotherapy for the treatment of a disease or condition. In some examples, the immunotherapy is cancer immunotherapy.

[0397] cancer In some embodiments, the compositions or pharmaceutical preparations described herein are used to treat cancer. In some embodiments, the cancer is a solid tumor. In some instances, the cancer is a hematological malignancy. In some instances, the cancer is a recurrent or refractory cancer, or a metastatic cancer. In some instances, the solid tumor is a recurrent or refractory solid tumor, or a metastatic solid tumor. In some instances, the hematological malignancy is a recurrent or refractory hematological malignancy, or a metastatic hematological malignancy.

[0398] In some embodiments, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, 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.

[0399] In some examples, the compositions or pharmaceutical preparations described herein that include polynucleic acid molecules and binding moieties attached to polymers are used to treat solid tumors. In some examples, the compositions or pharmaceutical preparations described herein that include polynucleic acid molecules and binding moieties attached to polymers 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.

[0400] In some instances, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is leukemia, lymphoma, myeloma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma. In some instances, the hematological malignancy is 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, or idiopathic ... These include: large B-cell lymphoma, Burkitt lymphoma, non-Burkitt 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.

[0401] In some examples, the compositions or pharmaceutical formulations described herein that include a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat hematological malignancies. In some examples, the compositions or pharmaceutical formulations described herein that include a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat leukemia, lymphoma, myeloma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma. In some examples, the hematological malignancies are 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, or leukemia. In some cases, the hematologic malignancy is a recurrent or refractory hematologic malignancy or a metastatic hematologic malignancy.

[0402] 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 comprising a polynucleic acid molecule and a binding moiety attached to a polymer 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 comprising a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat KRAS-related cancer. In some examples, the compositions or pharmaceutical preparations described herein comprising a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat EGFR-related cancer. In some examples, the compositions or pharmaceutical preparations described herein comprising a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat AR-related cancer. In some examples, the compositions or pharmaceutical preparations described herein comprising a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat HPRT1-related cancer. In some examples, the compositions or pharmaceutical preparations described herein comprising a polynucleic acid molecule and a binding moiety attached to a polymer are used to treat β-catenin-related 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, renal 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 comprises a PIK3C-associated cancer and / or a MYC-associated cancer.

[0403] immunotherapy 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. In some examples, cancer immunotherapy is classified as active, passive, or combined (active and passive) methods. In active cancer immunotherapy, for example, tumor-associated antigens (TAA) are presented to the immune system, triggering an attack on cancer cells presenting these TAAs. In some examples, active cancer immunotherapy includes tumor targeting and / or immune targeting agents (e.g., checkpoint inhibitors such as monoclonal antibodies), and / or vaccines, for example, in situ vaccination and / or cell-based or non-cell-based (e.g., dendritic cell-based, tumor cell-based, antigen, anti-idiotype, DNA, or vector-based) vaccines. In some examples, cell-based vaccines are vaccines generated using activated immune cells obtained from the patient's own immune system and then activated by the patient's own cancer. In some examples, active cancer immunotherapy is further subdivided into non-specific active immunotherapy and specific active immunotherapy. In some instances, non-specific active immunotherapy utilizes cytokines and / or other cell signaling components to induce a general immune system response. In some cases, specific active immunotherapy utilizes specific TAAs to induce an immune response.

[0404] In some embodiments, the compositions or pharmaceutical preparations described herein are used as active cancer immunotherapy for the treatment of a disease or disorder (e.g., cancer). In some embodiments, the compositions or pharmaceutical preparations described herein include a tumor targeting agent. In some examples, the tumor targeting agent is encompassed by the binding moiety A. In other examples, the tumor targeting agent is an additional agent used in combination with the molecule of formula (I). In some examples, the tumor targeting agent is a tumor-targeting polypeptide (e.g., a tumor-targeting antibody). In some examples, the tumor targeting agent is a tumor-targeting antibody that exerts its anti-tumor activity by mechanisms such as direct killing (e.g., signal transduction-induced apoptosis), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In further examples, the tumor targeting agent induces anti-tumor T cells to elicit an adaptive immune response.

[0405] In some embodiments, binding moiety A is a tumor-targeting polypeptide (e.g., a tumor-targeting antibody). In some examples, binding moiety A is a tumor-targeting antibody that exerts its anti-tumor activity by mechanisms such as direct killing (e.g., signal transduction-induced apoptosis), complement-dependent cytotoxicity (CDC), and / or antibody-dependent cellular cytotoxicity (ADCC). In further examples, binding moiety A induces anti-tumor T cells to elicit an adaptive immune response.

[0406] In some embodiments, the compositions or pharmaceutical formulations described herein include an immune targeting agent. In some examples, the immune targeting agent is encompassed by binding moiety A. In other examples, the immune targeting agent is an additional agent used in combination with the molecule of formula (I). In some examples, the immune targeting agent includes a cytokine, a checkpoint inhibitor, or a combination thereof.

[0407] 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 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.

[0408] 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, scFvs, minibodies, diabodies, antisense oligonucleotides, siRNAs, aptamers, or peptides. In some embodiments, immune checkpoint inhibitors include those that inhibit or inhibit 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, An inhibitor of 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.

[0409] In some embodiments, exemplary checkpoint inhibitors include:

[0410] PD-L1 inhibitors, such as MPDL3280A (RG7446) from Genentech, anti-mouse PD-L1 antibody clone 10F.9G2 (Cat# BE0101) from BioXcell, anti-PD-L1 monoclonal antibodies MDX-1105 (BMS-936559) and BMS-935559, MSB0010718C, mouse anti-PD-L1 clone 29E.2A3 and MEDI4736 from AstraZeneca;

[0411] PD-L2 inhibitors, e.g., GlaxoSmithKline's AMP-224 (Amplimmune), and rHIgM12B7;

[0412] PD-1 inhibitors, such as anti-mouse PD-1 antibody clone J43 (Cat# BE0033-2) from BioXcell, anti-mouse PD-1 antibody clone RMP1-14 (Cat# BE0146) from BioXcell, mouse anti-PD-1 antibody clone EH12, MK-3475 anti-mouse PD-1 antibody from Merck (Keytruda, pembrolizumab, lambrolizumab), anti-PD-1 antibody from AnaptysBio known as ANB011, antibody MDX-1 106 (ONO-4538), human IgG4 monoclonal antibody nivolumab from Bristol-Myers Squibb (Opdivo®, BMS-936558, MDX1106, AMP-514 and AMP-224 from AstraZeneca, and pidilizumab (CT-011) from CureTech Ltd;

[0413] 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;

[0414] LAG3 inhibitors, such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016 and LAG-3 chimeric antibody A9H12;

[0415] B7-H3 inhibitors, e.g., MGA271;

[0416] KIR inhibitors, e.g., Lirilumab (IPH2101);

[0417] CD137 (41BB) inhibitors, such as urelumab (BMS-663513 Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

[0418] PS inhibitors, such as Bavituximab;

[0419] As well as inhibitors, such as antibodies or fragments thereof (e.g., monoclonal antibodies, human, humanized or chimeric antibodies), RNAi molecules, or small molecules against TIM3, CD52, CD30, CD20, CD33, CD27, OX40 (CD134), GITR, icosa, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[0420] In some embodiments, binding moiety A comprising an immune checkpoint inhibitor is used to treat a disease or condition (e.g., cancer). In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. In some examples, binding moiety A is a bispecific antibody or binding fragment thereof comprising an immune checkpoint inhibitor. 3. Binding moiety A comprising an inhibitor of GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof, is used to treat a disease or disorder (e.g., cancer).

[0421] In some embodiments, the molecule of formula (I) in combination with an immune checkpoint inhibitor is used to treat a disease or condition (e.g., cancer). In some examples, the immune checkpoint inhibitor is an inhibitor of programmed death-ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD1 37, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulatory molecule), KIR, LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or any combination thereof. In some cases, the molecule of formula (I) is used in combination with ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C, MK-3475, or BMS-936559 for the treatment of a disease or condition (e.g., cancer).

[0422] In some embodiments, the immune targeting agent is a cytokine. In some cases, cytokines are further divided into subgroups: 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 are from 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, CCL27, CCL28, CCL29, CCL30, CCL31, CCL32, CCL33, CCL34, CCL35, CCL36, CCL37, CCL38, CCL39, CCL40, CCL41, CCL42, CCL43, CCL44, CCL45, CCL46, CCL47, CCL48, CCL49, CCL50, CCL51, CCL52, CCL53, CCL54, CCL55, CCL56, CCL57, CCL58, CCL59, CCL60, CCL61, CCL62, CCL63, CCL64, CCL65, CCL66, CCL67, CCL68, CCL69, CCL70, CCL71, CCL72, CCL73, CCL74, CCL75, CCL76, CCL77, CCL78, CCL79, CCL80, C chemokines from the CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16 and CXCL17; the XC subfamily: XCL1 and XCL2; and the CX3C subfamily: CX3CL1.

[0423] Interferons (IFNs) include type I interferons (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), type II interferons (e.g., IFN-γ), and type III interferons. In some embodiments, IFN-α is further classified into about 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0424] 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.

[0425] Tumor necrosis factors (TNFs) are a group of cytokines that regulate apoptosis. In some instances, there are about 19 members within the TNF family, including, but not limited to, TNFα, lymphotoxin-α (LT-α) and lymphotoxin-β (LT-β), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

[0426] In some embodiments, a molecule of formula (I) in combination with a cytokine is used to treat a disease or condition (e.g., cancer). Optionally, a molecule of formula (I) in combination with a chemokine is used to treat a disease or condition (e.g., cancer). Optionally, a molecule of formula (I) in combination with an interferon is used to treat a disease or condition (e.g., cancer). Optionally, a molecule of formula (I) in combination with an interleukin is used to treat a disease or condition (e.g., cancer). Optionally, a molecule of formula (I) in combination with a tumor necrosis factor is used to treat a disease or condition (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).

[0427] In some embodiments, the compositions or pharmaceutical formulations described herein include a vaccine. In some examples, the vaccine is an in situ vaccination. In some examples, the vaccine is a cell-based vaccine. In some examples, the vaccine is a non-cell-based vaccine. In some examples, the molecule of formula (I) in combination with a dendritic cell-based vaccine is used to treat a disease or illness (e.g., cancer). In some examples, the molecule of formula (I) in combination with a tumor cell-based vaccine is used to treat a disease or illness (e.g., cancer). In some examples, the molecule of formula (I) in combination with an antigen vaccine is used to treat a disease or illness (e.g., cancer). In some examples, the molecule of formula (I) in combination with an anti-idiotype vaccine is used to treat a disease or illness (e.g., cancer). In some examples, the molecule of formula (I) in combination with a DNA vaccine is used to treat a disease or illness (e.g., cancer). In some examples, the molecule of formula (I) in combination with a vector-based vaccine is used to treat a disease or illness (e.g., cancer).

[0428] In some embodiments, the compositions or pharmaceutical formulations 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 T cells (natural killer (NK) T cells) and / or exogenously generated chimeric antigen receptor (CAR) T cells, to attack cancer cells.

[0429] In some embodiments, a molecule of formula (I) in combination with a T cell-based therapeutic is used to treat a disease or condition (e.g., cancer). In some cases, the T cell-based therapeutic 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 comprises 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, molecules 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 are used to treat a disease or disorder (e.g., cancer).

[0430] In some embodiments, a molecule of formula (I) in combination with a natural killer (NK) T cell based therapeutic is used to treat a disease or condition (e.g., cancer). In some examples, the NK based therapeutic 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 is an activating NK agent that recognizes one or more of the CD4 cell surface markers, such as CD2, CD11a, CD11b, CD16, CD56, CD58, CD49, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD69, CD61, CD62, CD63, CD64, CD65, CD65, CD66, CD67, CD68, CD69 ... 6 2L, CD85j, CD158a / b, CD158c, CD158e / f / k, CD158h / j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319. In some instances, the activating NK agent recognizes one or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD 6Molecules of formula (I) in combination with activating NK agents that recognize one or more of 2L, CD85j, CD158a / b, CD158c, CD158e / f / k, CD158h / j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244, or CD319 are used to treat diseases or disorders (e.g., cancer).

[0431] In some embodiments, molecules of Formula (I) in combination with CAR-T cell-based therapeutics are used to treat a disease or condition (e.g., cancer).

[0432] In some embodiments, the molecules of formula (I) in combination with an additional agent that destabilizes the endosomal membrane (or interferes with endosomal-lysosomal membrane trafficking) are used to treat a disease or condition (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, vinorelbine, 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.

[0433] I...

Claims

1. A polynucleotide molecule comprising at least one 5'-vinylphosphonate modified non-natural nucleotide, The at least one 5'-vinylphosphonate modified non-natural nucleotide is selected from: 【Chemistry 1】 B is a heterocyclic base moiety; R ６ is selected from hydrogen, halogen, alkyl, alkoxy, or aminoalkyl, and A polynucleotide molecule, wherein J is an internucleotide linkage group that connects adjacent nucleotides of said polynucleotide molecule.

2. 2. The polynucleotide molecule of claim 1, wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is located at the 5'-terminus of the polynucleotide molecule.

3. The polynucleotide molecule of claim 1 or 2, wherein the at least one 5'-vinylphosphonate modified non-natural nucleotide is further modified at the 2'-position.

4. 4. The polynucleotide molecule of claim 3, wherein the 2'-modification is 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), 2'-O-N-methylacetamide (2'-O-NMA), 2'-O-ethyloxyethyl (2'-O-EOE), or 2'-O-(2-N-methylcarbamoylethyl) modified nucleotides.

5. 5. The polynucleotide molecule of claim 1, further comprising at least one modified internucleotide linkage or at least one inverted abasic portion.

6. 6. The polynucleotide molecule of claim 5, wherein the at least one modified internucleotide linkage comprises a phosphorothioate linkage, a phosphorodithioate linkage, a phosphorodiamidate linkage, a methylphosphonate linkage, or an amide linkage.

7. The polynucleotide molecule of claim 5 , wherein the at least one inverted abasic portion is at least one terminus.

8. The polynucleotide molecule of claim 1 , wherein the polynucleotide molecule comprises a single-stranded polynucleic acid molecule.

9. 8. The polynucleotide molecule of claim 1, wherein the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.

10. The polynucleotide molecule of claim 9 , wherein the first polynucleotide and the second polynucleotide are RNA molecules.

11. A polynucleotide conjugate molecule comprising a polynucleotide molecule according to any one of claims 1 to 10 and a binding moiety.

12. The binding moiety and the polynucleotide molecule may be a single bond, C １ -C ６ Alkyl group, or homobifunctional linker or C １ -C ６ The polynucleotide conjugate molecule of claim 11, which is linked by a heterobifunctional linker optionally attached to an alkyl group.

13. The binding moiety may be a humanized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab ２ 13. The polynucleotide conjugate molecule of claim 11 or 12, comprising a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or antigen-binding fragment thereof.

14. The polynucleotide conjugate molecule of claim 11 or 12, wherein the binding moiety comprises a peptide or a small molecule.

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