Nucleic acid-polypeptide compositions and methods for inducing exon skipping

Abstract

To provide nucleic acid-polypeptide compositions and methods for inducing exon skipping.SOLUTION: Disclosed herein are molecules and pharmaceutical compositions that induce an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion. Also described herein are methods for treating a disease or disorder that comprises a molecule or a pharmaceutical composition that induces an insertion, deletion, duplication, or alteration in an incorrectly spliced mRNA transcript to induce exon skipping or exon inclusion.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 561,939, filed September 22, 2017, and U.S. Provisional Patent Application No. 62 / 696,766, filed July 11, 2018, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Modulation of RNA function is a developing area of ​​therapeutic interest. Drugs that affect mRNA stability, such as antisense oligonucleotides and small interfering RNA, are one way to modulate RNA function. Another group of oligonucleotides can modulate RNA function by altering pre-mRNA processing to include or exclude specific regions of the pre-mRNA from the final gene product: the encoded protein. Oligonucleotide therapeutics therefore represent a means of modulating protein expression in disease states and thus have utility as therapeutic agents. Summary of the Invention

[0003] In certain embodiments, disclosed herein are molecules and pharmaceutical compositions for modulating RNA processing. In some embodiments, also disclosed herein are molecules and pharmaceutical compositions for treating muscular dystrophy.

[0004] Disclosed herein, in certain embodiments, is a method for treating a disease or disorder caused by a mis-spliced ​​mRNA transcript in a subject, the method comprising administering to the subject a polynucleic acid molecule conjugate, wherein the polynucleic acid molecule conjugate is conjugated to a cell target binding moiety, wherein the polynucleotide optionally comprises at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic portion, and wherein the polynucleic acid molecule conjugate induces an insertion, deletion, duplication, or alteration of the mis-spliced ​​mRNA transcript to induce exon skipping or exon inclusion in the mis-spliced ​​mRNA transcript to generate a fully processed mRNA transcript, wherein the fully processed mRNA transcript encodes a functional protein, thereby treating the disease or disorder in the subject. In some embodiments, the disease or disorder is further characterized by one or more mutations in the mRNA. In some embodiments, the disease or disorder includes a neuromuscular disease, a genetic disease, cancer, a hereditary disease, or a cardiovascular disease. In some embodiments, the disease or disorder is muscular dystrophy. In some embodiments, the disease or disorder is Duchenne muscular dystrophy. In some embodiments, the exon skipping is of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some embodiments, the exon skipping is of exon 23 of the DMD gene. In some embodiments, the polynucleic acid molecule conjugate is of Formula (I): AXB Formula I During the ceremony, A is a binding moiety; B is a polynucleotide; and X is a single bond or a first linker. In some embodiments, the polynucleic acid molecule conjugate has formula (II): AXBYC Formula II 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 single bond or a second linker. In some embodiments, the polynucleic acid molecule conjugate has formula (III): AXCYB Formula III 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 single bond or a second linker. In some embodiments, at least one 2'-modified nucleotide comprises a morpholino, 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-methylacetamido (2'-O-NMA) modified nucleotide. In some embodiments, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA), ethylene nucleic acid (ENA), or peptide nucleic acid (PNA). In some embodiments, at least one 2'-modified nucleotide comprises a morpholino. In some embodiments, at least one inverted base moiety is at least one terminal. In some embodiments, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleic acid molecule is at least about 10 to about 30 nucleotides in length. In some embodiments, the polynucleic acid molecule is at least one of 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. In some embodiments, the polynucleic acid molecule is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 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 embodiments, the polynucleic acid molecule comprises at least one of the following: about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications. In some embodiments, the polynucleic acid molecule comprises at least one of the following: about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications. In some embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications. In some embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications. In some embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications. In some embodiments, polynucleic acid molecules contain at least one of the following: about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications. In some embodiments, polynucleic acid molecules contain at least one of the following: about 10% to about 30% modifications, and about 20% to about 30% modifications. In some embodiments, polynucleic acid molecules contain about 10% to about 20% modifications. In some embodiments, polynucleic acid molecules contain about 15% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% modifications. In some embodiments, polynucleic acid molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.In some embodiments, a polynucleic acid molecule comprises at least 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 embodiments, a 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 embodiments, a polynucleic acid molecule comprises a single strand. In some embodiments, a polynucleic acid molecule comprises two or more strands. In some embodiments, the polynucleic acid molecule 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 and the second polynucleotide are siRNA molecules. In some embodiments, X and Y are independently a single bond, a degradable linker, a non-degradable linker, a cleavable linker, or a non-polymeric linker group. In some embodiments, X is a single bond. In some embodiments, X is a C1-C6 alkyl group. In some embodiments, Y is a C1-C6 alkyl group. In some embodiments, X is a homobifunctional linker or a heterobifunctional linker, optionally linked to a C1-C6 alkyl group. In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the binding moiety is an antibody or a binding fragment thereof.In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 5,000 Da. In some embodiments, AX is conjugated to the 5' end of B and YC is conjugated to the 3' end of B. In some embodiments, YC is conjugated to the 5' end of B and AX is conjugated to the 3' end of B. In some embodiments, AX, YC, or a combination thereof are conjugated to an internucleotide linkage group. In some embodiments, the method further comprises D. In some embodiments, D is conjugated to C or A. In some embodiments, D is conjugated to a molecular conjugate of Formula (II) according to Formula (IV): (AXBYC c )-LD Formula IV 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; 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; wherein the polynucleotide comprises at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety, and D is conjugated to any of A, B, or C. In some embodiments, D is INF7 or melittin. In some embodiments, L is a C1-C6 alkyl group. In some embodiments, L is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the method further comprises at least a second binding moiety, A. In some embodiments, at least a second binding moiety, A, is conjugated to A, B, or C.

[0005] In some embodiments, disclosed herein are methods for inducing insertions, deletions, duplications, or alterations in mis-spliced ​​mRNA transcripts to induce exon skipping or exon inclusion in the mis-spliced ​​mRNA transcript, the methods comprising: contacting a target cell with a polynucleic acid molecule conjugate, wherein the polynucleotide comprises at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; hybridizing the polynucleic acid molecule conjugate to the mis-spliced ​​mRNA transcript in the target cell to induce insertions, deletions, duplications, or alterations in the mis-spliced ​​mRNA transcript to induce exon skipping or exon inclusion, wherein the mis-spliced ​​mRNA transcript can encode a functional form of a protein; and translating the functional form of the protein from the fully processed mRNA transcript of the previous step. In some embodiments, the target cell is a target cell of a subject. In some embodiments, the mis-spliced ​​mRNA transcript further induces a disease or disorder. In some embodiments, the disease or disorder is further characterized by one or more mutations in mRNA. In some embodiments, the disease or disorder comprises a neuromuscular disease, a genetic disease, cancer, a hereditary disease, or a cardiovascular disease. In some embodiments, the disease or disorder is a muscular dystrophy. In some embodiments, the disease or disorder is Duchenne muscular dystrophy. In some embodiments, the exon skipping is of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some embodiments, the exon skipping is of exon 23 of the DMD gene. In some embodiments, the polynucleic acid molecule conjugate is of Formula (I): AXB Formula I During the ceremony, A is a binding moiety; B is a polynucleotide; and X is a single bond or a first linker. In some embodiments, the polynucleic acid molecule conjugate has formula (II): AXBYC Formula II 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 single bond or a second linker. In some embodiments, the polynucleic acid molecule conjugate has formula (III): AXCYB Formula III 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 single bond or a second linker. In some embodiments, at least one 2'-modified nucleotide comprises a morpholino, 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-methylacetamido (2'-O-NMA) modified nucleotide. In some embodiments, at least one 2'-modified nucleotide comprises a locked nucleic acid (LNA), ethylene nucleic acid (ENA), or peptide nucleic acid (PNA). In some embodiments, at least one 2'-modified nucleotide comprises a morpholino. In some embodiments, at least one inverted base moiety is at least one terminal. In some embodiments, at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleic acid molecule is at least about 10 to about 30 nucleotides in length. In some embodiments, the polynucleic acid molecule is at least one of the following: 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. In some embodiments, the polynucleic acid molecule is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 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 embodiments, the polynucleic acid molecule comprises at least one of the following: about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications. In some embodiments, the polynucleic acid molecule comprises at least one of the following: about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications. In some embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications. In some embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications. In some embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications. In some embodiments, polynucleic acid molecules contain at least one of the following: about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications. In some embodiments, polynucleic acid molecules contain at least one of the following: about 10% to about 30% modifications, and about 20% to about 30% modifications. In some embodiments, polynucleic acid molecules contain about 10% to about 20% modifications. In some embodiments, polynucleic acid molecules contain about 15% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% modifications. In some embodiments, polynucleic acid molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.In some embodiments, a polynucleic acid molecule comprises at least 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 embodiments, a 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 embodiments, a polynucleic acid molecule comprises a single strand. In some embodiments, a polynucleic acid molecule comprises two or more strands. In some embodiments, the polynucleic acid molecule 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 and the second polynucleotide are siRNA molecules. In some embodiments, X and Y are independently a single bond, a degradable linker, a non-degradable linker, a cleavable linker, or a non-polymeric linker group. In some embodiments, X is a single bond. In some embodiments, X is a C1-C6 alkyl group. In some embodiments, Y is a C1-C6 alkyl group. In some embodiments, X is a homobifunctional linker or a heterobifunctional linker, optionally linked to a C1-C6 alkyl group. In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the binding moiety is an antibody or a binding fragment thereof.In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody, or binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 5,000 Da. In some embodiments, AX is conjugated to the 5' end of B and YC is conjugated to the 3' end of B. In some embodiments, YC is conjugated to the 5' end of B and AX is conjugated to the 3' end of B. In some embodiments, AX, YC, or a combination thereof are conjugated to an internucleotide linkage group. In some embodiments, the method further comprises D. In some embodiments, D is conjugated to C or A. In some embodiments, D is conjugated to a molecular conjugate of Formula (II) according to Formula (IV): (AXBYC c )-LD Formula IV 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; 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; wherein the polynucleotide comprises at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety, and D is conjugated to any of A, B, or C. In some embodiments, D is INF7 or melittin. In some embodiments, L is a C1-C6 alkyl group. In some embodiments, L is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the method further comprises at least a second binding moiety A. In some embodiments, at least a second binding moiety A is conjugated to A, B, or C. In some embodiments, the method is an in vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the subject is a human.

[0006] In certain embodiments, disclosed herein is a pharmaceutical composition comprising a molecule obtained by any one of the methods disclosed herein and a pharmaceutically 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.

[0007] In certain embodiments, disclosed herein are compositions comprising a polynucleic acid molecule conjugate, wherein the polynucleic acid molecule conjugate comprises a polynucleotide comprising 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:45-963. In certain embodiments, disclosed herein are compositions comprising a polynucleic acid molecule conjugate, wherein the polynucleic acid molecule conjugate comprises a polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:45-963. In certain embodiments, the polynucleic acid molecule conjugate is of Formula (I): AXB Formula I During the ceremony, A is a binding moiety; B is a polynucleotide; and X is a single bond or a first linker. In certain embodiments, the polynucleic acid molecule conjugate has formula (II): AXBYC Formula II 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 single bond or a second linker. In certain embodiments, the polynucleic acid molecule conjugate has formula (III): AXCYB Formula III 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 single bond or a second linker.

[0008] In certain embodiments, at least one 2'-modified nucleotide comprises a morpholino, 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-methylacetamido (2'-O-NMA) modified nucleotide. In certain embodiments, at least one 2'-modified nucleotide comprises a morpholino.

[0009] In some embodiments, disclosed herein is a polynucleic acid conjugate comprising a target cell binding moiety that binds to at least one polynucleic acid molecule that hybridizes to a target region of a pre-mRNA transcript of a DMD gene, wherein the at least one polynucleic acid molecule induces the splicing out of an exon from the pre-mRNA transcript to generate an mRNA transcript encoding a functional dystrophin protein. In some embodiments, the functional dystrophin protein is a truncated form of the dystrophin protein. In some embodiments, the target region is at an exon-intron junction, where the exon is the exon to be spliced ​​out. In some embodiments, the exon is exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55. In some embodiments, the exon-intron junction is located 5' of the exon to be spliced ​​out. In some embodiments, the target region is an intron region upstream of the exon-intron junction. In some embodiments, the target region is about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides upstream of the exon-intron junction. In some embodiments, the exon-intron junction is located 3' of the exon to be spliced ​​out. In some embodiments, the target region is an intron region downstream of the exon-intron junction. In some embodiments, the target region is about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides downstream of the exon-intron junction. In some embodiments, the target cell binding moiety binds to two or more, three or more, four or more, five or more, six or more, or eight or more polynucleic acid molecules. In some embodiments, the polynucleic acid molecule is about 10 to about 50 nucleotides in length.In some embodiments, the polynucleic acid molecule comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule further comprises 1, 2, 3, or 4 mismatches. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1056-1094, 1147-1162, or 1173-1211. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1056-1076. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1077-1094. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1077-1094. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1173-1211. In some embodiments, the binding moiety comprises an antibody. In some embodiments, the antibody comprises an anti-transferrin antibody. In some embodiments, the binding moiety comprises a plasma protein. In some embodiments, the polynucleic acid conjugate comprises A-(X. 1 -B) ncomprising the formula (∨), wherein A comprises a binding moiety; B comprises a polynucleic acid molecule; X 1wherein n is a single bond or a first non-polymeric linker; and n is an average value selected from 1-12. In some embodiments, the polynucleic acid molecule comprises a passenger strand and a guide strand. In some embodiments, the guide strand comprises at least one modified internucleotide linkage, at least one inverted abasic moiety, at least one 5'-vinylphosphonate-modified unnatural nucleotide, or a combination thereof. In some embodiments, the guide strand comprises about 2, 3, 4, 5, 6, 7, 8, or 9 phosphorothioate-modified unnatural nucleotides. In some embodiments, the guide strand comprises one phosphorothioate-modified unnatural nucleotide. In some embodiments, the phosphorothioate-modified unnatural nucleotide is positioned at an internucleotide linkage of the polynucleotide. In some embodiments, the at least one 5'-vinylphosphonate-modified unnatural nucleotide is positioned about 1, 2, 3, 4, or 5 bases away from the 5'-end of the guide strand. In some embodiments, at least one 5'-vinylphosphonate-modified non-natural nucleotide is further modified at the 2'-position. In some embodiments, 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), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modified nucleotides. In some embodiments, the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorodiamidate morpholino oligomer-modified non-natural nucleotides, hi some embodiments, the passenger strand comprises 100% phosphorodiamidate morpholino oligomer-modified non-natural nucleotides.In some embodiments, the passenger strand is shorter in length than the guide strand, thereby generating a 5' overhang, a 3' overhang, or a combination thereof. In some embodiments, the passenger strand is equal in length to the guide strand, thereby generating blunt ends at each end of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule is a phosphorodiamidate morpholino oligomer / RNA heteroduplex. In some embodiments, the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptide nucleic acid modified unnatural nucleotides. In some embodiments, the passenger strand comprises 100% peptide nucleic acid modified unnatural nucleotides. In some embodiments, the passenger strand is shorter in length than the guide strand, thereby generating a 5' overhang, a 3' overhang, or a combination thereof. In some embodiments, the passenger strand is equal in length to the guide strand, thereby generating blunt ends at each end of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule is a peptide nucleic acid / RNA heteroduplex. In some embodiments, the passenger strand is AX. 1 In some embodiments, AX 1 is conjugated to the 5' end of the passenger strand. 1 is conjugated to the 3' end of the passenger strand. 1 is a single bond. In some embodiments, X 1 is a C1-C6 alkyl group. In some embodiments, X 1 is a homobifunctional or heterobifunctional linker, optionally attached to a C1-C6 alkyl group. In some embodiments, the polynucleic acid conjugate further comprises C. In some embodiments, C is polyethylene glycol. In some embodiments, C is X 2 In some embodiments, X is directly conjugated to B via 2comprises a single bond or a second non-polymeric linker. In some embodiments, X 2 is a single bond. In some embodiments, X 2 is a C1-C6 alkyl group. In some embodiments, X 2 is a homobifunctional or heterobifunctional linker optionally attached to a C1-C6 alkyl group. In some embodiments, the passenger chain is 1 and X 2 In some embodiments, AX is conjugated to -C. 1 is attached to the 5' end of the passenger strand, and X 2 -C is conjugated to the 3' end of the passenger strand. 2 -C is attached to the 5' end of the passenger strand, and AX 1 is conjugated to the 3' end of the passenger strand. In some embodiments, the polynucleic acid conjugate is AX 1 -(BX 2 -C) n comprising formula (VI); wherein A comprises a linking moiety; B comprises a polynucleic acid molecule; C comprises a polymer; X 1 is a single bond or a first non-polymeric linker; X 2 and n is an average value selected from 1-12. In some embodiments, the polynucleic acid conjugate further comprises D. In some embodiments, D is an endosomolytic moiety.

[0010] In certain embodiments, disclosed herein are polynucleic acid molecules comprising at least 23 contiguous bases of a base sequence selected from SEQ ID NOs: 1056-1058 or 1087-1089, wherein the polynucleic acid molecule comprises a length of 50 or less nucleotides.

[0011] In certain embodiments, disclosed herein is a polynucleic acid molecule comprising SEQ ID NOs: 1056-1058, wherein the polynucleic acid molecule comprises a length of 50 or less nucleotides.

[0012] In certain embodiments, disclosed herein are polynucleic acid molecules comprising SEQ ID NOs: 1087-1089, wherein the polynucleic acid molecule comprises a length of 50 or less nucleotides.

[0013] In some embodiments, pharmaceutical compositions are disclosed herein, comprising a polynucleic acid conjugate described herein or a polynucleic acid molecule described herein; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0014] In certain embodiments, disclosed herein are methods for treating a disease or disorder characterized by defective mRNA in a subject, the method comprising administering to the subject a polynucleic acid conjugate described herein or a polynucleic acid molecule described herein to induce exon skipping that causes the defective mRNA to generate a processed mRNA encoding a functional protein, thereby treating the disease or disorder in the subject. In some embodiments, the disease or disorder is a neuromuscular disease, a genetic disease, cancer, a hereditary disease, or a cardiovascular disease. In some embodiments, the neuromuscular disease is a muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some embodiments, the subject is a human.

[0015] In certain embodiments, disclosed herein are methods of treating muscular dystrophy in a subject, the methods comprising administering to the subject a polynucleic acid conjugate described herein or a polynucleic acid molecule described herein, thereby treating the muscular dystrophy in the subject. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. In some embodiments, the subject is human.

[0016] In certain embodiments, disclosed herein are kits that include the polynucleic acid conjugates described herein or the polynucleic acid molecules described herein.

[0017] Disclosed herein, in certain embodiments, is a kit comprising a molecule obtained by any one of the methods disclosed herein. [Brief explanation of the drawings]

[0018] [Figure 1] Depicts phosphorodiamidate morpholino oligomer (PMO) sequences with extended terminal nucleotides. [Figure 2A] The phosphorothioate antisense oligonucleotide (PS ASO) sequence with extended terminal nucleotides is depicted. [Figure 2B-1] A fully extended phosphorothioate antisense oligonucleotide (PS ASO) sequence is depicted. [Figure 2B-2] A fully extended phosphorothioate antisense oligonucleotide (PS ASO) sequence is depicted. [Figure 2B-3] A fully extended phosphorothioate antisense oligonucleotide (PS ASO) sequence is depicted. [Figure 2B-4] A fully extended phosphorothioate antisense oligonucleotide (PS ASO) sequence is depicted. [Figure 3] 1 depicts the method used to quantify skipped DMD mRNA in total RNA using Taqman qPCR. [Figure 4] 1 depicts a chromatogram of the anti-CD71 mAb-PMO reaction mixture produced by hydrophobic interaction chromatography (HIC) method 2. [Figure 5A] 1 depicts a chromatogram of anti-CD71 mAb produced using size exclusion chromatography (SEC) method 1. [Figure 5B]Figure 1 depicts a chromatogram of anti-CD71 mAb-PMO DAR1,2 generated using size exclusion chromatography (SEC) method 1. [Figure 5C] 1 depicts a chromatogram of anti-CD71 mAb-PMO DAR>2 generated using size exclusion chromatography (SEC) method 1. [Figure 6A] 1 depicts a chromatogram of anti-CD71 mAb produced using hydrophobic interaction chromatography (HIC) method 2. [Figure 6B] 1 depicts a chromatogram of purified anti-CD71 mAb-PMO DAR1,2 conjugate produced using hydrophobic interaction chromatography (HIC) method 2. [Figure 6C] 1 depicts a chromatogram of purified anti-CD71 mAb-PMO DAR>2 conjugate produced using hydrophobic interaction chromatography (HIC) method 2. [Figure 7A] Figure 1 depicts the chromatogram of fast protein liquid chromatography (FPLC) purification of anti-CD71 Fab-PMO using hydrophobic interaction chromatography (HIC) method 3. [Figure 7B] Chromatogram of anti-CD71 Fab generated using SEC method 1 is depicted. [Figure 7C] 1 depicts a chromatogram of an anti-CD71 Fab-PMO DAR1 conjugate produced using SEC method 1. [Figure 7D] 1 depicts a chromatogram of an anti-CD71 Fab-PMO DAR2 conjugate produced using SEC method 1. [Figure 7E] 1 depicts a chromatogram of an anti-CD71 Fab-PMO DAR3 conjugate produced using SEC method 1. [Figure 7F] Chromatogram of anti-CD71 Fab generated using HIC method 4 is depicted. [Figure 7G] 1 depicts a chromatogram of an anti-CD71 Fab-PMO DAR1 conjugate produced using HIC method 4. [Figure 7H]1 depicts a chromatogram of an anti-CD71 Fab-PMO DAR2 conjugate produced using HIC method 4. [Figure 7I] 1 depicts a chromatogram of an anti-CD71 Fab-PMO DAR3 conjugate produced using HIC method 4. [Figure 8A] 1 depicts a chromatogram of an anti-CD71 mAb-PS ASO reaction mixture generated using SAX method 2. [Figure 8B] 1 depicts a chromatogram of anti-CD71 mAb produced using SEC method 1. [Figure 8C] 1 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR1 conjugate produced using SEC method 1. [Figure 8D] 1 depicts a chromatogram of an anti-CD71 mAb-PS ASO DAR2 conjugate produced using SEC method 1. [Figure 8E] 1 depicts a chromatogram of an anti-CD71 mAb-PS ASO DAR3 conjugate produced using SEC method 1. [Figure 8F] 1 depicts a chromatogram of an anti-CD71 mAb-PS ASO DAR1 conjugate produced using SAX method 2. [Figure 8G] 1 depicts a chromatogram of an anti-CD71 mAb-PS ASO DAR2 conjugate produced using SAX method 2. [Figure 8H] 1 depicts a chromatogram of an anti-CD71 mAb-PS ASO DAR3 conjugate produced using SAX method 2. [Figure 9] Agarose gel from nested PCR detecting exon 23 skipping in differentiated C2C12 cells using PMO and anti-CD71 mAb-PMO conjugate. [Figure 10] Agarose gel from nested PCR detecting exon 23 skipping in differentiated C2C12 cells using PMO, anti-CD71 mAb-PMO, and anti-CD71 mAb-PMO conjugate. [Figure 11]1 depicts an agarose gel from nested PCR detecting exon 23 skipping in differentiated C2C12 cells using PMO, ASO, DAR1 ("ASC-DAR1")-conjugated anti-CD71 mAb-ASO, DAR2 ("ASC-DAR2")-conjugated anti-CD71 mAb-ASO, and DAR3 ("ASC-DAR3")-conjugated anti-CD71 mAb-ASO. [Figure 12A] 1 depicts an agarose gel from nested PCR detecting exon 23 skipping in the gastrocnemius muscle of wild-type mice administered a single intravenous injection of anti-CD71 mAb-PMO conjugate. [Figure 12B] 1 is a graph of quantification of PCR products from gastrocnemius muscle. [Figure 12C] 13 is a graph of quantification of in vivo exon skipping using Taqman qPCR in gastrocnemius muscle from wild-type mice. [Figure 13A] 1 depicts an agarose gel from nested PCR detecting exon 23 skipping in wild-type mouse cardiac muscle after a single intravenous injection. [Figure 13B] 1 is a graph of quantification of PCR products from cardiac muscle. [Figure 14] Depicts sequencing data of DNA fragments from skipping and wild-type PCR products. [Figure 15] 1 illustrates the exon skipping activity of exon skipping PMOs of various lengths targeting exon 45 in the human DMD pre-mRNA in transfected primary human skeletal muscle cells. [Figure 16] 1 illustrates the binding of hTfR1.mAb-PMO conjugates to the human transferrin receptor in vitro. [Figure 17] 1 illustrates the exon skipping activity of hTfR1.mAb-PMO conjugates in primary human skeletal muscle cells. [Figure 18] 1 illustrates the exon skipping activity of hTfR1.mAb-PMO conjugates in primary immortalized human skeletal muscle myotubes. DETAILED DESCRIPTION OF THE INVENTION

[0019] Nucleic acid (e.g., RNAi) therapy is a targeted therapy that boasts high selectivity and specificity. However, in some cases, nucleic acid therapy is also hindered by poor cellular uptake, insufficient intracellular concentration in target cells, and low efficacy. To address these issues, various modifications of nucleic acid compositions have been explored, such as novel linkers for better stabilization and / or lower toxicity, optimization of binding moieties for increased target specificity and / or targeted delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0020] In some cases, one such area that oligonucleotides are used for is to treat muscular dystrophy.Muscular dystrophy encompasses a variety of diseases that affect muscles.Duchenne muscular dystrophy is a severe form of muscular dystrophy, and is caused by mutation in DMD gene.In some cases, mutation in DMD gene disrupts translation reading frame, resulting in non-functional dystrophin protein.

[0021] In certain embodiments, disclosed herein are methods and compositions related to nucleic acid therapy for inducing insertions, deletions, duplications, or alterations in mis-spliced ​​mRNA transcripts to induce exon skipping or exon inclusion, which are used to restore the translation reading frame. Also described herein, in some embodiments, are methods and compositions for treating diseases or disorders characterized by mis-processed mRNA transcripts, where, after exon removal, the mRNA can encode a functional protein, thereby treating the disease or disorder. In further embodiments, described herein are pharmaceutical compositions and kits for treating the above-mentioned diseases or disorders.

[0022] RNA processing RNA plays a central role in regulating gene expression and cellular physiology. Proper processing of RNA is important for the translation of functional proteins. Alterations in RNA processing, such as those resulting from RNA mis-splicing, can lead to disease. For example, splice site mutations can expose premature stop codons, exon loss, or intron inclusion. In some instances, alterations in RNA processing result in insertions, deletions, or duplications. In some instances, alterations in RNA processing result in exon insertions, deletions, or duplications. In some instances, alterations in RNA processing result in intron insertions, deletions, or duplications.

[0023] Exon skipping Exon skipping is a form of RNA splicing. In some cases, exon skipping occurs when an exon is skipped in or spliced ​​out of a processed mRNA. As a result of exon skipping, the processed mRNA does not contain the skipped exon. In some instances, exon skipping results in the expression of an altered product.

[0024] In some instances, antisense oligonucleotides (AONs) are used to induce exon skipping. In some instances, AONs are short nucleic acid sequences that bind to specific mRNA or pre-mRNA sequences. For example, AONs bind to splice sites or exon enhancers. In some instances, binding of AONs to specific mRNA or pre-mRNA sequences generates double-stranded regions. In some instances, the formation of double-stranded regions occurs at sites where spliceosomes or spliceosome-associated proteins would normally bind, resulting in exon skipping. In some instances, exon skipping results in restoration of the transcript reading frame, allowing for the production of a partially functional protein.

[0025] Exon inclusion In some instances, the mutation in the RNA causes exon skipping. In some instances, the mutation is at least one of a splice site, a splice site proximal, and a splice site distant from the splice site. In some instances, the mutation results in at least one of inactivating or weakening a splice site, disrupting an exon splice enhancer or an intron splice enhancer, and creating an exon splice silencer or an intron splice enhancer. In some instances, the mutation alters RNA secondary structure. In some instances, the mutation alters RNA secondary structure, causing disruption of the accessibility of signals important for exon recognition.

[0026] In some instances, use of the AON results in inclusion of a skipped exon. In some instances, the AON binds to at least one of a splice site, a site near a splice site, and a site distant from a splice site. In some instances, the AON binds at a site in the RNA to prevent destruction of an exon splice enhancer or an intron splice enhancer. In some instances, the AON binds at a site in the RNA to prevent generation of an exon splice silencer or an intron splice silencer.

[0027] index In some embodiments, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat diseases or disorders characterized by defective mRNA. In some embodiments, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat diseases or disorders by inducing insertions, deletions, duplications, or alterations of misspliced ​​mRNA transcripts to induce exon skipping or exon inclusion.

[0028] The majority of human protein-coding genes are alternatively spliced. In some instances, mutations result in improperly spliced ​​or partially spliced ​​mRNA. For example, the mutation is in at least one splice site in a protein-coding gene, a silencer or enhancer sequence, an exon sequence, or an intron sequence. In some instances, the mutation causes gene dysfunction. In some instances, the mutation causes a disease or disorder.

[0029] In some examples, diseases or disorders resulting from improperly spliced ​​or partially spliced ​​mRNA include, but are not limited to, neuromuscular diseases, genetic diseases, cancer, inherited diseases, or cardiovascular diseases.

[0030] In some examples, the genetic disease or disorder comprises an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial genetic disorder, or a multifactorial or multigenic disorder.

[0031] In some instances, cardiovascular diseases such as hypercholesterolemia are caused by improperly spliced ​​or partially spliced ​​mRNA. In hypercholesterolemia, a single nucleotide polymorphism in exon 12 of the low-density lipoprotein receptor (LDLR) has been shown to promote exon skipping.

[0032] In some instances, improperly spliced ​​or partially spliced ​​mRNA causes cancer. For example, improperly spliced ​​or partially spliced ​​mRNA affects cellular processes involved in cancer, including, but not limited to, proliferation, motility, and drug response. In some instances, the cancer is a solid cancer or a blood cancer. In some instances, the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin's lymphoma, cervical cancer, ovarian cancer, colon cancer, pancreatic cancer, esophageal cancer, prostate cancer, kidney cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer.

[0033] In some cases, improperly spliced ​​or partially spliced ​​mRNA causes neuromuscular diseases or disorders. Exemplary neuromuscular diseases include muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some cases, muscular dystrophy is genetic. In some cases, muscular dystrophy is caused by spontaneous mutations. Becker muscular dystrophy and Duchenne muscular dystrophy have been shown to be associated with mutations in the DMD gene, which encodes the protein dystrophin. Facioscapulohumeral muscular dystrophy has been shown to be associated with mutations in the double homeobox 4 (DUX4) gene.

[0034] In some cases, improperly spliced ​​or partially spliced ​​mRNA causes Duchenne muscular dystrophy, which is caused by a mutation in the DMD gene that causes severe muscle weakness and eliminates the production of functional dystrophin. In some cases, Duchenne muscular dystrophy is the result of an exon mutation in the DMD gene. In some instances, Duchenne muscular dystrophy is the result of a mutation in at least one of exons 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, and 79 in the DMD gene. In some instances, Duchenne muscular dystrophy is the result of a mutation in at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 in the DMD gene. In some instances, Duchenne muscular dystrophy is the result of a mutation in at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, and 55 in the DMD gene. In some instances, multiple exons are mutated. For example, mutations in exons 48-50 are common in patients with Duchenne muscular dystrophy. In some instances, Duchenne muscular dystrophy is the result of a mutation in exon 51. In some instances, Duchenne muscular dystrophy is the result of a mutation in exon 23. In some instances, the mutation involves one or more multiple exon deletions. In some instances, the mutation involves one or more multiple duplications. In some instances, the mutation involves an exon point mutation.For example, some patients have been shown to harbor nonsense point mutations in exon 51 of the DMD gene.

[0035] In some examples, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat muscular dystrophy. In some examples, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some examples, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat Duchenne muscular dystrophy.

[0036] Polynucleic acid molecule In some embodiments, polynucleic acid molecules are described herein that induce insertions, deletions, duplications, or alterations of misspliced ​​mRNA transcripts to induce exon skipping or exon inclusion. In some instances, the polynucleic acid molecules restore the translational reading frame. In some instances, the polynucleic acid molecules result in functional, truncated proteins.

[0037] In some examples, the polynucleic acid molecule targets an mRNA sequence. In some examples, the polynucleic acid molecule targets a splice site. In some examples, the polynucleic acid molecule targets a cis-regulatory element. In some examples, the polynucleic acid molecule targets a trans-regulatory element. In some examples, the polynucleic acid molecule targets an exon splice enhancer or an intron splice enhancer. In some examples, the polynucleic acid molecule targets an exon splice silencer or an intron splice silencer.

[0038] In some instances, the polynucleic acid molecule targets a sequence found in an intron or exon. For example, the polynucleic acid molecule targets a sequence found in an exon that mediates splicing of said exon. In some instances, the polynucleic acid molecule targets an exon recognition sequence. In some instances, the polynucleic acid molecule targets a sequence upstream of an exon. In some instances, the polynucleic acid molecule targets a sequence downstream of an exon.

[0039] As described above, the polynucleic acid molecule targets the misprocessed mRNA transcripts that cause diseases or disorders, such as, but not limited to, neuromuscular diseases, genetic diseases, cancer, genetic diseases, or cardiovascular diseases.In some cases, the polynucleic acid molecule targets the misprocessed mRNA transcripts that cause neuromuscular diseases or disorders.In some cases, the neuromuscular diseases or disorders are Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy.In some cases, the polynucleic acid molecule targets the misprocessed mRNA transcripts that cause Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy.In some cases, the polynucleic acid molecule targets the misprocessed mRNA transcripts that cause Duchenne muscular dystrophy.

[0040] In some examples, the polynucleic acid molecule targets an exon that is mutated in the DMD gene that causes Duchenne muscular dystrophy. Exemplary exons mutated in the DMD gene causing Duchenne muscular dystrophy include, but are not limited to, exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78. In some examples, the polynucleic acid molecule targets sequences adjacent to the mutated exon. For example, if there is a deletion of exon 50, the polynucleic acid molecule targets a sequence in exon 51 such that exon 51 is skipped. In another example, if there is a mutation in exon 23, the polynucleic acid molecule targets a sequence in exon 22 such that exon 23 is skipped.

[0041] In some examples, the polynucleic acid molecules described herein target regions at the exon-intron junctions of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at the exon-intron junction of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at the exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 35 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 43 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 45 of the DMD gene.In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 55 of the DMD gene.

[0042] In some examples, the polynucleic acid molecule hybridizes to a target region at a 5' intron-exon junction or a 3' exon-intron junction of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region at a 5' intron-exon junction or a 3' exon-intron junction of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region at the 5' intron-exon junction or the 3' exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

[0043] In some cases, the polynucleic acid molecule is selected from exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 hybridize to a target region at the 5' intron-exon junction of at least one of (e.g., the 5' intron-exon junction of exon 3 is the intron 2-exon junction). In some cases, the polynucleic acid molecule hybridizes to a target region at a 5' intron-exon junction of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene (e.g., the 5' intron-exon junction of exon 3 is the intron 2-exon 3 junction). In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 23 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 35 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 43 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 44 of the DMD gene.In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 45 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 50 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 52 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 53 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 55 of the DMD gene.

[0044] In some cases, the polynucleic acid molecule is selected from exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 hybridize to a target region at the 3' exon-intron junction of at least one of (e.g., the 3' exon-intron junction of exon 3 is the junction of exon 3-intron 3). In some cases, the polynucleic acid molecule hybridizes to a target region at a 3' exon-intron junction of at least one of 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene (e.g., the 3'-intron junction of exon 3 is the exon 3-intron 3 junction). In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 23 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 35 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 43 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 44 of the DMD gene.In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 45 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 50 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 52 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 53 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 55 of the DMD gene.

[0045] In some examples, the polynucleic acid molecules described herein target splice sites of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a splice site in exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a splice site in exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 8 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 23 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 35 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 43 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 44 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 45 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 48 splice site of the DMD gene. In some cases, the polynucleic acid molecules described herein target the exon 49 splice site of the DMD gene.In some cases, the polynucleic acid molecules described herein target the splice site of exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 55 of the DMD gene. As used herein, splice site includes canonical splice sites, cryptic splice sites, or alternative splice sites that can induce insertions, deletions, duplications, or alterations of incorrectly spliced ​​mRNA transcripts to induce exon skipping or exon inclusion.

[0046] In some embodiments, the polynucleic acid molecule targets a partially spliced ​​mRNA sequence that contains additional exons involved in Duchenne muscular dystrophy, such as exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63.

[0047] In some examples, the polynucleic acid molecules hybridize to a region proximal to an exon-intron junction. In some examples, the polynucleic acid molecules described herein hybridize to exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 1 Target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5' from) of 9, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.In some examples, the polynucleic acid molecules described herein target a region that is at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 8 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 23 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 35 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 43 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 44 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5 nt upstream (or 5') of exon 45 of the DMD gene.In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 48 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 49 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 50 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 51 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 52 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 53 of the DMD gene.In some examples, the polynucleic acid molecules described herein target a region that is at least 1000 nt, 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') of exon 55 of the DMD gene.

[0048] In some examples, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region that is upstream (or 5') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region that is upstream (or 5') of at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region that is about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene.

[0049] In some examples, the polynucleic acid molecules described herein may be selected from the group consisting of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 Target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3' from) of 9, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 8 of the DMD gene.In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 23 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 35 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 43 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 44 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 45 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 48 of the DMD gene.In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 49 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 50 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 51 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 52 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 53 of the DMD gene. In some examples, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200 nt, 100 nt, 80 nt, 60 nt, 50 nt, 40 nt, 30 nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of exon 55 of the DMD gene.

[0050] In some examples, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 5') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 3') of at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

[0051] In some examples, the polynucleic acid molecule targets an internal region within exon 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 8 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 23 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 35 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 43 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 44 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 45 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 48 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 49 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 50 of the DMD gene.In some examples, the polynucleic acid molecules described herein target an internal region within exon 51 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 52 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 53 of the DMD gene. In some examples, the polynucleic acid molecules described herein target an internal region within exon 55 of the DMD gene.

[0052] In some examples, the polynucleic acid molecule hybridizes to at least one target region of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region within at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region within at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

[0053] In some embodiments, the polynucleic acid molecules described herein target a partially spliced ​​mRNA sequence comprising exon 44 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region upstream (or 5') to exon 44. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of exon 44. In some examples, the polynucleic acid molecule hybridizes to a target region downstream (or 3') to exon 44. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 3') of exon 44.

[0054] In some examples, the polynucleic acid molecule hybridizes to a target region within exon 44 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region at the 5' intron-exon 44 junction or the 3' exon 44-intron junction.

[0055] In some embodiments, the polynucleic acid molecules described herein target a partially spliced ​​mRNA sequence that includes exon 45 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region upstream (or 5') to exon 45. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of exon 45. In some examples, the polynucleic acid molecule hybridizes to a target region downstream (or 3') to exon 45. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 3') of exon 45.

[0056] In some examples, the polynucleic acid molecule hybridizes to a target region within exon 45 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region at the 5' intron-exon 45 junction or the 3' exon 45-intron junction.

[0057] In some embodiments, the polynucleic acid molecules described herein target a partially spliced ​​mRNA sequence that includes exon 51 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region upstream (or 5') to exon 51. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of exon 51. In some examples, the polynucleic acid molecule hybridizes to a target region downstream (or 3') to exon 51. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 3') of exon 51.

[0058] In some examples, the polynucleic acid molecule hybridizes to a target region within exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region at the 5' intron-exon 51 junction or the 3' exon 51-intron junction.

[0059] In some embodiments, the polynucleic acid molecules described herein target a partially spliced ​​mRNA sequence comprising exon 53 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region upstream (or 5') to exon 53. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of exon 53. In some examples, the polynucleic acid molecule hybridizes to a target region downstream (or 3') to exon 53. In some examples, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 3') of exon 53.

[0060] In some examples, the polynucleic acid molecule hybridizes to a target region within exon 53 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to a target region at the 5' intron-exon 53 junction or the 3' exon 53-intron junction.

[0061] 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 the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule consists of the target sequence of interest.

[0062] In some embodiments, the polynucleic acid molecule 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 a target sequence of interest. 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 a target sequence of interest. 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 a target sequence of interest, 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 the target sequence of interest.

[0063] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:964-1285. In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:964-1285.

[0064] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1056-1094.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1056-1094.

[0065] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1147-1162.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1147-1162.

[0066] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1173-1211.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1173-1211.

[0067] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1056-1076.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1056-1076.

[0068] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1077-1094.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1077-1094.

[0069] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1056-1058.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1056-1058.

[0070] In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NOs:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NOs:1087-1089.In some embodiments, the polynucleic acid molecule consists of SEQ ID NOs:1087-1089.

[0071] In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 964-1285. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1056-1094, 1147-1162, or 1173-1211. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1056-1076. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1077-1094. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1147-1162. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1173-1211. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1056-1058. In some examples, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more contiguous bases of a base sequence selected from SEQ ID NOs: 1087-1089. In some cases, the polynucleic acid molecule further comprises 1, 2, 3, or 4 mismatches.

[0072] In some embodiments, the polynucleic acid molecule comprises a guide strand and a passenger strand. In some examples, the guide strand 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:964-1285. In some cases, the guide strand comprises a sequence selected from SEQ ID NOs:964-1285.

[0073] In some embodiments, the polynucleic acid molecules described herein comprise RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some examples, the RNA comprises small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneous nuclear RNA (hnRNA). In some examples, the RNA comprises shRNA. In some examples, the RNA comprises miRNA. In some examples, the RNA comprises dsRNA. In some examples, the RNA comprises tRNA. In some examples, the RNA comprises rRNA. In some examples, the RNA comprises hnRNA. In some examples, the RNA comprises siRNA. In some examples, the polynucleic acid molecule comprises siRNA. In some examples, the polynucleic acid molecule is an antisense oligonucleotide (ASO).

[0074] In some embodiments, the nucleic acid polymer is about 10 to about 50 nucleotides in length. In some examples, the polynucleic acid molecule is about 10 to about 30, about 15 to about 30, about 18 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 20 to about 30, about 20 to about 25, about 20 to about 24, about 20 to about 23, or about 20 to about 22 nucleotides in length.

[0075] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 19 nucleotides in length. In some instances, the polynucleic acid molecule is about 18 nucleotides in length. In some instances, the polynucleic acid molecule is about 17 nucleotides in length. In some instances, the polynucleic acid molecule is about 16 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 nucleotides in length. In some instances, the polynucleic acid molecule is about 14 nucleotides in length. In some instances, the polynucleic acid molecule is about 13 nucleotides in length. In some instances, the polynucleic acid molecule is about 12 nucleotides in length. In some instances, 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. In some examples, the polynucleic acid molecule is about 19 to about 30 nucleotides in length. In some examples, the polynucleic acid molecule is about 20 to about 30 nucleotides in length.In some instances, the polynucleic acid molecule is about 19 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 to about 25 nucleotides in length.

[0076] In some embodiments, a polynucleic acid molecule is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides in length. In some examples, a polynucleic acid molecule is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some examples, a polynucleic acid molecule is at least 15 nucleotides in length. In some examples, a polynucleic acid molecule is at least 18 nucleotides in length. In some examples, a polynucleic acid molecule is at least 19 nucleotides in length. In some examples, a polynucleic acid molecule is at least 20 nucleotides in length. In some examples, a polynucleic acid molecule is at least 21 nucleotides in length. In some examples, a polynucleic acid molecule is at least 22 nucleotides in length. In some examples, a polynucleic acid molecule is at least 23 nucleotides in length. In some examples, a polynucleic acid molecule is at least 24 nucleotides in length. In some instances, the polynucleic acid molecule is at least 25 nucleotides in length. In some instances, the polynucleic acid molecule is at least 30 nucleotides in length.

[0077] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 29 nucleotides in length. In some instances, the polynucleic acid molecule is about 28 nucleotides in length. In some instances, the polynucleic acid molecule is about 27 nucleotides in length. In some instances, the polynucleic acid molecule is about 26 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 24 nucleotides in length. In some instances, the polynucleic acid molecule is about 23 nucleotides in length. In some instances, the polynucleic acid molecule is about 22 nucleotides in length. In some instances, the polynucleic acid molecule is about 21 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, 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.

[0078] 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 passenger strand. In some examples, the second polynucleotide is an antisense strand or guide strand.

[0079] 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 examples, the first polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 20 to about 30, about 20 to about 25, about 20 to about 24, about 20 to about 23, or about 20 to about 22 nucleotides in length.

[0080] 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.

[0081] 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 examples, the second polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 20 to about 30, about 20 to about 25, about 20 to about 24, about 20 to about 23, or about 20 to about 22 nucleotides in length.

[0082] 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 second polynucleotide is about 15 to about 25 nucleotides in length.In some examples, the second polynucleotide is about 15 to about 30 nucleotides in length. In some examples, the second polynucleotide is about 12 to about 30 nucleotides in length.

[0083] 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.

[0084] In some embodiments, the sequence of the polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule is 100% complementary to the target sequence described herein.

[0085] In some embodiments, the sequence of the polynucleic acid molecule has five or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has four or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has three or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has two or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has one or fewer mismatches to the target sequences described herein.

[0086] In some embodiments, the specificity of a polynucleic acid molecule that hybridizes to a target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some instances, hybridization is under highly stringent hybridization conditions.

[0087] In some embodiments, the polynucleic acid molecule has reduced off-target effects. In some instances, "off-target" or "off-target effect" refers to any instance in which a polynucleic acid polymer directed against 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.

[0088] In some embodiments, polynucleic acid molecules comprise natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules comprise a combination of DNA, RNA, and / or nucleotide analogs. In some instances, the synthetic or artificial nucleotide analogs or bases comprise modifications at one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0089] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some examples, the modification includes H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some examples, the alkyl moiety further comprises a modification. In some examples, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some examples, the alkyl moiety further comprises a heterosubstitution. In some examples, a carbon of the heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In some examples, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0090] In some cases, the modification of the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. Exemplary chemical structures of a 2'-O-methyl modification of an adenosine molecule and a 2'O-methoxyethyl modification of a uridine are illustrated below.

[0091] [ka]

[0092] 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 connects 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. A typical chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below.

[0093] [ka]

[0094] In some instances, the modification of the 2' hydroxyl group is a locked or bridged ribose modification (e.g., Locked Nucleic Acid or LNA), in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C, 4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. Representative examples of the chemical structure of LNA are illustrated below. The representative example shown on the left highlights the chemical bond nature of the LNA monomer. The representative example shown on the right shows a locked 3'-endo( 3 E) emphasizes structure.

[0095] [ka]

[0096] In some embodiments, the modification at the 2' hydroxyl group includes ethylene nucleic acid (ENA), such as 2'-4'-ethylene bridged nucleic acid, which locks the sugar structure into a 3'-endo sugar puckering conformation. ENA is part of the bridged nucleic acid class of modified nucleic acids, which also includes LNA. Typical chemical structures of ENA and bridged nucleic acids are illustrated below.

[0097] [ka]

[0098] 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).

[0099] In some embodiments, nucleotide analogs include, but are 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) Deazanucleotides such as 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, 7-deazaadenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyluridine, 5-methyl- Modified bases include thyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, queusine, 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 alkylcarbonylalkylated nucleotides. Modified nucleotides further include nucleotides modified in the sugar moiety, as well as 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 also 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.

[0100] In some embodiments, the nucleotide analog further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics natural nucleic acid structures by deviating from 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, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some cases, backbone modifications remove all positive and negative charges, making morpholino neutral molecules capable of crossing cell membranes without the aid of cellular delivery agents, such as those used by charged oligonucleotides.

[0101] [ka]

[0102] In some embodiments, peptide nucleic acids (PNAs) contain no sugar backbone rings or phosphate linkages, and the bases are linked and appropriately spaced by molecules such as oligoglycines, thus eliminating backbone charge.

[0103] [ka]

[0104] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some examples, the modified internucleotide bond includes, but is not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, borontrifluorides, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkylphosphonates, alkylphosphonothioates, arylphosphorothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, aminoalkylphosphoramidates, thionophospho Phosphorothioate antisense oligonucleotides include amides, phosphoropiperazidates, phosphoroanilothioates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazoates, methylenedimethylhydrazoates, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamidates, riboacetyl groups, aminoethylglycine, silyl, or siloxane bonds, saturated or unsaturated and / or substituted and / or heteroatom-containing alkyl or cycloalkyl bonds of 1 to 10 carbon atoms with or without heteroatoms, morpholino bonds, amides, polyamides in which bases are directly or indirectly bound to the aza nitrogens of the backbone, or combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate bonds. Exemplary PS ASOs are described below.

[0105] [ka]

[0106] In some instances, the modification is a methyl or thiol modification, such as a methyl phosphonate or thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left) and methyl phosphonate nucleotides (right) are illustrated below.

[0107] [ka]

[0108] In some examples, modified nucleotides include, but are not limited to, 2'-fluoro N3-P5'-phosphoramidites, exemplified as follows:

[0109] [ka]

[0110] In some examples, modified nucleotides include, but are not limited to, hexitol nucleic acids (alternatively, 1',5'-anhydrohexitol nucleic acids (HNA)), exemplified as follows:

[0111] [ka]

[0112] 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, wherein X is O or S; and B is a heterocyclic base moiety.

[0113] [ka]

[0114] 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 connects 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.

[0115] In some instances, 5'-vinylphosphonate is further modified with a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thus 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 X is O or S; B is a heterocyclic base moiety; and J is an internucleotide linking group that connects adjacent nucleotides of a polynucleotide.

[0116] [ka]

[0117] 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).

[0118] In some embodiments, the nucleotide analogs contain 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, e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine, 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups, e.g., 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 alkylcarbonylalkylated nucleotides. 5'-vinylphosphonate-modified nucleotides further include those nucleotides with modifications on the sugar moiety, such as, in some cases, mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles, as well as 5'-vinylphosphonate-modified nucleotides having non-ribosyl sugars or analogs thereof.The term nucleotide also includes what are 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.

[0119] In some embodiments, the 5'-vinylphosphonate-modified nucleotide analogs further include morpholinos, peptide nucleic acids (PNAs), methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoroN3-P5' phosphoramidites, or 1',5'-anhydrohexitol nucleic acids (HNAs). Morpholinos or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics that of natural nucleic acids 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, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In such cases, backbone modification removes 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, where X is O or S; and B is a heterocyclic base moiety.

[0120] [ka]

[0121] In some embodiments, the 5'-vinylphosphonate-modified morpholino or PMO described above is a PMO containing a positive or cationic charge. In some examples, the PMO is PMOplus (Sarepta). PMOplus refers to a phosphorodiamidate morpholino oligomer containing any number of (1-piperazino)phosphinylideneoxy, 1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy bonds (such as those described in PCT Publication WO2008 / 036127). In some examples, the PMO is a PMO described in U.S. Patent No. 7,943,762.

[0122] In some embodiments, the morpholino or PMO described above is PMO-X (Sarepta). In some instances, PMO-X refers to a phosphorodiamidate morpholino oligomer containing at least one bond or at least one of the disclosed terminal modifications, such as those described in PCT Publication WO 2011 / 150408 and U.S. Publication No. 2012 / 0065169.

[0123] In some embodiments, the morpholino or PMO described above is a PMO as described in Table 5 of U.S. Publication No. 2014 / 0296321.

[0124] An exemplary representation of the chemical structure of a 5'-vinylphosphonate modified nucleic acid is illustrated below, where X is O or S; B is a heterocyclic base moiety; and J is an internucleotide linkage.

[0125] [ka]

[0126] 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 backbone charge.

[0127] [ka]

[0128] In some embodiments, one or more modifications of the 5'-vinylphosphonate modified oligonucleotide are optionally 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' linked boranophosphate and selenophosphate; phosphotriester; thionoalkylphosphotriester; hydrogen phosphonate bond; alkyl phosphonate; alkyl phosphonothioate; aryl phosphonothioate; phosphoroselenoate; phosphorodiselenoate; phosphinate; phosphoramidate; 3'-alkyl phosphoramidate; aminoalkyl phosphoramidate; thionophosphoramidate; phosphinate. Examples of suitable linkers include holopiperazidates, phosphoroanilothioates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethyldimethylhydrazo, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamidates, bonds with riboacetyl groups, aminoethylglycines, silyl or siloxane bonds, alkyl or cycloalkyl bonds, for example, saturated or unsaturated and / or substituted and / or containing heteroatoms, with or without heteroatoms, from 1 to 10 carbons, bonds with morpholino structures, amides, polyamides in which bases are attached directly or indirectly to the aza nitrogens of the backbone, or combinations thereof.

[0129] In some examples, the modification is a methyl or thiol modification, such as a methylphosphonate or thiolphosphonate modification. Exemplary thiolphosphonate nucleotides (left), phosphorodithioate (center), and methylphosphonate nucleotides (right) are illustrated below.

[0130] [ka]

[0131] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, phosphoramidites exemplified as follows:

[0132] [ka]

[0133] In some instances, the modified internucleotide linkage is a phosphorodiamidate linkage. Non-limiting examples of phosphorodiamidate linkages using morpholino systems are shown below:

[0134] [ka]

[0135] In some instances, the modified internucleotide linkage is a methylphosphonate linkage. Non-limiting examples of methylphosphonate linkages are shown below:

[0136] [ka]

[0137] In some instances, the modified internucleotide bond is an amide bond. Non-limiting examples of amide bonds are shown below:

[0138] [ka]

[0139] In some examples, 5'-vinylphosphonate modified nucleotides include, but are not limited to, the modified nucleic acids exemplified below.

[0140] In some embodiments, the one or more modifications include a modified phosphate backbone, where the modification produces a neutral or uncharged backbone. In some instances, the phosphate backbone is modified by alkylation, which produces an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some instances, alkyl group, as used herein in the context of alkylation, refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms. In some instances, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl groups. In some instances, the modified phosphate is a phosphate group described in U.S. Pat. No. 9,481,905.

[0141] In some embodiments, the additional modified phosphate backbone comprises a methyl phosphonate, an ethyl phosphonate, a methylthiophosphonate, or a methoxyphosphonate. In some embodiments, the modified phosphate is a methyl phosphonate. In some embodiments, the modified phosphate is an ethyl phosphonate. In some embodiments, the modified phosphate is a methylthiophosphonate. In some embodiments, the modified phosphate is a methoxyphosphonate.

[0142] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or a nucleotide analog modification 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' linkage. In another alternative, the 3' end is optionally conjugated with an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In a further alternative, the 3' end is optionally conjugated with an abasic site, such as an apurinic or apyrimidinic acid site. In some examples, the 5' end is conjugated with an aminoalkyl group, such as a 5'-O-aminoalkyl substituent. In some cases, the 5' end is conjugated with an abasic site, such as an apurinic or apyrimidinic acid site.

[0143] In some embodiments, the polynucleic acid molecules comprise one or more of the artificial nucleotide analogs described herein. In some examples, the polynucleic acid molecules described herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more of the artificial nucleotide analogs 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 modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or combinations thereof. In some embodiments, the polynucleic acid molecule 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 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more artificial nucleotide analogs selected from modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof. In some embodiments, the 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 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, a polynucleic acid molecule described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more thiolphosphonate nucleotides.

[0144] In some embodiments, a polynucleic acid molecule comprises a plurality of phosphorodiamidate morpholino oligomer- or peptide nucleic acid-modified non-natural nucleotides, and optionally comprises at least one inverted abasic moiety. In some examples, the polynucleic acid molecule comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorodiamidate morpholino oligomer-modified non-natural nucleotides. In some examples, the polynucleic acid molecule comprises 100% phosphorodiamidate morpholino oligomer-modified non-natural nucleotides.

[0145] In some examples, the polynucleic acid molecule comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptide nucleic acid modified non-natural nucleotides, hi some examples, the polynucleic acid molecule comprises 100% peptide nucleic acid modified non-natural nucleotides.

[0146] In some embodiments, a polynucleic acid molecule comprises one or more nucleotide analogs, each of which is a stereochemically isomeric form. In such instances, the polynucleic acid molecule is a chiral molecule. In some cases, the nucleotide analog comprises a backbone stereochemistry. In further cases, the nucleotide analog comprises a chiral analog such as those described in U.S. Patents 9,982,257, 9,695,211, or 9,605,019.

[0147] In some examples, the 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.

[0148] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications.

[0149] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications.

[0150] In some examples, the polynucleic acid molecule comprises at least one of the following: about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications.

[0151] In some examples, the polynucleic acid molecule comprises at least one of the following: about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications.

[0152] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications.

[0153] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0154] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 30% modifications, and about 20% to about 30% modifications.

[0155] Optionally, the polynucleic acid molecule contains about 10% to about 20% modifications.

[0156] In some cases, the polynucleic acid molecule contains from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

[0157] In further instances, the polynucleic acid molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

[0158] In some embodiments, the 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 modifications.

[0159] In some examples, the 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.

[0160] In some examples, about 5 to about 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 10% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 15% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 20% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 25% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 30% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 35% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 40% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 45% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 50% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 55% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 60% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 65% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 70% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 75% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 80% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 85% of the polynucleic acid molecules comprise the artificial nucleotide analogs described herein.In some examples, about 90% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 95% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 96% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 97% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 98% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 99% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some examples, about 100% of the polynucleic acid molecules comprise an artificial nucleotide analog 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 modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidite, or combinations thereof.

[0161] In some embodiments, the polynucleic acid molecule comprises from about 1 to about 25 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 1 modification, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 2 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 3 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 4 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 5 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 6 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 7 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 8 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 9 modifications, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 10 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, wherein the modifications comprise an artificial nucleotide analog described herein.In some embodiments, the polynucleic acid molecule comprises about 15 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 16 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 17 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 18 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 19 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 20 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 21 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 22 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 23 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 24 modifications, wherein the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 25 modifications, wherein the modifications include artificial nucleotide analogs described herein.

[0162] In some embodiments, the polynucleic acid molecule is assembled from two separate polynucleotides, where one polynucleotide comprises the sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule, hi other embodiments, the sense strand is connected to the antisense strand by a linker molecule, which in some instances is a polynucleotide linker or a non-nucleotide linker.

[0163] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides in the sense strand comprise 2'-O-methylpyrimidine nucleotides and the purine nucleotides in the sense strand comprise 2'-deoxypurine nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoropyrimidine nucleotides and the purine nucleotides present in the sense strand comprise 2'-deoxypurine nucleotides.

[0164] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, are 2'-O-methyl purine nucleotides.

[0165] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, comprise 2'-deoxy-purine nucleotides.

[0166] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a cap moiety at the 5'-end, the 3'-end, or both the 5' and 3' ends of the sense strand, hi other embodiments, the terminal cap moieties are inverted deoxyabasic moieties.

[0167] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a phosphate backbone modification at the 3' end of the antisense strand. In some instances, the phosphate backbone modification is phosphorothioate. In some instances, the passenger strand comprises more phosphorothioate modifications than the guide strand. In other instances, the guide strand comprises more phosphorothioate modifications than the passenger strand. In additional instances, the passenger strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications. In additional instances, the guide strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications.

[0168] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

[0169] In some embodiments, a polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides with and / or universal base modifications, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 10, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, or with one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or with or without end cap molecules at the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0170] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand has about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or end cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0171] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-end, 5'-end, 6'-end, 7'-end, 8'-end, 9'-end, 10'-end, 11'-end, 12'-end, 13'-end, 14'-end, 15'-end, 16'-end, 17'-end, 18'-end, 19'-end, 20 ... or terminal cap molecules at both the 3'- and 5'-ends; and the antisense strand comprises about 1 to about 10, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally terminal cap molecules at the 3'-end, 5'-end, or both the 3'- and 5'-ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkages, and / or end cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0172] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand has from about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base-modified nucleotides, and optionally, terminal cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoro nucleotides, with or without about 1 to about 5, e.g., about 1, 2, 3, 4, 5, or more, phosphorothioate internucleotide linkages, and / or end cap molecules at the 3'-terminus, 5'-terminus, or both the 3'- and 5'-terminus, present on the same or different strands.

[0173] In some embodiments, the polynucleic acid molecule is a double-stranded polynucleic acid molecule with one or more of the following properties: high hepatocyte stability, reduced overall charge, reduced hepatocyte uptake, or enhanced pharmacokinetics. In some embodiments, the double-stranded polynucleic acid molecule comprises a passenger strand (e.g., sense strand) and a guide strand (e.g., antisense strand) that contain multiple modifications.

[0174] In some embodiments, a double-stranded polynucleic acid molecule comprises a guide strand (e.g., antisense strand) having one or more of the modifications described above, and a passenger strand (e.g., sense strand) having a plurality of phosphorodiamidate morpholino oligomers or a plurality of peptide nucleic acid modified non-natural nucleotides.

[0175] In some embodiments, the polynucleic acid molecules described herein are chemically modified short interfering nucleic acid molecules having about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, phosphorothioate internucleotide linkages in each strand of the polynucleic acid molecule.

[0176] In another embodiment, the polynucleic acid molecules described herein comprise a 2'-5' internucleotide linkage. In some examples, the 2'-5' internucleotide linkage is at the 3'-terminus, the 5'-terminus, or both the 3'-terminus and the 5'-terminus of one or both strands of the sequence. In additional examples, the 2'-5' internucleotide linkage is present at various other positions within one or both strands of the sequence, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of all internucleotide linkages of pyrimidine nucleotides in one or both strands of the polynucleic acid molecule comprise a 2'-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of all internucleotide linkages of purine nucleotides in one or both strands of the polynucleic acid molecule comprise a 2'-5' internucleotide linkage.

[0177] In some embodiments, the polynucleic acid molecule is a single-stranded polynucleic acid molecule that mediates RNAi activity in a cell or is reconstituted in an in vitro system, wherein the polynucleic acid molecule comprises a single-stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides, or alternatively, a plurality of pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and any purine nucleotides present in the polynucleic acid are 2'-deoxy purine nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides, or alternatively, a plurality of purine nucleotides are 2'-deoxy purine nucleotides), and terminal cap modifications are optionally present at the 3'-end, 5'-end, or both the 3' and 5'-ends of the antisense sequence. The polynucleic acid molecule optionally further comprises about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxyribonucleotides at the 3' end of the polynucleic acid molecule, wherein the terminal nucleotides further comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages, and the polynucleic acid molecule optionally further comprises a terminal phosphate group, such as a 5'-terminal phosphate group.

[0178] In some cases, one or more of the artificial nucleotide analogs described herein are more resistant to nucleases, such as ribonucleases, e.g., RNase H, deoxyribonucleases, e.g., DNase, or exonucleases, e.g., 5'-3' exonucleases and 3'-5' exonucleases, compared to naturally occurring polynucleic acid molecules. In some examples, the artificial nucleotide analogs are 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 modified 2'-O-N-methylacetamide (2'-O-N-methylacetamide). Artificial nucleotide analogs, including 2'-O-methyl nucleotides (NMA), 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, e.g., RNase H, deoxyribonucleases, e.g., DNase, or exonucleases, e.g., 5'-3' exonucleases or 3'-5' exonucleases. In some examples, the 2'-O-methyl modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some examples, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-deoxy modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, 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., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, 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., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, LNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, ENA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, HNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, the morpholino is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the PNA modified polynucleic acid molecule is resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the methylphosphonate nucleotide modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the thiolphosphonate nucleotide modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease 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.

[0179] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity for their mRNA targets compared to a comparable naturally occurring polynucleic acid molecule. Polynucleic acid molecules containing one or more 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), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidite, have increased binding affinity for their mRNA targets compared to comparable naturally occurring polynucleic acid molecules. In some instances, 2'-O-methyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-aminopropyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-deoxy modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, T-deoxy-2'-fluoro modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules.In some instances, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, LNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, ENA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, PNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some examples, HNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, morpholino-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, methylphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, thiolphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, the increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0180] In some embodiments, the polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules comprising a single enantiomer. In some examples, the polynucleic acid molecules comprise L-nucleotides. In some examples, the polynucleic acid molecules comprise D-nucleotides. In some examples, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some cases, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some examples, the polynucleic acid molecule is a polynucleic acid molecule described in U.S. Patent Application Publication Nos. 2014 / 194610 and 2015 / 211006; WO2015107425.

[0181] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugated moiety. In some examples, the aptamer-conjugated moiety is a DNA aptamer-conjugated moiety. In some examples, the aptamer-conjugated moiety is Alphamer (Centauri Therapeutics), which includes an aptamer moiety that recognizes a specific cell surface target and a moiety that displays a specific epitope for binding to circulating antibodies. In some examples, the polynucleic acid molecules described herein are further modified to include an aptamer-conjugated moiety as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975:

[0182] In additional embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some examples, the polynucleic acid molecule is modified with 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), T-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, methyl phosphonate nucleotide, thiol phosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability. In some examples, the polynucleic acid molecule is a chiral pure (or stereopure) polynucleic acid molecule. In some examples, the chiral pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications of RNA to increase delivery stability will be apparent to those skilled in the art.

[0183] In some embodiments, the polynucleic acid molecules described herein have RNAi activity that regulates the expression of RNA encoded by genes involved in muscular dystrophy, such as, but not limited to, DMD, DUX4, DYSF, EMD, or LMNA. In some examples, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein one strand of the double-stranded siRNA molecule comprises a nucleotide sequence complementary to at least one nucleotide sequence of DMD, DUX4, DYSF, EMD, or LMNA, or the RNA encoded by at least one of DMD, DUX4, DYSF, EMD, or LMNA, or a portion thereof, and wherein the second strand of the double-stranded siRNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of at least one of DMD, DUX4, DYSF, EMD, or LMNA, or the RNA encoded by at least one of DMD, DUX4, DYSF, EMD, or LMNA, or a portion thereof. In some instances, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein each strand of the siRNA molecule comprises about 15-25, 18-24, or 19-23 nucleotides, and each strand comprises at least about 14, 17, or 19 nucleotides complementary to nucleotides in the other strand. In some instances, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein each strand of the siRNA molecule comprises about 19-23 nucleotides, and each strand comprises at least about 19 nucleotides complementary to nucleotides in the other strand. In some instances, RNAi activity occurs intracellularly. In other instances, RNAi activity occurs in a reconstituted in vitro system.

[0184] In some embodiments, the polynucleic acid molecules described herein have RNAi activity that regulates the expression of RNA encoded by the DMD gene. In some examples, the polynucleic acid molecules described herein are single-stranded siRNA molecules that downregulate the expression of DMD, wherein the single-stranded siRNA molecule comprises a nucleotide sequence complementary to the nucleotide sequence of DMD or the RNA encoded by DMD, or a portion thereof. In some cases, the polynucleic acid molecules described herein are single-stranded siRNA molecules that downregulate the expression of DMD, wherein the siRNA molecule comprises about 15-25, 18-24, or 19 to about 23 nucleotides. In some cases, the polynucleic acid molecules described herein are single-stranded siRNA molecules that downregulate the expression of DMD, wherein the siRNA molecule comprises about 19 to about 23 nucleotides. In some examples, the RNAi activity occurs intracellularly. In other examples, the RNAi activity occurs in a reconstituted in vitro system.

[0185] In some instances, the polynucleic acid molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some instances, the polynucleic acid molecule is assembled from two separate polynucleotides, one strand being the sense strand and the other strand being the antisense strand, wherein the antisense strand and the sense strand are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to the nucleotide sequence in the other strand; such as when the antisense strand and the sense strand form a duplex or double-stranded structure, e.g., the double-stranded region is about 19, 20, 21, 22, 23, or more base pairs); the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the polynucleic acid molecule can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the polynucleic acid molecule are joined by a nucleic acid-based or non-nucleic acid-based linker.

[0186] In some cases, the polynucleic acid molecule is a polynucleotide with a double, asymmetric double, hairpin, or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a part thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof.In other cases, the polynucleic acid molecule is a circular single-stranded polynucleotide with two or more loop structures and a base comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a part thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof, and the circular polynucleotide is processed in vivo or in vitro to generate an active polynucleic acid molecule that can mediate RNAi. In further cases, the polynucleic acid molecule further comprises a single-stranded polynucleotide having a nucleotide sequence complementary to that of a target nucleic acid molecule or a portion thereof (e.g., such a polynucleic acid molecule need not be present within the polynucleic acid molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-stranded polynucleotide further comprises a terminal phosphate group such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate.

[0187] In some instances, a linear polynucleic acid molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region is asymmetric, with the sense region containing fewer nucleotides than the antisense region, sufficient to base-pair with the antisense region and form a looped duplex. For example, an asymmetric hairpin polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in vitro system, and having a loop region comprising about 4 to about 8 nucleotides, and a sense region of about 3 to about 18 nucleotides complementary to the antisense region. In some instances, the asymmetric hairpin polynucleic acid molecule further comprises a chemically modified 5'-terminal phosphate group. In further instances, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0188] In some embodiments, an asymmetric duplex is a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region has sufficient complementary nucleotides to base-pair with the antisense region but contains fewer nucleotides than the antisense region, sufficient to form a duplex. For example, an asymmetric duplex polynucleic acid molecule may comprise an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in an in vitro system, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0189] In some cases, universal base refers to the nucleotide base analogue that forms base pairs with each of the few natural DNA / RNA bases.Non-limiting examples of universal base include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole, as known in the prior art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0190] Polynucleic acid molecule synthesis In some embodiments, the polynucleic acid molecule described herein is constructed by chemical synthesis and / or enzyme ligation reaction, using procedures known in the art.For example, polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides, or by using various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between polynucleic acid molecule and target nucleic acid.Exemplary methods include those described in U.S. Patent No. 5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT Publication No. WO2009099942; or European 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, polynucleic acid molecules can be 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 is in an antisense orientation relative to the desired target polynucleic acid molecule).

[0191] In some embodiments, the polynucleic acid molecule is synthesized by a tandem synthesis method, where both strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker, which is then cleaved to provide separate fragments or strands that hybridize to the duplex and allow for purification of the duplex.

[0192] In some instances, the polynucleic acid molecule is also assembled from two distinct nucleic acid strands or fragments, where one fragment comprises the sense region and the second fragment comprises the antisense region of the molecule.

[0193] Further modification methods, for example, to incorporate sugar, base, and phosphate modifications, include: Eckstein et al., International Publication PCT No. WO 92 / 07065; Perrault et al. al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93 / 15187; Sproat, US Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97 / 26270; Beigelman et al., US Pat. No. 5,716,824; Usman et al., US Pat. No. 5,627,053; Woolf et al., International PCT Publication No. WO 98 / 13526; Thompson et al., US Ser. No. 60 / 082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. This publication describes methods and strategies for determining the location for incorporating sugar, base, and / or phosphate modifications into nucleic acid molecules without modulating catalytic activity.

[0194] In some instances, chemical modification of internucleotide linkages of polynucleic acid molecules with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate linkages improves stability, while excessive modification often causes toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages is sometimes minimized. In such cases, reducing the concentration of these linkages reduces the toxicity of these molecules and increases their efficacy and specificity.

[0195] Nucleic Acid Polypeptide Conjugates In some embodiments, the polynucleic acid molecule is further conjugated to a polypeptide A, which is delivered to a desired site. In some cases, the polynucleic acid molecule is conjugated to polypeptide A and optionally to a polymer moiety.

[0196] In some examples, at least one polypeptide A is conjugated to at least one B. In some examples, at least one polypeptide A is conjugated to at least one B to form an AB conjugate. In some embodiments, at least one A is conjugated 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 polypeptide A is conjugated to at least two Bs. In some examples, at least one polypeptide A is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more Bs.

[0197] In some embodiments, at least one polypeptide A is conjugated at one end of at least one B, while at least one C is conjugated at the opposite end of at least one B to form an ABC conjugate. In some examples, at least one polypeptide A is conjugated at one end of at least one B, while at least one C is conjugated at an internal site of at least one B. In some examples, at least one polypeptide A is directly conjugated to at least one C. In some examples, at least one B is indirectly conjugated to at least one polypeptide A via at least one C to form an ACB conjugate.

[0198] In some examples, at least one B and / or at least one C, and optionally at least one D, are conjugated to at least one polypeptide A. In some examples, at least one B is conjugated to at least one polypeptide A at a terminal end (e.g., the 5' end or the 3' end) or conjugated to at least one polypeptide A via an internal site. Optionally, at least one C is conjugated to at least one polypeptide A directly or indirectly via at least one B. Indirectly, via at least one B, at least one C is conjugated at the same end on B as at least one polypeptide A, at the opposite end from at least one polypeptide A, or independently at an internal site. In some examples, at least one additional polypeptide A is further conjugated to at least one polypeptide A, B, or C. In a further example, at least one D is optionally conjugated, directly or indirectly, to at least one polypeptide A, at least one B, or at least one C. When conjugated directly to at least one polypeptide A, the at least one D is also optionally conjugated to at least one B to form an ADB conjugate, or optionally conjugated to at least one B and at least one C to form an ADBC ​​conjugate. In some examples, the at least one D is directly conjugated to at least one polypeptide A and indirectly conjugated to at least one B and at least one C to form a DABC conjugate. When conjugated indirectly to at least one polypeptide A, the at least one D is also optionally conjugated to at least one B to form an ABD conjugate, or optionally conjugated to at least one B and at least one C to form an ABDC conjugate. In some examples, at least one additional D is further conjugated to at least one polypeptide A, B, or C.

[0199] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0200] [ka]

[0201] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0202] [ka]

[0203] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0204] [ka]

[0205] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0206] [ka]

[0207] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0208] [ka]

[0209] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0210] [ka]

[0211] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0212] [ka]

[0213] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0214] [ka]

[0215] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0216] [ka]

[0217] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0218] [ka]

[0219] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0220] [ka]

[0221] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0222] [ka]

[0223] In some embodiments, the polynucleic acid molecule conjugate comprises a construct as exemplified:

[0224] [ka]

[0225] [ka] includes, by way of example only, a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0226] joining part In some embodiments, binding moiety A is a polypeptide. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some examples, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv (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.

[0227] In some examples, A is an antibody or binding fragment thereof. In some examples, A is a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a 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 Fab2. In some examples, A is a single chain variable fragment (scFv).

[0228] In some embodiments, binding moiety A is a bispecific antibody or binding fragment thereof. In some instances, the bispecific antibody is a trispecific antibody or a bispecific miniantibody. In some instances, the bispecific antibody is a trispecific antibody. In some instances, the trispecific antibody is a full-length monoclonal antibody that contains binding sites for two different antigens.

[0229] In some cases, the bispecific antibody is a bispecific miniantibody. In some examples, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single-chain variable fragments (scFvs), where the two scFvs target epitopes of two different antigens.

[0230] In some embodiments, binding moiety A is a bispecific miniantibody. In some instances, A is a bispecific Fab2. In some instances, A is a bispecific F(ab)'3 fragment. Optionally, A is a bispecific bis-scFv. Optionally, 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).

[0231] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody comprises a F(ab)'3 fragment or a trispecific antibody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, A is a trispecific antibody. In some embodiments, A is a trispecific antibody such as those 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).

[0232] In some embodiments, binding moiety A is an antibody or binding fragment thereof that recognizes a cell surface protein. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a cell surface protein on muscle cells. Exemplary cell surface proteins recognized by antibodies or binding fragments thereof include, but are not limited to, Sca-1, CD34, Myo-D, myogenin, MRF4, NCAM, CD43, and CD95 (Fas).

[0233] In some examples, the cell surface protein comprises a cluster of differentiation (CD) cell surface marker. 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, and 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, 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), etc.

[0234] In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. In some examples, binding moiety A is an antibody or binding fragment thereof that recognizes a CD cell surface marker. 34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, C D49b, CD49c, CD49d, CD49e, CD49f, 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, or a binding fragment thereof.

[0235] In some embodiments, binding moiety A is an anti-myosin antibody, an anti-transferrin antibody, and an antibody that recognizes muscle-specific kinase (MuSK).

[0236] In some instances, binding moiety A is an anti-myosin antibody. In some instances, the anti-myosin antibody is a humanized antibody. In other instances, the anti-myosin antibody is a chimeric antibody. In further instances, the anti-myosin antibody is a monovalent, bivalent, or multivalent antibody.

[0237] In some examples, binding moiety A is an anti-transferrin (anti-CD71) antibody. In some cases, the anti-transferrin antibody is a humanized antibody. In other cases, the anti-transferrin antibody is a chimeric antibody. In further cases, the anti-transferrin antibody is a monovalent, bivalent, or polyvalent antibody. In some embodiments, exemplary anti-transferrin antibodies include MAB5746 from R&D Systems, AHP858 from Bio-Rad Laboratories, A80-128A from Bethyl Laboratories, Inc., and T2027 from MilliporeSigma.

[0238] In some instances, binding moiety A is an antibody that recognizes MuSK. In some instances, the anti-MuSK antibody is a humanized antibody. In other instances, the anti-MuSK antibody is a chimeric antibody. In further instances, the anti-MuSK antibody is a monovalent, bivalent, or multivalent antibody.

[0239] In some embodiments, binding moiety A is non-specifically conjugated to polynucleic acid molecule (B). In some instances, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site-specific manner via a lysine or cysteine ​​residue. In some instances, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site-specific manner via a lysine residue. In some instances, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site-specific manner via a cysteine ​​residue.

[0240] In some embodiments, binding moiety A is conjugated to polynucleic acid molecule (B) in a non-site-specific manner. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner via a lysine residue, a cysteine ​​residue, at the 5'-end, at the 3'-end, at an unnatural amino acid, or at an enzyme-modified or enzyme-catalyzed residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner via a lysine residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner via a cysteine ​​residue. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner at the 5'-end. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner at the 3'-end. In some examples, binding moiety A is conjugated to polynucleic acid molecule (B) in a site-specific manner via an unnatural amino acid. In some instances, the binding moiety A is conjugated to the polynucleic acid molecule (B) via an enzyme-modified or enzyme-catalyzed residue in a site-specific manner.

[0241] In some embodiments, one or more polynucleic acid molecules (B) are conjugated to a binding moiety A. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 1 polynucleic acid molecule is conjugated to a single binding moiety A. In some examples, about 2 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 3 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 4 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 5 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 6 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 7 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 8 polynucleic acid molecules are conjugated to a single binding moiety A. In some examples, about 9 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 10 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 11 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 12 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 13 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 14 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 15 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 16 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, one or more polynucleic acid molecules are the same. In other examples, one or more polynucleic acid molecules are different.

[0242] In some embodiments, the number of polynucleic acid molecules (B) conjugated to binding moiety A 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 a polynucleic acid molecule (B). In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 2 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 3 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 4 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 5 or more. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 6 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 7 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 8 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 9 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 10 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 11 or greater. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 12 or greater.

[0243] In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 3. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 5. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 7. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 9. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 10. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 11. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 12. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 13. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 14. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 15. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is about 16.

[0244] In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 1. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 2. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 4. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 6. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 8. In some examples, the DAR ratio of polynucleic acid molecule (B) to binding moiety A is 12.

[0245] In some examples, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety A has improved activity compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some examples, the improved activity results in an enhancement of a biologically relevant function, e.g., improved stability, affinity, binding, functional activity, and efficacy in treating or preventing a disease condition. In some examples, the disease condition is the result of one or more mutated exons of a gene. In some examples, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety A results in increased exon skipping of one or more mutated exons compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some examples, exon skipping is increased by at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% in a conjugate comprising polynucleic acid molecule (B) and binding moiety A compared to a conjugate comprising polynucleic acid molecule (B) without binding moiety A.

[0246] 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 amino acid deletion, insertion, substitution, or addition, and / or by 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 an Fc receptor. In some examples, one or more modifications include, for example, those described in International Publication No. WO 97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into nucleic acid sequences underlying the amino acid sequence of an antibody or binding fragment thereof are well known to those of skill in the art.

[0247] In some instances, an antibody binding fragment further includes derivatives thereof, and comprises a polypeptide sequence comprising at least one CDR.

[0248] In some instances, the term "single chain" as used herein means that the first and second domains of the bispecific single chain construct are covalently linked, preferably in the form of a colinear amino acid sequence that can be encoded by a single nucleic acid molecule.

[0249] In some instances, bispecific single-chain antibody constructs relate to constructs comprising binding domains from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some instances, the scFv comprises a VH and a VL domain connected by a linker peptide. In some instances, the linker is of sufficient length and sequence to allow each of the first and second domains to retain their differential binding specificities independently of each other.

[0250] 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 instances, the antigen-interaction site defines a polypeptide motif that exhibits a specific interaction capacity with a specific antigen or a specific group of antigens. In some cases, binding / interaction is also understood to define specific recognition. In such cases, specific recognition refers to the ability of an antibody or binding fragment thereof to specifically interact and / or bind to at least two amino acids of each of the target molecules. For example, specific recognition relates to the specificity of an antibody molecule or its ability to distinguish a specific range of target molecules. In additional examples, the specific interaction of an antigen-interaction site with its specific antigen results in the initiation of a signal, for example, by inducing a conformational change in the antigen, antigen oligomerization, etc. In further embodiments, binding is exemplified by the specificity of the "key-lock principle." Thus, in some instances, specific motifs in the amino acid sequences of the antigen-interaction site and the antigen bind to each other as a result of their primary, secondary, or tertiary structure, as well as as a result of secondary modifications of the above structures. 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.

[0251] 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 binding fragment thereof that binds to a desired polypeptide / protein but does not essentially bind to any other polypeptides is considered specific for the desired polypeptide / protein. Specificity of the antigen-interaction site Examples of specific interactions with an antigen include the specificity of a ligand with its receptor, for example, the interaction of an antigenic determinant (epitope) with the antigen-binding site of an antibody.

[0252] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. In some examples, the plasma protein includes albumin. In some examples, binding moiety A is albumin. In some examples, the albumin is bound to the polynucleic acid molecule by one or more of the linking chemistries described herein. In some examples, the albumin is bound to the polynucleic acid molecule by native ligation chemistry. In some examples, the albumin is bound to the polynucleic acid molecule by a lysine bond.

[0253] In some examples, the conjugated moiety is a steroid. Exemplary steroids are cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (saturated, unsaturated, and substituted), or combinations thereof. In some examples, the steroid is cholesterol. In some examples, the conjugated moiety is cholesterol. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by native ligation chemistry. In some examples, the cholesterol is conjugated to the polynucleic acid molecule by a lysine bond.

[0254] In some examples, the binding moiety is a polymer, including but not limited to, a polynucleic acid molecule aptamer that binds to a specific surface marker on a cell. In this example, the binding moiety is a polynucleic acid that does not hybridize to the target gene or mRNA, but instead can selectively bind to the cell surface marker, similar to an antibody that binds to that specific epitope of the cell surface marker.

[0255] In some instances, the binding moiety is a peptide. In some instances, the peptide comprises about 1 to about 3 kDa. In some instances, the peptide comprises about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some instances, the peptide is a bicyclic peptide. In some instances, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the peptide moiety is a bicyclic peptide (e.g., Bicycles from Bicycle Therapeutics).

[0256] In further instances, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some instances, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, where the target-binding end is capable of recognizing and interacting with a cell surface receptor. For example, in some instances, the target-binding end comprises a glutamate urea compound, which allows interaction with PSMA, thereby enhancing antibody interaction with cells expressing PSMA. In some instances, the binding moiety comprises a target-binding end comprising ... as 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 a small molecule described in 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).

[0257] bond chemistry In some embodiments, polynucleic acid molecule B is conjugated to a binding moiety. In some examples, the binding moiety includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and all analogs or derivatives of these classes of substances. Additional examples of binding moieties include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, steroids such as 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.

[0258] In some embodiments, the polynucleic acid molecule is attached to the binding moiety by a chemical ligation process. In some instances, the polynucleic acid molecule is attached to the binding moiety by native ligation. In some examples, the conjugation is as described in Dawson, et al. "Synthesis of proteins by native chemical ligation," Science 1994, 266, 776-779; Dawson, et al. "Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives," J. Am. Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. "Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology," Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. "Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol," Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleic acid molecule is site-specifically or non-specifically conjugated to the binding moiety via native ligation chemistry.

[0259] 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 polynucleic acid molecule that contains an aldehyde group and is then coupled to a binding moiety. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS 134(13): 5887-5892 (2012)).

[0260] In some examples, polynucleic acid molecules are conjugated to binding moieties by site-directed methods utilizing unnatural amino acids incorporated into the binding moieties. 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)).

[0261] In some instances, polynucleic acid molecules are attached to binding moieties by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Redwood). In some instances, SMARTag™ technology involves the generation of a formylglycine (FGly) residue from cysteine ​​by formylglycine generating enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent attachment of FGly to an alkylhydrazine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51 (2013))

[0262] In some examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some examples, the polynucleic acid molecule is conjugated to a binding moiety using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some examples, mTG is produced by Streptomyces mobaraensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013)).

[0263] In some examples, the polynucleic acid molecule is conjugated to a binding moiety by the method described in PCT Publication No. WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0264] In some examples, the polynucleic acid molecule is attached to the binding moiety by the methods described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0265] Production of antibodies or their binding fragments In some embodiments, the polypeptides (e.g., antibodies and binding fragments thereof) described herein are produced using any method known in the art useful for the synthesis of polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression techniques.

[0266] 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 involve the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligation of those oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

[0267] 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 that expresses immunoglobulins) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0268] In some instances, antibodies or binding sites thereof may be isolated by immunizing animals such as rabbits to generate polyclonal antibodies, or more preferably by immunizing animals such as rabbits using, for example, Kohler and Milstein (1975, Nature 256:495-497) or by generating monoclonal antibodies as described 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) or antibody libraries (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937) for clones of Fab fragments that bind to a specific antigen.

[0269] In some embodiments, "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) are used. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region (e.g., humanized antibodies).

[0270] In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are 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, resulting in a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0271] In some embodiments, an expression vector containing an antibody nucleotide sequence or the antibody nucleotide sequence is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0272] In some embodiments, various host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent not only the vehicles in which the antibody coding sequences are produced and subsequently purified, but also cells that, 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., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or binding fragment thereof coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or binding fragment thereof coding sequence; insect cell systems (e.g., baculovirus) infected with recombinant viral expression vectors containing the antibody or binding fragment thereof coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or binding fragment thereof coding sequence; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0273] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, cells are engineered to grow in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes, grow, and form foci that are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or their binding fragments.

[0274] 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 cells, hgprt cells, or aprt cells, respectively. Similarly, antimetabolite resistance has been used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods 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).

[0275] In some instances, antibody expression levels are increased by vector amplification (for review, see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is related to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0276] In some examples, any method known in the art for purifying or analyzing antibodies or antibody conjugates is used, such as 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 purifying proteins. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and fast protein liquid chromatography.

[0277] Polymer-binding moiety In some embodiments, polymer moiety C is further attached to a polynucleic acid molecule described herein, a binding moiety described herein, or a combination thereof. In some instances, polymer moiety C is attached to a polynucleic acid molecule. In some instances, polymer moiety C is attached to a binding moiety. In other instances, polymer moiety C is attached to a polynucleic acid molecule binding moiety. In further instances, polymer moiety C is attached as illustrated above.

[0278] In some examples, polymer moiety C is a natural or synthetic polymer composed of long chains of branched or unbranched monomers and / or crosslinked networks of two- or three-dimensional monomers. In some examples, polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C includes, but is not limited to, alpha-dihydroxyl polyethylene glycol, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, a mixture refers to the use of various polymers within the same compound, as in the context of 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 polyalkylene oxide. In some examples, polymer portion C includes PEG. In some examples, polymer portion C includes polyethylene imide (PEI) or hydroxyethyl starch (HES).

[0279] 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 linking 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 linking moiety is attached at the 5' end of the polynucleic acid molecule. In some examples, the PEG moiety, the linking 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 attachment is via native ligation.

[0280] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some instances, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some instances, a monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight indicated represents the average molecular weight of the polyalkylene oxide (e.g., PEG) molecules.

[0281] 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.

[0282] 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, 2700, , 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 850 In some examples, C has a molecular weight of about 00, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some examples, C has a molecular weight of about 200 Da. In some examples, C has a molecular weight of about 300 Da. In some examples, C has a molecular weight of about 400 Da. In some examples, C has a molecular weight of about 500 Da. In some examples, C has a molecular weight of about 600 Da. In some examples, C has a molecular weight of about 700 Da. In some examples, the molecular weight of C is about 800 Da. In some examples, the molecular weight of C is about 900 Da. In some examples, the molecular weight of C is about 1000 Da. In some examples, the molecular weight of C is about 1100 Da. In some examples, the molecular weight of C is about 1200 Da. In some examples, the molecular weight of C is about 1300 Da. In some examples, the molecular weight of C is about 1400 Da.In some examples, the molecular weight of C is about 1450 Da. In some examples, the molecular weight of C is about 1500 Da. In some examples, the molecular weight of C is about 1600 Da. In some examples, the molecular weight of C is about 1700 Da. In some examples, the molecular weight of C is about 1800 Da. In some examples, the molecular weight of C is about 1900 Da. In some examples, the molecular weight of C is about 2000 Da. In some examples, the molecular weight of C is about 2100 Da. In some examples, the molecular weight of C is about 2200 Da. In some examples, the molecular weight of C is about 2300 Da. In some examples, the molecular weight of C is about 2400 Da. In some examples, the molecular weight of C is about 2500 Da. In some examples, the molecular weight of C is about 2600 Da. In some examples, the molecular weight of C is about 2700 Da. In some examples, the molecular weight of C is about 2800 Da. In some examples, the molecular weight of C is about 2900 Da. In some examples, the molecular weight of C is about 3000 Da. In some examples, the molecular weight of C is about 3250 Da. In some examples, the molecular weight of C is about 3350 Da. In some examples, the molecular weight of C is about 3500 Da. In some examples, the molecular weight of C is about 3750 Da. In some examples, the molecular weight of C is about 4000 Da. In some examples, the molecular weight of C is about 4250 Da. In some examples, the molecular weight of C is about 4500 Da. In some examples, the molecular weight of C is about 4600 Da. In some examples, the molecular weight of C is about 4750 Da. In some examples, the molecular weight of C is about 5000 Da. In some examples, the molecular weight of C is about 5500 Da. In some examples, the molecular weight of C is about 6000 Da. In some examples, the molecular weight of C is about 6500 Da. In some examples, the molecular weight of C is about 7000 Da. In some examples, the molecular weight of C is about 7500 Da. In some examples, the molecular weight of C is about 8000 Da. In some examples, the molecular weight of C is about 10,000 Da. In some examples, the molecular weight of C is about 12,000 Da. In some examples, the molecular weight of C is about 20,000 Da. In some examples, the molecular weight of C is about 35,000 Da. In some examples, the molecular weight of C is about 40,000 Da.In some examples, the molecular weight of C is about 50,000 Da. In some examples, the molecular weight of C is about 60,000 Da. In some examples, the molecular weight of C is about 100,000 Da.

[0283] In some embodiments, the polyalkylene oxide (e.g., PEG) is a dispersed PEG, which is a polymeric PEG containing one or more repeating ethylene oxide units. 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 stepwise as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting materials. In some examples, the dPEG has a specific molecular weight rather than an average molecular weight. In some examples, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

[0284] In some embodiments, the polymer portion C comprises a cationic mucic acid-based polymer (cMAP). In some examples, the cMAP comprises one or more subunits of at least one repeating subunit, and the subunit structure is represented as formula (V):

[0285] [ka]

[0286] 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 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

[0287] 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, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

[0288] In some examples, polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, polymer moiety C is a cMAP-PEG copolymer. In other cases, polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. In further cases, polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0289] In some embodiments, the polymer moiety C is conjugated to a polynucleic acid molecule, a binding moiety, and optionally an endosomolytic moiety, as exemplified above.

[0290] Endosomolytic moiety In some embodiments, the molecule of Formula (I):AXBYC further comprises an additional conjugated moiety. In some examples, the additional conjugated moiety is an endosomolytic moiety. In some cases, the endosomolytic moiety is a compound capable of releasing a cellular compartment-releasing component, e.g., any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum comprising a cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer.

[0291] Endosomolytic Polypeptides In some embodiments, the molecule of formula (I): AXBYC is further conjugated to an endosomolytic polypeptide. In some embodiments, the molecule of formula (V): A-(X 1 -B) n Or formula (II): AX 1 -(BX 2 -C) n The molecule further binds 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 (meucin), or a derivative thereof. In some examples, the endosomolytic polypeptide comprises INF or a derivative thereof. In other cases, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further cases, the endosomolytic polypeptide comprises a mucin or a derivative thereof.

[0292] In some examples, INF7 is a 24-residue polypeptide, and these sequences include CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 1), or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2). In some examples, INF7 or a derivative thereof includes the following sequence: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 3), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 4), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 5).

[0293] In some instances, melittin is a 26-residue polypeptide, and the sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 6), or alternatively, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 7). In some instances, melittin comprises a polypeptide sequence described in U.S. Patent No. 8,501,930.

[0294] In some instances, the mucins are antimicrobial peptides (AMPs) derived from the venom glands of the scorpion Mesobuthus eupeus. In some instances, the mucins are comprised of mucin-13 (these sequences include IFGAIAGLLKNIF-NH2 (SEQ ID NO:8)) and mucin-18 (these sequences include FFGHLFKLATKIIPSLFQ (SEQ ID NO:9)).

[0295] In some examples, the endosomolytic polypeptide comprises a polypeptide having 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.

[0296] 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:1-5. 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:1. 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:1. Optionally, 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:1. Optionally, the endosomolytic portion comprises SEQ ID NO:2-5. Optionally, the endosomolytic portion consists of SEQ ID NO:1. Optionally, the endosomolytic portion consists of SEQ ID NO:2-5.

[0297] 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:6 or 7. 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:6. 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:7. Optionally, the endosomolytic portion comprises SEQ ID NO:6. Optionally, the endosomolytic portion comprises SEQ ID NO:7. Optionally, the endosomolytic portion consists of SEQ ID NO:6. Optionally, the endosomolytic portion consists of SEQ ID NO:7.

[0298] 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:8 or 9. 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:8. 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:9. Optionally, the endosomolytic moiety comprises SEQ ID NO:8. 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:9. Optionally, the endosomolytic moiety comprises SEQ ID NO:8. Optionally, the endosomolytic moiety consists of SEQ ID NO:9.

[0299] In some examples, the endosomolytic moiety comprises a sequence shown in Table 1 below.

[0300] [Table 1-1]

[0301] [Table 1-2]

[0302] In some cases, the endosomolytic moiety inhibits Bcl-2 and / or Bcl-x LIn 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).

[0303] In some examples, the endosomolytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) described in PCT Publication Nos. WO2013 / 166155 and WO2015 / 069587.

[0304] Endosomolytic polymers In some embodiments, the compound of formula (V): A-(X 1 -B) n Or formula (VI): AX 1 -(BX 2 -C) n The molecule is further conjugated to an endosomolytic polymer. As used herein, endosomolytic polymers include linear, branched network, star, comb, or ladder polymers. In some instances, the endosomolytic polymer is a homopolymer or copolymer comprising two or more different types of monomers. In some instances, the endosomolytic polymer is a polycationic polymer. In other instances, the endosomolytic polymer is a polyanionic polymer.

[0305] In some instances, polycationic polymers contain positively, neutrally, or negatively charged monomer units, resulting in a net positive charge. In other instances, polycationic polymers contain non-polymeric molecules containing 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).

[0306] In some cases, polyanionic polymers contain positively charged, neutrally charged, or negatively charged monomer units, resulting in a net negative charge. In other cases, polyanionic polymers contain non-polymeric molecules containing two or more negative charges. Exemplary anionic polymers include p(alkyl acrylates) (e.g., poly(propylacrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIPAM). Further examples include PP75, Khormaee, et al., "Edosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications,” Advanced Functional Materials 23: 565-574 (2013), including the L-phenylalanine-poly(L-lysine isophthalamide) polymer described therein.

[0307] In some embodiments, the endosomolytic polymers described herein are pH-responsive endosomolytic polymers. pH-responsive polymers include polymers that increase in size (swell) or collapse depending on the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers.

[0308] In some examples, the endosomolytic moieties described herein are membrane-disruptive polymers. In some cases, the membrane-disruptive polymers include cationic polymers, neutral or hydrophobic polymers, or anionic polymers. In some examples, the membrane-disruptive polymers are hydrophilic polymers.

[0309] In some examples, the endosomolytic moieties described herein are pH-responsive membrane-disruptive polymers. Exemplary pH-responsive membrane-disruptive polymers include p(alkylacrylic acid), poly(N-isopropylacrylamide) (NIPAM) copolymers, succinylated p(glycidol), and p(β-malic acid) polymers.

[0310] 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).

[0311] In some embodiments, the pH-responsive membrane-disruptive polymer comprises p(butyl acrylate-co-methacrylic acid). (Bulmus, et al., Journal of Controlled Release 93: 105-120 (2003); and Yessine, et al., Biochimica et Biophysica Acta 1613: 28-38 (2003))

[0312] In some embodiments, the pH-responsive membrane-disruptive polymer comprises p(styrene-alt-maleic anhydride). (See Henry, et al., Biomacromolecules 7: 2407-2414 (2006))

[0313] In some embodiments, the pH-responsive membrane-disruptive polymer comprises a pyridyldisulfide 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 (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))

[0314] In some embodiments, the pH-responsive membrane disruptive polymer comprises a cytolytic polymer comprising the following basic structure:

[0315] [ka]

[0316] In some examples, the endosomolytic moieties described herein are further conjugated to an additional conjugate, such as a polymer (e.g., PEG) or a modified polymer (e.g., a cholesterol-modified polymer).

[0317] In some examples, the additional conjugate comprises a surfactant (e.g., Triton X-100). In some examples, the endosomolytic moiety described herein comprises a polymer (e.g., poly(amidoamine)) conjugated with a surfactant (e.g., Triton X-100). In some examples, the endosomolytic moiety described herein comprises a poly(amidoamine)-Triton X-100 conjugate (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)).

[0318] Endosomolytic lipids In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the endosomolytic moiety is a lipid having formula (V):A-(X 1 -B) n Or formula (VI): AX 1 -(BX 2 -C) nThe molecule further binds 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), and 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).

[0319] In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in PCT Publication No. WO 09 / 126,933.

[0320] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, the endosomolytic moiety is a small molecule of Formula (I): A-(X 1 -B) n Or formula (II): AX 1 -(BX 2 -C) nis 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), ampicillin, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, quinoline endosomolytic moieties include, but are not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino)quinoline; )quinoline;7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline;7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine);7-fluoro-4-(4-diethylamino-1-butylamino)quinoline;4-(4-diethylamino-1-butylamino)quinoline;7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4- 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 -diethyl-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino-)quinoline;7-Hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;Hydroxychloroquine phosphate;7-Chloro-4-(4-ethyl-(2-hydroxyethyl)-1-amino-1-butylamino)quinoline (desmethylhydroxychloroquine);7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(4-Ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-Hydroxy-4-(4 -Ethyl-(2-hydroxyethyl)-amino-1-butylamino(quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline 7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline Amino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline;1-Acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-Butyryl-1,2,3,4-tetrahydroquinoline;3-Chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline)(4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline (4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline); 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde; 1,1'-pentamethylenequinolinium iodide; 8-quinolinol sulfate and amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs thereof. In some examples, the endosomolytic moiety is selected from the group consisting of quinolinol 8-sulfate, ... 280:884-893) and U.S. Patent No. 5,736,557. In some embodiments, the endosomolytic moiety is nigericin or a conjugate thereof, such as a folate-nigericin ester conjugate, a folate-nigericin amide conjugate, or a folate-nigericin carbamate linkage. In some examples, the endosomolytic moiety is nigericin, as described in Rangasamy, et. al., "New mechanism for release of endosomal contents: osmotic lysis via nigericin-mediated K / H exchange," Bioconjugate Chem. 29:1047-1059 (2018).

[0321] Linker In some embodiments, the linkers described herein are cleavable or non-cleavable linkers. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker.

[0322] In some cases, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain repeating units of a monomer produced by a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In some cases, the non-polymeric linker includes a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In further cases, the non-polymeric linker does not include more than two linkers of the same type, for example, more than two homobifunctional cross-linkers or more than two peptide linkers. In further instances, the non-polymeric linker optionally includes one or more reactive functional groups.

[0323] In some examples, the non-polymeric linker does not include a polymer as described above. In some examples, the non-polymeric linker does not include a polymer encompassed by polymer moiety C. In some cases, the non-polymeric linker does not include a polyalkylene oxide (e.g., PEG). In some cases, the non-polymeric linker does not include PEG.

[0324] 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 (sulfosuccinimidyl tartrate), and the like. DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridinyl) (Dithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene and 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED ), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0325] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-bridged 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 amines 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, 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-maleimidecarbonyl reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate reactive and photoreactive cross-linkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine reactive and photoreactive cross-linkers such as ρ-azidophenylglyoxal (APG).

[0326] 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.

[0327] In some embodiments, the linker comprises a maleimide group. In some examples, the maleimide group is also referred to as a maleimide spacer. In some examples, the maleimide group further comprises caproic acid to form maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), described above.

[0328] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing retro-Michael elimination. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0329] In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide moiety comprises at least 1, 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety comprises at most 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 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, 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, or Gly-Phe-Leu-Gly. In some examples, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, 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, or Gly-Phe-Leu-Gly. In some instances, the linker comprises Val-Cit. In some instances, the linker is Val-Cit.

[0330] 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).

[0331] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0332] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some examples, the linker includes a linker described in U.S. Pat. No. 9,089,614 or PCT Publication WO2015038426.

[0333] 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.

[0334] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or linker group) on the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D after cleavage. Exemplary traceless linkers include, but are not limited to, a germanium linker, a selenium linker, a sulfur linker, a selenium linker, a nitrogen linker, a phosphorus linker, a boron linker, a chromium linker, or a phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker 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.

[0335] In some embodiments, 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.

[0336] 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.

[0337] In some examples, X is a single bond or a linker. In some examples, X is a single bond. In some examples, X is a linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X is a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. When used in the context of a linker, particularly in the context of X, alkyl refers to a saturated, straight- or branched-chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X is a non-polymeric linker. In some examples, X comprises a homobifunctional linker or a heterobifunctional linker described above. In some cases, X comprises a heterobifunctional linker. In some examples, X comprises sMCC. In other examples, X comprises a heterobifunctional linker optionally bonded to a C1-C6 alkyl group. In other examples, X comprises sMCC optionally bonded to a C1-C6 alkyl group. In some additional examples, X does not include a homobifunctional linker or a heterobifunctional linker described above.

[0338] In some examples, Y is a single bond or a linker. In some examples, Y is a single bond. In other cases, Y is a linker. In some embodiments, Y is a C1-C6 alkyl group. In some examples, Y is a homobifunctional linker or a heterobifunctional linker described above. In some examples, Y is a homobifunctional linker described above. In some examples, Y is a heterobifunctional linker described above. In some examples, Y comprises a maleimide group, such as maleimidocaproyl (mc), 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 an additional example, Y comprises a mc-val-cit-PABA group.

[0339] In some examples, L is a single bond or a linker. In some examples, L is a single bond. In some examples, L is a linker. In some embodiments, L is a C1-C6 alkyl group. In some examples, L is a homobifunctional linker or a heterobifunctional linker described above. In some examples, L is a homobifunctional linker described above. In some examples, L is a heterobifunctional linker described above. In some examples, L comprises a maleimide group, such as maleimidocaproyl (mc), 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 a further example, L comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In a further example, L comprises an mc group. In a further example, L comprises an mc-val-cit group. In a further example, L comprises a val-cit-PABA group. In a further example, L comprises a mc-val-cit-PABA group.

[0340] In some embodiments, X 1 and X 2 are each independently a single bond or a non-polymeric linker. 1 and X 2 are each independently a single bond. 1 and X 2 are each independently a non-polymeric linker.

[0341] In some instances, X 1 may include a single bond or a non-polymeric linker. In some examples, X 1 is a single bond. In some instances, X 1 is a non-polymeric linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X 1 is a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. When used in the context of a linker, particularly in the context of X1, alkyl refers to a saturated, straight- or branched-chain hydrocarbon radical containing up to six carbon atoms. In some examples, X 1 comprises a homobifunctional linker or heterobifunctional linker as described above. 1 comprises a heterobifunctional linker. 1 In another example, X 1 comprises a heterobifunctional linker optionally attached to a C1-C6 alkyl group. 1 In some additional examples, X comprises sMCC optionally bonded to a C1-C6 alkyl group. 1 does not include the homobifunctional or heterobifunctional linkers described above.

[0342] In some instances, X 2 is a single bond or a linker. In some examples, X 2 is a single bond. In other cases, X 2is a linker. In a further case, X 2 is a non-polymeric linker. In some embodiments, X 2 is a C1-C6 alkyl group. In some examples, X 2 is a homobifunctional linker or heterobifunctional linker as described above. In some examples, X 2 is a homobifunctional linker as described above. In some examples, X 2 is a heterobifunctional linker as described above. In some examples, X 2 contains a maleimide group, such as maleimidocaproyl (mc), described above, or a self-stabilizing maleimide group. In some examples, X 2 contains a peptide moiety such as Val-Cit. In some instances, X 2 contains a benzoic acid group, such as PABA. 2 contains a combination of maleimide groups, peptide moieties, and / or benzoic acid groups. 2 In a further example, X 2 contains the mc-val-cit group. 2 contains a val-cit-PABA group. 2 contains the mc-val-cit-PABA group.

[0343] Pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal routes of administration. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet other examples, the pharmaceutical compositions described herein are formulated for nasal administration.

[0344] In some embodiments, pharmaceutical compositions include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and combined immediate and controlled release formulations.

[0345] In some examples, the pharmaceutical formulation comprises a multiparticulate formulation. In some examples, the pharmaceutical formulation comprises a nanoparticle formulation. In some examples, the nanoparticle comprises cMAP, cyclodextrin, or lipid. In some cases, the nanoparticle comprises a solid lipid nanoparticle, a polymeric nanoparticle, a self-emulsifying nanoparticle, a liposome, a microemulsion, or a micellar solution. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those with covalently bound metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, the nanoparticles are metal nanoparticles, such as nanoparticles of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys, or oxides thereof.

[0346] In some instances, the nanoparticles comprise a core or a core and a shell, such as core-shell nanoparticles.

[0347] In some examples, the nanoparticles are further coated with molecules for attachment of functional elements, such as one or more of the polynucleic acid molecules or binding moieties described herein. In some examples, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyldextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, or dextrin or cyclodextrin. In some examples, the nanoparticles comprise graphene-coated nanoparticles.

[0348] In some cases, the nanoparticles have at least one dimension that is less than about 500 nm, 400 nm, 300 nm, 200 nm, or even 100 nm.

[0349] In some examples, the nanoparticle formulation comprises a paramagnetic nanoparticle, a superparamagnetic nanoparticle, a metal nanoparticle, a fullerene-like material, an inorganic nanotube, a dendrimer (such as one with a covalently bound metal chelate), a nanofiber, a nanohorn, a nano-onion, a nanorod, a nanorope, or a quantum dot. In some examples, a polynucleic acid molecule or binding moiety described herein is directly or indirectly attached to the nanoparticle. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more polynucleic acid molecules or binding moieties described herein are directly or indirectly attached to the nanoparticle.

[0350] In some embodiments, the pharmaceutical formulation comprises a delivery vector, e.g., a recombinant vector for delivery of a polynucleic acid molecule into a cell. In some examples, the recombinant vector is a DNA plasmid. In other examples, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. In some examples, the recombinant vector capable of expressing a polynucleic acid molecule provides stable expression in target cells. In further examples, viral vectors are used that provide transient expression of a polynucleic acid molecule.

[0351] In some embodiments, pharmaceutical formulations include a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier substances include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, humectants, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholate, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See (e.g., Remington): The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0352] In some examples, the pharmaceutical formulation further comprises a pH adjusting or buffering agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0353] In some examples, the pharmaceutical formulation includes one or more salts in an amount necessary to bring the osmolality of the composition to an acceptable range. Such salts include sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0354] In some cases, pharmaceutical formulations further comprise a diluent that is used to stabilize the compound, since the diluent can provide a more stable environment.Salts dissolved in buffers, including but not limited to phosphate buffered saline, (which also control or maintain pH) are used as diluents in the art.In certain cases, diluents increase the size of the composition to facilitate compression or create sufficient bulk for homogeneous mixing for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; calcium hydrogen phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0355] In some cases, pharmaceutical formulations contain disintegration agents or disintegrants that promote the breakdown or disintegration of substances. The term "disintegrate" includes both the dissolution and dispersion of the dosage form when it comes into contact with gastrointestinal fluids. Examples of disintegrants are starches, such as natural starches, e.g., corn starch or potato starch, pregelatinized starches, e.g., National 1551 or Amijel®, or sodium starch glycolate, e.g., Promogel® or Explotab®, celluloses, e.g., wood products, methyl crystalline cellulose, ...

Claims

[Claim 1] The invention described in the specification.

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