Nucleic acid polypeptide compositions and methods for inducing exon skipping - Patents.com

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 is a continuation of U.S. Provisional Patent Application No. 62 / 561,939, filed September 22, 2017. No. 62 / 696,766, filed July 11, 2018. No. 6,393,635, each of which is incorporated herein by reference in its entirety. Can be enjoyed. [Background technology]

[0002] Modulation of RNA function is a developing area of ​​therapeutic interest. Drugs that affect mRNA stability, such as nucleotides and small interfering RNA, can disrupt RNA function. Another group of oligonucleotides is one way to regulate the function of the final gene product: To include or exclude specific regions of the pre-mRNA from the encoded protein, By altering pre-mRNA processing, RNA function can be modulated. Thus, oligonucleotide therapeutics provide a means to modulate protein expression in disease states. and therefore have utility as therapeutic agents. Summary of the Invention

[0003] Provided herein, in certain embodiments, are molecules for regulating RNA processing and In some embodiments, the present invention relates to a method for treating muscular dystrophy, and a pharmaceutical composition thereof. Molecules and pharmaceutical compositions for the same are also disclosed herein.

[0004] In one embodiment, the present invention relates to a method for detecting an incorrectly spliced ​​mRNA in a subject. A method of treating a disease or disorder caused by a transcript is disclosed, the method comprising: The method includes administering to a subject a polynucleic acid molecule conjugate, wherein the polynucleic acid molecule conjugate is a cell The polynucleotide is optionally linked to a target binding moiety, where the polynucleotide optionally has at least one 2' modification. modified nucleotide, at least one modified internucleotide bond, or at least a single inverted abasic portion, wherein the polynucleic acid molecule conjugate is a fully processed mRNA An exon in an mRNA transcript that is incorrectly spliced ​​to produce a transcript Incorrectly spliced ​​to induce exon skipping or exon inclusion Inducing insertions, deletions, duplications, or alterations of the mRNA transcripts, and The fully processed mRNA transcript encodes a functional protein, thereby In some embodiments, the disease or disorder further comprises treating a disease or disorder of the body. In some embodiments, the disease or The conditions or disorders may include neuromuscular diseases, genetic diseases, cancer, hereditary diseases, or cardiovascular diseases. In some embodiments, the disease or disorder is muscular dystrophy. In some embodiments, the disease or disorder is Duchenne muscular dystrophy. In this study, exon skipping was performed on exons 8, 23, 35, 43, and 50 of the DMD gene. 44, 45, 50, 51, 52, 53, or 55. In this study, the exon skipping was of exon 23 of the DMD gene. In an embodiment, the polynucleic acid molecule conjugate has 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 bond or a first linker; and Y is a 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 bond or a first linker; and Y is a bond or a second linker. In some embodiments, at least one 2' modified nucleotide is a morpholino, 2'-O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Amino Propyl, 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-Dimethylaminoethyl Oxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotides. Another 2' modified nucleotide is Locked Nucleic Acid (LNA), Ethylene Nucleic Acid (ENA), Alternatively, it comprises a peptide nucleic acid (PNA). In some embodiments, at least one In some embodiments, the 2' modified nucleotide comprises a morpholino. In some embodiments, at least one inverted base portion is at least one terminal end. At least one modified internucleotide linkage is a phosphorothioate linkage or a phospho In some embodiments, the polynucleic acid molecule comprises at least about 1 In some embodiments, the polynucleic acid molecule is from 0 to about 30 nucleotides in length. About 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 20 to about In some embodiments, the polynucleic acid is at least one of about 22 nucleotides in length. The molecule is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, or In some embodiments, the polynucleic acid molecule has at least At least one of: about 5% to about 100% modification, about 10% to about 100% modification, about 2 0% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modified, about 50% to about 100% modified, about 60% to about 100% modified, 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: % to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modified, about 50% to about 90% modified, about 60% to about 90% modified , about 70% to about 90% modified, and about 80% to about 100% modified. In embodiments, the polynucleic acid molecules comprise at least one of the following: about 10% to about 80% modified, about 20% to about 80% modified, about 30% to about 80% modified, about 40% to about 80% modified % modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% modification. % to about 80% modified. In some embodiments, the polynucleic acid molecule comprises at least Including one of: about 10% to about 70% modified, about 20% to about 70% modified, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and About 60% to about 70% modified. In some embodiments, the polynucleic acid molecule has at least At least one of: about 10% to about 60% modification, about 20% to about 60% modification, about 30 % to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification. In some embodiments, the polynucleic acid molecule comprises at least one of the following: % to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and and about 40% to about 50% modifications. In some embodiments, the polynucleic acid molecule comprises At least one of: about 10% to about 40% modification, about 20% to about 40% modification, and and about 30% to about 40% modifications. In some embodiments, the polynucleic acid molecule comprises At least one of: about 10% to about 30% modification, and about 20% to about 30% modification. In some embodiments, the polynucleic acid molecule contains about 10% to about 20% modifications. In some embodiments, the polynucleic acid molecules are from about 15% to about 90%, from about 20% to about 80%, 0%, about 30% to about 70%, or about 40% to about 60% modifications. In embodiments, the polynucleic acid molecule is at least about 15%, 20%, 30%, 40%, 50%, %, 60%, 70%, 80%, 90%, 95%, or 99% modifiers. In embodiments, the polynucleic acid molecule comprises at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, approx. 10, approx. 11, approx. 12, approx. 13, approx. 14, approx. 15, approx. 16, approx. 17, approx. 18, approx. In some embodiments, the amino acid sequence includes about 9, about 20, about 21, about 22, or more modifications. The polynucleic acid molecule may comprise 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 embodiments, the polynucleic acid molecule comprises a single strand. In some embodiments, the polynucleic acid molecule comprises two or more strands. A first polynucleotide and a nucleic acid molecule comprising a nucleic acid sequence that is a nucleic acid fragment of the first polynucleotide. In some embodiments, the second polynucleotide is a hybridized second polynucleotide. The polynucleotide comprises at least one modification. In some embodiments, the first polynucleotide The nucleotide and the second polynucleotide are RNA molecules. In one embodiment, 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, or a In some embodiments, the linker group is a polymer linker, a cleavable linker, or a non-polymer linker group. In some embodiments, X is C 1 -C 6 is an alkyl group In some embodiments, Y is C 1 -C 6 In some embodiments, the alkyl group is an alkyl group. In the formula, X is optionally C 1 -C 6A homobifunctional linker is attached to an alkyl group. In some embodiments, Y is a homobifunctional linker. In some embodiments, the linking moiety is In some embodiments, the antibody or binding fragment thereof is an antibody or binding fragment thereof. The fragment may be a humanized antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof. fragment, monoclonal antibody or binding fragment thereof, monovalent Fab', bivalent Fa b 2 , single chain variable fragment (scFv), diabody, minibody dy), nanobody, single domain antibody (sdAb), or camelid antibody, or In some embodiments, C is a polyethylene glycol. In some embodiments, C has a molecular weight of about 5,000 Da. In this embodiment, 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 is In some embodiments, the method further comprises conjugating the internucleotide linkage group to D. In some embodiments, D is conjugated to C or A. wherein D is linked 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 bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer between 0 and 1; wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one or at least one inverted abasic moiety, and and D is conjugated to any position of A, B, or C. In some embodiments, D is INF7 or melittin. , L is C 1 -C 6 In some embodiments, L is a homobifunctional linker. In some embodiments, the method further comprises: In some embodiments, the at least one binding moiety is a second binding moiety A. The binding moiety A is conjugated to A, B, or C.

[0005] In some embodiments, the exons in the misspliced ​​mRNA transcripts Missplicing to induce exon skipping or exon inclusion Methods for inducing insertions, deletions, duplications, or alterations in a synthesized mRNA transcript are described herein. The method disclosed in the publication includes the steps of: contacting a target cell with a polynucleic acid molecule conjugate, The polynucleotide comprises at least one 2' modified nucleotide, at least one modified or at least one inverted abasic moiety; In order to induce exon skipping or exon inclusion, to induce insertions, deletions, duplications or alterations of the encoded mRNA transcript in target cells. Hybridization of polynucleic acid molecule conjugates to incorrectly spliced ​​mRNA transcripts in the a process of synthesizing the mis-spliced ​​mRNA transcripts to determine the protein function; The step is capable of encoding the fully processed mRNA transcript of the previous step; In some embodiments, the method further comprises translating a functional form of the protein of interest. The cell is a target cell of the subject. In some embodiments, the misspliced The resulting mRNA transcript further induces a disease or disorder. The disease or disorder may further be characterized by one or more mutations in the mRNA. In an embodiment, the disease or disorder is a neuromuscular disease, a genetic disease, a cancer, a hereditary disease, or a cardiac disorder. 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, exon skipping is performed by skipping an exon of the DMD gene. 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 In some embodiments, exon skipping is performed on 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 bond or a first linker; and Y is a 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 bond or a first linker; and Y is a bond or a second linker. In some embodiments, at least one 2' modified nucleotide is a morpholino, 2'-O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Amino Propyl, 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-Dimethylaminoethyl Oxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotides. Another 2' modified nucleotide is Locked Nucleic Acid (LNA), Ethylene Nucleic Acid (ENA), In some embodiments, the nucleic acid comprises at least one 2' modified peptide nucleic acid (PNA). In some embodiments, the modified nucleotide comprises a morpholino. In some embodiments, the inverted base portion is at least one terminal. The modified internucleotide linkages are phosphorothioate or phosphorodithioate. In some embodiments, the polynucleic acid molecule comprises at least about 10 to about 10 ethyl ester linkages. In some embodiments, the polynucleic acid molecule has at least At least one: about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about In some embodiments, the length of the nucleic acid sequence is at least one of about 20 to about 22 nucleotides. In some embodiments, the polynucleic acid molecule comprises at least about 16, 17, 18, 19, 20, 21, 22, In some embodiments, the polynucleotide is 23, 24, or 25 nucleotides in length. The acid molecules include at least one of the following: about 5% to about 100% modification, about 10% to about 1 00% 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% modified; about 80% to about 100% modified; and about 90% In some embodiments, the polynucleic acid molecule comprises at least one of the following: Including one of: about 10% to about 90% modified, about 20% to about 90% modified, about 30% to About 90% modified, about 40% to about 90% modified, about 50% to about 90% modified, about 60% from about 90% modified, from about 70% to about 90% modified, and from about 80% to about 100% modified. Modifications. In some embodiments, the polynucleic acid molecule comprises at least one of the following: 0% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, About 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification In some embodiments, the polynucleic acid molecule contains at least one of the following: about 10% to about 70% modification, about 20% to about 70% modified, about 30% to about 70% modified, about 40% to about 70% modified, about 50% to about 70% modified % modification, and from about 60% to about 70% modification. The acid molecules include at least one of the following: about 10% to about 60% modified, about 20% to about 6 0% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% modification. % to about 60% modified. In some embodiments, the polynucleic acid molecule comprises at least Including one of: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to About 50% modification, and about 40% to about 50% modification. The nucleic acid molecule comprises at least one of the following: about 10% to about 40% modifications, about 20% to about About 40% modification, and about 30% to about 40% modification. The nucleic acid molecule comprises at least one of the following: about 10% to about 30% modifications, and about 20 % to about 30% modified. In some embodiments, the polynucleic acid molecule has about 10% to about 20% modified. In some embodiments, the polynucleic acid molecule comprises about 15% to about 90% , about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% modified. In some embodiments, the polynucleic acid molecule comprises at least about 15%, 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% qualified In some embodiments, the polynucleic acid molecule comprises at least about 3, about 4, about 5, about 6, approx. 7, approx. 8, approx. 9, approx. 10, approx. 11, approx. 12, approx. 13, approx. 14, approx. 15, approx. 16, approx. About 17, about 18, about 19, about 20, about 21, about 22, or more modifications. In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, Approx. 7, Approx. 8, Approx. 9, Approx. 10, Approx. 11, Approx. 12, Approx. 13, Approx. 14, Approx. 15, Approx. 16, Approx. 17 , about 18, about 19, about 20, about 21, about 22 or more modified nucleotides. In some embodiments, the polynucleic acid molecule comprises a single strand. In some embodiments, a polynucleic acid molecule comprises two or more strands. The acid molecule reacts with the first polynucleotide to form a double stranded polynucleic acid molecule. and a second polynucleotide hybridized to the oligonucleotide. In some embodiments, the second polynucleotide comprises at least one modification. In one embodiment, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide and the second polynucleotide are siRNA. In some embodiments, X and Y are independently a single bond, a degradable phosphorus The linker may be a polymeric linker, a non-degradable linker, a cleavable linker, or a non-polymeric linker group. In some embodiments, X is a single bond. 1 -C 6 In some embodiments, Y is an alkyl group. 1 -C 6 It is an alkyl group. In some embodiments, X is optionally C 1 -C 6 Homodicyclic alkyl groups In some embodiments, the linker is a heterobifunctional linker. Y is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the binding moiety is an antibody or a binding fragment thereof. The antibody or binding fragment thereof may be a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab', bivalent Fab 2 , single chain variable fragment (scFv), diabody, minibody Minibodies, nanobodies, single domain antibodies (sdAbs), or camelids In some embodiments, C is a 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 A- X is conjugated to the 3' end of B. In some embodiments, AX, YC, or The combination is conjugated to an internucleotide linkage group. The method further comprises D. In some embodiments, D is conjugated to C or A. In some embodiments, D is attached to a molecular conjugate of formula (II) according to formula (IV). R, (AXBYC c )-LD Formula IV During the ceremony, A is a binding moiety; B is a polynucleotide; C is a polymer; X is a bond or a first linker; Y is a bond or a second linker; L is a bond or a third linker; D is an endosomolytic moiety; and c is an integer between 0 and 1; wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one or at least one inverted abasic moiety, and and D is conjugated to any position of A, B, or C. In some embodiments, D is INF7 or melittin. , L is C 1 -C 6 In some embodiments, L is a homobifunctional linker. In some embodiments, the method further comprises: In some embodiments, the at least one binding moiety is a second binding moiety A. is conjugated to A, B, or C. In some embodiments, the method comprises In some embodiments, the method is an in vivo method. In some embodiments, the subject is a human.

[0006] In certain embodiments, a method for producing a medicament for use in a pharmaceutical composition comprising administering to said patient a therapeutically effective amount of said medicament, ... Disclosed is a pharmaceutical composition comprising a molecule and a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. The pharmaceutical compositions may be administered parenterally, orally, intranasally, bucally, rectally, or intravenously. Formulated for dermal administration.

[0007] In certain embodiments, compositions comprising the polynucleic acid molecule conjugates disclosed herein are provided. and the polynucleic acid molecule conjugate has at least 60 amino acids corresponding to SEQ ID NO:45-963. %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% %, 99%, or 100% sequence identity to the In certain embodiments, compositions comprising the polynucleic acid molecule conjugates disclosed herein are wherein the polynucleic acid molecule conjugate has a sequence similar to that of SEQ ID NO:45-963. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% % sequence identity. wherein the polynucleic acid molecule conjugate has 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 bond or a first linker; and Y is a 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 bond or a first linker; and Y is a bond or a second linker.

[0008] In certain embodiments, at least one 2' modified nucleotide is a morpholino, 2' -O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Aminopropanol Pyr, 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-Dimethylaminoethyloxy Diethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2 In one embodiment, at least one 2'-O-NMA) modified nucleotide is 'Modified nucleotides include morpholinos.

[0009] In one embodiment, a target region of the pre-mRNA transcript of the DMD gene is hybridized to the target region of the pre-mRNA transcript of the DMD gene. A polynucleic acid comprising a target cell binding moiety that binds to at least one polynucleic acid molecule that is to be soybean. Conjugates are disclosed herein, wherein at least one polynucleic acid molecule is a functional dystrophin. Transcribing the precursor-mRNA to generate an mRNA transcript encoding the lophin protein In some embodiments, the function is to induce splicing out of an exon from the product. Functional dystrophin protein is a truncated form of the dystrophin protein. In some embodiments, the target region is at an exon-intron junction, And the exon is the exon that is going to be spliced ​​out. In some embodiments, the exon is exon 8, 23, 35, 43, 44, 45, 50 , 51, 52, 53, or 55. A junction is located 5' of an exon that is to be spliced ​​out. In some embodiments, the target region is an intron upstream of an exon-intron junction. In some embodiments, the target region is an exon-intron region. Approx. 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides upstream In some embodiments, the exon-intron junction is a splice-out site. In some embodiments, the target exon is located 3' of the exon that is to be targeted. The target region is the intron region downstream of the exon-intron junction. In this embodiment, the target region is about 500, 450, 400, 300, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2 50, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40 In some embodiments, the target The target cell binding portion may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, or 8 or more polynuclear. In some embodiments, the polynucleic acid molecule is about 10 to about 50 nucleotides long. In some embodiments, the polynucleic acid molecule is a sequence of SEQ ID NO. For sequences selected from 964-1285, approximately 80%, 85%, 90%, 95%, In some embodiments, the sequence identity is 96%, 97%, 98%, or 99%. In the present invention, the polynucleic acid molecule is a nucleic acid sequence selected from SEQ ID NO: 964-1285. At least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2 In some embodiments, the polynuclear nucleic acid comprises 5, 26, or more contiguous bases. The acid molecule further comprises one, two, three, or four mismatches. In the present invention, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs: 1056-1094, 1147-116 2, or at least 10, 15, or 16 of a sequence selected from 1173-1211 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more neighbors In some embodiments, the polynucleic acid molecule comprises SEQ ID NO: At least 10, 15, 16, 17, or 1 of the sequence selected from 1056-1076 8, 19, 20, 21, 22, 23, 24, 25, 26, or more adjacent bases In some embodiments, the polynucleic acid molecule comprises SEQ ID NO:1077 At least 10, 15, 16, 17, 18, 19, or 20 of a sequence selected from -1094 Contains 20, 21, 22, 23, 24, 25, 26 or more contiguous bases. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1077-1094 At least 10, 15, 16, 17, 18, 19, 20, 21 of the nucleotide sequence selected from , 22, 23, 24, 25, 26, or more adjacent bases. In embodiments, the polynucleic acid molecule is selected from SEQ ID NOs: 1173-1211. At least 10, 15, 16, 17, 18, 19, 20, 21, 22, or 24 of the sequence In some embodiments, the sequence includes 3, 24, 25, 26, or more contiguous bases. In some embodiments, the binding moiety comprises an antibody. In some embodiments, the binding moiety comprises a plasma protein. In an embodiment, the polynucleic acid conjugate is A-(X 1 -B) n ; containing the formula (∨), where A comprises a binding moiety; B comprises a polynucleic acid molecule; X1 is a single bond or the 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 one embodiment, the guide strand comprises at least one modified internucleotide bond, at least One reverse abasic moiety, at least one 5'-vinylphosphonate modified non-natural nucleic acid In some embodiments, the nucleic acid comprises a nucleic acid sequence that is a nucleotide sequence, ... The do chain is modified with approximately 2, 3, 4, 5, 6, 7, 8, or 9 phosphorothioate residues. In some embodiments, the guide strand comprises one phospho In some embodiments, the nucleotide sequence includes a phosphatase-modified non-natural nucleotide. The phosphorothioate modified non-natural nucleotide is a nucleotide of a polynucleotide. In some embodiments, at least one 5'-vinylphospho The nate-modified non-natural nucleotides are located approximately 1, 2, 3, or 4 positions from the 5' end of the guide strand. , or 5 bases apart. In some embodiments, at least one 5'-vinylphosphonate modified unnatural nucleotides are further modified at the 2'-position. In some embodiments, the 2'-modification is 2'-O-methyl, 2'-O-methyl, Toxoethyl (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- O-Dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-O -N-methylacetamide (2'-O-NMA) modified nucleotides In some embodiments, the passenger strand has at least 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Sulfodiamidate morpholino oligomers contain modified non-natural nucleotides. In some embodiments, the passenger strand is 100% phosphorodiamidate morpholino In some embodiments, the oligomer comprises a modified non-natural nucleotide. The sensor strand is shorter in length than the guide strand, thereby forming a 5' overhang, a 3' In some embodiments, the nucleotide sequence of ... The passenger strand is equal in length to the guide strand, thereby providing a polynucleotide sequence at each end of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule is a phosphorodiamidin In some embodiments, the morpholino oligomer / RNA heteroduplex is The stimulator chain is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, Peptide nucleic acid modified non-natural nucleic acids having 16, 17, 18, 19, 20 or more In some embodiments, the passenger strand comprises 100% peptide. In some embodiments, the nucleic acid contains modified non-natural nucleotides. The guide strand is shorter than the 5' overhang, 3' overhang, and In some embodiments, the pass-through generates a The guide strand is equal in length to the guide strand, thereby creating blunt ends at each end of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule is a peptide nucleic acid / RNA heterodimer. In some embodiments, the passenger strand is AX 1 Conjugate to. In some embodiments, AX 1 is conjugated to the 5' end of the passenger strand. In some embodiments, AX 1 is conjugated to the 3' end of the passenger strand. In the embodiment of the present invention, X 1 is a single bond. In some embodiments, X 1 is C 1 - C 6 In some embodiments, X is an alkyl group. 1 is C 1 -C 6 Alkyl group Optionally, the linker may be a homobifunctional or heterobifunctional linker. In some embodiments, the polynucleic acid conjugate further comprises C. In some embodiments, C is polyethylene glycol. In some embodiments, C is X 2 Through B In some embodiments, X 2 is a single bond or a second non-polymeric linker. In some embodiments, X 2 is a single bond. In this state, X 2 is C 1 -C 6 In some embodiments, X is an alkyl group. 2 teeth , C 1 -C 6 A homobifunctional linker or heterobifunctional linker optionally attached to an alkyl group. In some embodiments, the passenger strand is a AX 1 and X 2 In some embodiments, AX 1is the 5' end of the passenger strand is combined with X 2 -C is conjugated to the 3' end of the passenger strand. Hey, X 2 -C is attached to the 5' end of the passenger strand, and AX 1 is a passenger In some embodiments, the polynucleic acid conjugate is AX 1 -( BX 2 -C) n Formula (VI): wherein A comprises a binding moiety; B comprises a polynucleic acid molecule; C is a polymer; X is 1 comprises a single bond or a first non-polymeric linker ;X 2 comprises a single bond or a second non-polymeric linker; and n is selected from 1-12. In some embodiments, the polynucleic acid conjugate further comprises D. In some embodiments, D is an endosomolytic moiety.

[0010] In a specific embodiment, SEQ ID NO: 1056-1058 or 1087 -1089, comprising at least 23 contiguous bases of a base sequence selected therefrom. Disclosed herein are polynucleic acid molecules comprising a length of 50 or less nucleotides.

[0011] In certain embodiments, the polynucleic acid sequence comprises SEQ ID NO:1056-1058. Disclosed herein are polynucleic acid molecules, wherein the polynucleic acid molecule comprises a length of 50 or less nucleotides.

[0012] In certain embodiments, the polynucleic acid sequence comprises SEQ ID NO:1087-1089. Disclosed herein are polynucleic acid molecules, wherein the polynucleic acid molecule comprises a length of 50 or less nucleotides.

[0013] In certain embodiments, a pharmaceutical composition is disclosed herein, the pharmaceutical composition comprising a polynucleic acid conjugate as described herein or a polynucleic acid molecule as described herein; and In some embodiments, the pharmaceutical composition is for systemic delivery and comprises a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. It is formulated.

[0014] In one embodiment, the subject is treated for a disease or disorder characterized by defective mRNA. Disclosed herein is a method, comprising: Induce exon skipping that causes a defective mRNA to generate a deleted mRNA To this end, the polynucleic acid conjugates described herein or the polynucleic acids described herein may be used. Administering a molecule to a subject, thereby treating a disease or disorder in the subject. In some embodiments, the disease or condition is a neuromuscular disease, a genetic disease, In some embodiments, the disease is a neuromuscular disease, cancer, a genetic disease, or a cardiovascular disease. In some embodiments, the disease is muscular dystrophy. Chenne muscular dystrophy, Becker muscular dystrophy, Facioscapulohumeral muscular dystrophy Fibe, congenital muscular dystrophy, or myotonic dystrophy. In embodiments, the subject is a human.

[0015] Provided herein, in certain embodiments, is a method for treating muscular dystrophy in a subject. The method is disclosed, which comprises the steps of: administering to the subject a polynucleic acid molecule that is capable of inhibiting muscular dystrophy in the subject, thereby In some embodiments, the muscular dystrophy is duodenal dystrophy. In some embodiments, the subject is a human.

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

[0017] In one embodiment, the composition obtained by any one of the methods disclosed herein is Disclosed herein are kits that include the [Brief description of the drawings]

[0018] [Figure 1] A phosphorodiamidate morpholino oligomer (PMO) sequence with extended terminal nucleotides is depicted. [Figure 2A] Phosphorothioate antisense oligonucleotide (PS ASO) sequences with terminal nucleotide extensions are 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. [Diagram 3] FIG. 1 depicts the method used to quantify skipped DMD mRNA in total RNA using Taqman qPCR. [Figure 4]FIG. 2 depicts a chromatogram of an anti-CD71 mAb-PMO reaction mixture produced by hydrophobic interaction chromatography (HIC) method 2. [Figure 5A] 1 depicts a chromatogram of anti-CD71 mAb generated using size exclusion chromatography (SEC) method 1. [Figure 5B] FIG. 2 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] 2 depicts a chromatogram of anti-CD71 mAb produced using hydrophobic interaction chromatography (HIC) method 2. [Figure 6B] FIG. 2 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 2 depicts a 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] 2 depicts a chromatogram of anti-CD71 Fab-PMO DAR1 conjugate produced using SEC method 1. [Figure 7D] 2 depicts a chromatogram of anti-CD71 Fab-PMO DAR2 conjugate produced using SEC method 1. [Figure 7E] 2 depicts a chromatogram of 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 anti-CD71 Fab-PMO DAR1 conjugate generated using HIC method 4. [Figure 7H] 1 depicts a chromatogram of anti-CD71 Fab-PMO DAR2 conjugate generated using HIC method 4. [Figure 7I] 1 depicts a chromatogram of anti-CD71 Fab-PMO DAR3 conjugate generated 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] Chromatogram of anti-CD71 mAb generated using SEC method 1 is depicted. [Figure 8C] 2 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR1 conjugate generated using SEC method 1. [Figure 8D] 2 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR2 conjugate generated using SEC method 1. [Figure 8E] 2 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR3 conjugate generated using SEC method 1. [Figure 8F] 1 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR1 conjugate generated using SAX method 2. [Figure 8G] 1 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR2 conjugate generated using SAX method 2. [Figure 8H] 1 depicts a chromatogram of anti-CD71 mAb-PS ASO DAR3 conjugate generated 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, anti-CD71 mAb-ASO conjugated to DAR1 ("ASC-DAR1"), anti-CD71 mAb-ASO conjugated to DAR2 ("ASC-DAR2"), and anti-CD71 mAb-ASO conjugated to DAR3 ("ASC-DAR3"). [Figure 12A] 1 depicts an agarose gel from nested PCR detecting exon 23 skipping in gastrocnemius muscle of wild-type mice receiving a single intravenous injection of anti-CD71 mAb-PMO conjugate. [Figure 12B] 13 is a graph of quantification of PCR products from gastrocnemius muscle. [Figure 12C] FIG. 13 is a graph of quantification of exon skipping in vivo 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] 13 is a graph of quantification of PCR products from cardiac muscle. [Figure 14] Figure 1 depicts sequencing data of DNA fragments from skipping and wild-type PCR products. [Figure 15] FIG. 1 illustrates the exon skipping activity of exon skipping PMOs of various lengths targeting exon 45 in 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 the hTfR1.mAb-PMO conjugate 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 cell myotubes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Nucleic acid (e.g., RNAi) therapy is a targeted therapy that boasts high selectivity and specificity. However, in some cases, nucleic acid therapeutics also suffer from poor cellular uptake and insufficient expression in target cells. To address these issues, nucleic acid Various modifications of the composition, e.g., new compounds for better stabilization and / or lower toxicity, New linkers, increased target specificity and / or optimization of the binding moiety for targeted delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects. is being explored.

[0020] In some instances, one such region in which oligonucleotides are used is a muscular dystrophy. Muscular dystrophies are a group of diseases that affect the muscles. Duchenne muscular dystrophy is a severe form of muscular dystrophy. It is caused by a mutation in the DMD gene. Mutations in the gene disrupt the translation reading frame and produce non-functional dystrophin. Provides protein.

[0021] In one embodiment, a nucleic acid sequence that is used to restore the translation reading frame is To induce exon skipping or exon inclusion, To induce insertions, deletions, duplications, or alterations in lysed mRNA transcripts Disclosed herein are methods and compositions relating to nucleic acid therapy. In order to treat a disease or disorder characterized by a misprocessed mRNA transcript, Also described herein are methods and compositions for the purpose of providing a method for the production of a polypeptide comprising the steps of: The polypeptide can encode a protein, thereby treating a disease or disorder. In some embodiments, pharmaceutical compositions and kits for treating the above diseases or disorders are provided herein. It will be published.

[0022] RNA processing RNA plays a central role in regulating gene expression and cell physiology. Processing is important for the translation of functional proteins. Alterations in RNA processing, such as those resulting from sequencing, can result in disease. Splice site mutations can result in exposure of premature stop codons, loss of exons, or insertion of endogenous codons. In some cases, changes in RNA processing lead to the inclusion of , resulting in insertions, deletions, or duplications. In some cases, alterations in RNA processing Alterations in RNA processing can result in the insertion, deletion, or duplication of exons. In some cases, this can result in the insertion, deletion, or duplication of introns.

[0023] Exon skipping Exon skipping is a form of RNA splicing. Either the nucleotide sequence is skipped in the processed mRNA or the nucleotide sequence is deleted in the processed mRNA. Exon skipping occurs when a gene is spliced ​​out of a target gene. As a result of the 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 inhibit exon skipping. In some instances, AONs are used to induce expression of a specific mRNA or AONs are short nucleic acid sequences that bind to pre-mRNA sequences. For example, AONs are splice sites or In some cases, it binds to specific mRNAs or exon enhancers. Binding of AONs to the RNA precursor sequence produces double-stranded regions. In some instances, the formation of the double-stranded region is associated with the spliceosome or a protein associated with the spliceosome. This occurs at the site where the protein would normally bind, causing exon skipping. In this example, exon skipping caused restoration of the transcript reading frame. , allowing the production of a partially functional protein.

[0025] Exon inclusion In some cases, mutations in the RNA lead to exon skipping. Thus, mutations may occur at, near, or at splice sites. In some instances, the mutation is at least one location away from the Inactivation or weakening of splice sites, exon splice enhancers or introns Disruption of splice enhancers, and exon splice silencers or intros In some instances, the method results in at least one of the following: Mutations alter RNA secondary structure. In some cases, mutations alter RNA secondary structure. This disrupts the accessibility of signals important for exon recognition.

[0026] In some instances, the use of AONs may allow for the inclusion of skipped exons. In some instances, AONs may be located at or near splice sites. Optionally, the AO binds to at least one of the sites distant from the splice site. N indicates the presence of exon or intron splice enhancers. In some cases, AONs bind to exons in the RNA to prevent destruction. To prevent the generation of splice silencers or intronic splice silencers, It binds to a certain site in RNA.

[0027] index In some embodiments, the polynucleic acid molecules or pharmaceutical compositions described herein The method is used to treat a disease or disorder characterized by defective mRNA. In one embodiment, the polynucleic acid molecules or pharmaceutical compositions described herein may be mis-spliced ​​to induce exon pruning or exon inclusion by inducing insertions, deletions, duplications, or alterations in mRNA transcripts, resulting in disease or is used to treat disorders.

[0028] The majority of human protein-coding genes are alternatively spliced. In the example, the mutations were either improperly spliced ​​or partially spliced. For example, mutations can cause RNA mutations in protein-coding genes, silencers or The sequence of the splice site in the enhancer, exon, or intron sequence is In some cases, the mutation causes a gene to malfunction. In some instances, the mutation causes a disease or disorder.

[0029] In some cases, it is improperly spliced ​​or partially spliced Diseases or disorders caused by mRNA include, but are not limited to, neuromuscular diseases, genetic diseases, and cancer. , genetic disorders, or cardiovascular diseases.

[0030] In some instances, the genetic disease or disorder may be an autosomal dominant disorder, an autosomal recessive disorder, X-linked dominant, X-linked recessive, Y-linked, mitochondrial, or multifactorial or polygenic disorders.

[0031] In some cases, cardiovascular diseases such as hypercholesterolemia are caused by improper splicing. This is due to mismatched or partially spliced ​​mRNA. A monosodium salt in exon 12 of the low-density lipoprotein receptor (LDLR) in rheumatoid arthritis Base polymorphisms have been shown to promote exon skipping.

[0032] In some cases, it is improperly spliced ​​or partially spliced mRNA can cause cancer. For example, it can be improperly spliced ​​or partially spliced. Lysed mRNAs can be used to regulate a variety of physiological functions, including, but not limited to, proliferation, motility, and drug response. In some cases, it affects cellular processes involved in solid tumors or hematologic cancers. In some instances, the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-Hodgkin's Breast cancer, 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, it is improperly spliced ​​or partially spliced The mRNA causes a neuromuscular disease or disorder. Exemplary neuromuscular diseases include Duchenne Becker muscular dystrophy, Facioscapulohumeral muscular dystrophy , congenital muscular dystrophy, or myotonic dystrophy In some instances, the muscular dystrophy is genetic. Muscular dystrophies are caused by spontaneous mutations. Becker muscular dystrophy and Duchenne muscular dystrophy, which are caused by the DMD gene that encodes the protein dystrophin. Mutations in the gene have been shown to be involved in facioscapulohumeral muscular dystrophy. has been shown to be associated with mutations in the double homeobox 4 (DUX4) gene. There are.

[0034] In some cases, it is improperly spliced ​​or partially spliced mRNA causes Duchenne muscular dystrophy. Duchenne muscular dystrophy Fie is a type of DMD that causes severe muscle weakness and abolishes the production of functional dystrophin. It is caused by a mutation in a gene. In some cases, Duchenne muscular dystrophy Trophies are the result of mutations in exons in the DMD gene. In some instances, Duchenne muscular dystrophy is caused by the deletion of exons 1, 2, 3, 4, 5, and 6 in the DMD gene. , 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3 0, 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, 7 At least one of 0, 71, 72, 73, 74, 75, 76 77, 78, and 79 In some cases, Duchenne muscular dystrophy is the result of a genetic mutation. 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, 3 8, 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 It is the result of at least one mutation. In some cases, it is called Duchenne muscular dystrophy. Fee has been identified in exons 8, 23, 35, 43, 44, 45, 50, 51, and 60 of the DMD gene. 52, 53, and / or 55. Many exons are mutated. For example, mutations in exons 48-50 cause Duchenne In some cases, Duchenne muscular dystrophy is more common in people with The strophy is the result of a mutation in exon 51. In some cases, Duchenne Type 2 muscular dystrophies are the result of mutations in exon 23. In some cases, the mutation The mutation may include a deletion of one or more exons. In some instances, the mutation may include a deletion of one or more exons. In some cases, the mutation involves an exon-specific point mutation. For example, some patients harbor nonsense point mutations in exon 51 of the DMD gene. It has been shown that some people are

[0035] In some instances, the polynucleic acid molecules or pharmaceutical compositions described herein are useful in treating muscular dysplasia. In some instances, the polynucleic acid molecules described herein are used to treat trophies. Alternatively, the pharmaceutical composition may be used for treating Duchenne muscular dystrophy, Becker muscular dystrophy, , facioscapulohumeral muscular dystrophy, congenital muscular dystrophy or myotonic dystrophy In some instances, the polynucleic acid molecules described herein are used to treat Alternatively, the pharmaceutical composition is used for the treatment of Duchenne muscular dystrophy.

[0036] Polynucleic acid molecule In some embodiments, exon skipping or exon inclusion Insertions, deletions, duplications, and deletions of misspliced ​​mRNA transcripts induce Alternatively, polynucleic acid molecules that induce the changes are described herein. The nucleic acid molecule restores the translation reading frame. In some instances, the polynucleic acid molecule results in a functional and truncated protein.

[0037] In some instances, the polynucleic acid molecule targets an mRNA sequence. In some instances, the polynucleic acid molecule targets a splice site. Targeting a factor In some instances, the polynucleic acid molecule targets a transregulatory factor. In some instances, the polynucleic acid molecule comprises an exon splice enhancer or an intron splice enhancer. In some instances, the polynucleic acid molecule targets an exon splice enhancer. Targeting rice silencers or intron splice silencers.

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

[0039] As described above, polynucleic acid molecules can be used to treat, but are not limited to, neuromuscular diseases, genetic diseases, cancer, Misprocessed proteins that lead to diseases or disorders, such as genetic or cardiovascular diseases In some cases, the polynucleic acid molecule may be used to treat a neuromuscular or Targets the misprocessed mRNA transcripts that cause the disorder. In this regard, the neuromuscular disease or disorder is Duchenne muscular dystrophy, Becker muscular dystrophy, muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or muscle contraction Optionally, the polynucleic acid molecule is a nucleic acid molecule that is capable of inhibiting the expression of a gene that is involved in Duchenne muscular dystrophy. Roffey, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy misprocessed proteins that cause myotonic dystrophy or myotonic dystrophy In some cases, the polynucleic acid molecule targets an mRNA transcript. It targets the misprocessed mRNA transcripts that cause the strophy.

[0040] In some instances, the polynucleic acid molecule is an antibody that encodes a gene encoding a D dystrophy gene that causes Duchenne muscular dystrophy. Targeting the mutated exon in the MD gene. Exemplary exons that are mutated in the causative DMD gene include, but are not limited to, 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, 5 2, 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 instances, the polynucleic acid molecule may include a mutated exon adjacent For example, if there is a deletion of exon 50, exon 51 is skipped. As shown, the polynucleic acid molecule targets a sequence in exon 51. If there is a mutation in exon 23, the polynucleotide sequence is modified so that exon 23 is skipped. The offspring targets a sequence in exon 22.

[0041] In some examples, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to exon 2 of the DMD gene. , 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, 5 4, 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 exos In some instances, the method targets a region at an intron-intron junction. The polynucleic acid molecule is a nucleic acid sequence encoding exons 3, 4, 5, 6, 7, 18, 19, 20, and 21 of the DMD gene. 1, 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, 6 Targeting regions at exon-intron junctions 1, 62, or 63. In one example, the polynucleic acid molecule described herein is selected from exons 8, 23, 3 and 4 of the DMD gene. 5, 43, 44, 45, 50, 51, 52, 53, or 55 exon-intron junctions In some cases, the polynucleic acid molecules described herein target a region at the junction. Targets the region at the exon-intron junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein may comprise a nucleic acid sequence encoding exon 23 of the DMD gene. In some cases, the method targets a region at an intron-intron junction. The polynucleic acid molecule is 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 the DMD gene. Targeting the region at the exon-intron junction of exon 43 of The polynucleic acid molecules described herein comprise an exon-intron sequence of exon 44 of the DMD gene. In some cases, the polynucleic acid described herein targets a region at the thoron junction. The molecule targets a region at the exon-intron junction of exon 45 of the DMD gene. In some cases, the polynucleic acid molecules described herein may be selected from the group consisting of exons of the DMD gene. 48 exon-intron junctions. The polynucleic acid molecule described in is an exon-intron junction of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located in the D Targets the region at the exon-intron junction of exon 50 of the MD gene. In some cases, the polynucleic acid molecules described herein may comprise a nucleic acid sequence encoding exon 51 of the DMD gene. In some cases, the method targets a region at an intron-intron junction. The polynucleic acid molecule is 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 the DMD gene. Targeting the region at the exon-intron junction of exon 53 of The polynucleic acid molecules described herein comprise an exon-intron sequence of exon 55 of the DMD gene. It targets a region at the Tron junction.

[0042] In some instances, the polynucleic acid molecule comprises exons 2, 3, 4, 5, 6, 7 of the DMD gene. , 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, 4 4, 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, At least one 5' end of 71, 72, 73, 74, 75, 76, 77, and 78 The hybridization sequence is located at the 3' exon-intron junction or 3' exon-intron junction. In some examples, the polynucleic acid molecule is a nucleic acid molecule that ligates exons 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, , 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, 5 7, 58, 59, 60, 61, 62, and at least one 5' intron of 63 Hybridizes to target regions at exon junctions or 3' exon-intron junctions In some examples, the polynucleic acid molecule comprises a nucleic acid sequence encoding exons 8, 23, 35, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 43, 44, 45, 50, 51, 52, 53, or 55 5' intron-exon junctions It hybridizes to a target region at the junction or 3' exon-intron junction.

[0043] In some cases, the polynucleic acid molecule comprises a nucleic acid sequence selected from exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 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, 7 At least one 5' intro of 1, 72, 73, 74, 75, 76, 77, and 78 hybridizes to a target region at a 5' end of exon junction (e.g., the 5' end of exon 3) Intron-exon junctions are the junctions of intron 2 and exon 3. In accordance with the present invention, the polynucleic acid molecule is selected from exons 3, 4, 5, 6, 7, 18, 19, and 20 of the DMD gene. , 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, 4 7, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, and 63, which have target regions in at least one 5' intron-exon junction. (e.g., the 5' intron-exon junction of exon 3 is In some cases, the polynucleic acid molecule is a nucleic acid sequence that is a sequence that is a junction of exon 2-exon 3 of the DMD gene. Exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 5 of the gene 5 hybridizes to a target region at the 5' intron-exon junction of In one embodiment, the polynucleic acid molecule is a 5' intron-exon junction of exon 8 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to a target region of the DMD gene. It hybridizes to a target region at the 5' intron-exon junction of exon 23. Optionally, the polynucleic acid molecule comprises a 5' intron-exon of exon 35 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to a target region at the Son junction. Hybridizes to a target region in the 5' intron-exon junction of exon 43 of the MD gene. Optionally, the polynucleic acid molecule is a 5' end of exon 44 of the DMD gene. Hybridizes to a target region at a tron-exon junction. The acid molecule targets the target region at the 5' intron-exon junction of exon 45 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to the exon 5 of the DMD gene. 0. In one embodiment, the polynucleic acid molecule is a 5' intron-exon junction of exon 51 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to a target region in the DMD gene. Hybridizes to a target region at the 5' intron-exon junction of exon 52 of Optionally, the polynucleic acid molecule comprises a 5' intron-encoding region of exon 53 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to a target region at a chon junction. The target region located at the 5' intron-exon junction of exon 55 of the DMD gene was hybridized to Resize.

[0044] In some cases, the polynucleic acid molecule comprises a nucleic acid sequence selected from exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 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, 7 At least one 3' exon of 1, 72, 73, 74, 75, 76, 77, and 78 - hybridizes to a target region at an intron junction (e.g., the 3' exon of exon 3) (The exon-intron junction is the exon 3-intron 3 junction.) For example, the polynucleic acid molecule may be a nucleic acid sequence encoding 3, 4, 5, 6, 7, 18, 19, 20, 21, or 22 of the DMD gene. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3 5, 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 hybridized to the target region at at least one 3' exon-intron junction. (e.g., the 3'-intron junction of exon 3 is hybridized with the exon 3-intron In some cases, the polynucleic acid molecule is a nucleic acid molecule that is a nucleic acid sequence that is a junction of exon 8 of the DMD gene. , 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 3' exons -hybridizes to a target region at an intron junction. The offspring targeted the target region at the 3' exon-intron junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to the 3′ end of exon 23 of the DMD gene. 'Hybridizes to a target region at an exon-intron junction. The polynucleic acid molecule is located at the 3' exon-intron junction of exon 35 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to the target region of the DMD gene. It hybridizes to a target region at the 3' exon-intron junction of gene 43. In some cases, the polynucleic acid molecule is a 3' exon-intron sequence of exon 44 of the DMD gene. The polynucleic acid molecule hybridizes to a target region at the junction. Hybridizes to a target region located at the 3' exon-intron junction of exon 45 of the gene Optionally, the polynucleic acid molecule comprises a 3' exon of exon 50 of the DMD gene. -hybridizes to a target region at an intron junction. The offspring targeted the target region at the 3' exon-intron junction of exon 51 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to exon 52 of the DMD gene. Hybridizes to a target region at the 3' exon-intron junction. The polynucleic acid molecule is located at the 3' exon-intron junction of exon 53 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to a target region of the DMD gene. It hybridizes to a target region at the 3' exon-intron junction of xon 55.

[0045] In some examples, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to exon 2 of the DMD gene. , 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, 5 4, 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 Splats In some examples, the polynucleic acid molecules described herein target a DM Exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24 of the D gene 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 3 8, 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 In some instances, the polynucleic acid molecules described herein target splice sites. , exons 8, 23, 35, 43, 44, 45, 50, 51, 52, and 53 of the DMD gene 55 splice site. The nucleic acid molecule targets a splice site in exon 8 of the DMD gene. The polynucleic acid molecule described herein is a polynucleotide that encodes a splice site of exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region of the DMD gene. Optionally, the method further comprises targeting the exon 35 splice site of The polynucleic acid molecule targets a splice site in exon 43 of the DMD gene. In particular, the polynucleic acid molecules described herein are intended to identify a splice of exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the DMD gene. Optionally, the method further comprises targeting a splice site in exon 45 of the gene. The polynucleic acid molecule targets the splice site of exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein may be a splice of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the DM Optionally, the method targets the splice site of exon 50 of the D gene. The carried polynucleic acid molecule targets a splice site in exon 51 of the DMD gene. Optionally, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to that of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites. Targeting the splice site of exon 53 of the DMD gene. The polynucleic acid molecule described in is a polynucleotide that targets the splice site of exon 55 of the DMD gene. As used herein, a splice site refers to an exon skipping or Incorrectly spliced ​​mRNA transcripts to induce son inclusion canonical splice sites, cryptic splice sites, which can induce insertions, deletions, duplications, or alterations of The present invention includes alternative splice sites, alternative splice sites, and alternative splice sites.

[0046] In some embodiments, the polynucleic acid molecule comprises exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 3 2, 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, Additional factors implicated in Duchenne muscular dystrophy, such as 60, 61, 62, or 63 It targets a partially spliced ​​mRNA sequence that contains the exon.

[0047] In some instances, the polynucleic acid molecule comprises a nucleic acid sequence that encodes a polypeptide that is hybridized to a region proximal to an exon-intron junction. In some examples, the polynucleic acid molecules described herein hybridize to a DMD gene. 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, 3 8, 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 at least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, Targeting regions 20nt, 10nt, or 5nt upstream (or 5' from) In some examples, the polynucleic acid molecules described herein comprise a nucleic acid sequence encoding exons 3 and 4 of the DMD gene. , 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, 4 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, or 63 at least 1,000 Leutide (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 8 0nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5n In some instances, the targeting of the region upstream (or 5') of the The polynucleic acid molecule identified is a nucleotide sequence encoding exons 8, 23, 35, 43, 44, 45, and 50 of the DMD gene. , 51, 52, 53, or 55 at least 1000 nt, 500 nt, 400 nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30 Targeting a region nt, 20nt, 10nt, or 5nt upstream (or 5') In some examples, the polynucleic acid molecules described herein comprise an exon of the DMD gene. At least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt or a region 5 nt upstream (or 5' from) the target. The polynucleic acid molecules described herein comprise at least 10 sequences of exon 23 of the DMD gene. 00nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream ( In some instances, the method may target a region located 5' to the target nucleic acid sequence described herein. The polynucleic acid molecule may comprise at least 1000 nt, 500 nt, or 1000 nt of exon 35 of the DMD gene. 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 4 0 nt, 30 nt, 20 nt, 10 nt, or 5 nt upstream (or 5') In some examples, the polynucleic acid molecules described herein target a region that is involved in DMD. At least 1000nt, 500nt, 400nt, or 300nt of exon 43 of the gene , 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20 It targets a region that is nt, 10nt, or 5nt upstream (or 5'). In some examples, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to that of exon 44 of the DMD gene. At least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt t, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or In some instances, the targeting gene may target a region 5 nt upstream (or 5') of the target gene. The polynucleic acid molecule described herein comprises at least 1000 nt of exon 45 of the DMD gene. , 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt , 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or In some instances, the polynucleic acid described herein targets a region located 5' from the The molecule comprises at least 1000 nucleotides (nt), 50 nucleotides (nt) of exon 48 of the DMD gene. 0nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50 nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5' In some instances, the polynucleic acid molecules described herein target a region in , at least 1000 nucleotides (nt) of exon 49 of the DMD gene, 500 nt , 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') In some instances, the polynucleic acid molecules described herein target a region. At least 1000 nucleotides (nt), 500 nt, 40 nt, or 100 nt of exon 50 of the D gene 0nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40n t, 30nt, 20nt, 10nt, or 5nt upstream (or 5') In some examples, the polynucleic acid molecules described herein target the DMD gene. At least 1000 nucleotides (nt), 500 nt, or 400 nt of exon 51 of the offspring , 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 3 Target the region 0nt, 20nt, 10nt, or 5nt upstream (or 5'). In some examples, the polynucleic acid molecules described herein target the end of the DMD gene. At least 1000 nucleotides (nt), 500 nt, 400 nt, 30 nt, or 0nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt , targeting a region 20nt, 10nt, or 5nt upstream (or 5' from) In some examples, the polynucleic acid molecules described herein are selected from the group consisting of exons of the DMD gene. 53 at least 1000nt, 500nt, 400nt, 300nt, 200nt, 1 00nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or targeting a region 5 nt upstream (or 5'). The polynucleic acid molecules described herein comprise at least 100 copies of exon 55 of the DMD gene. 0nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 6 0nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or The target region is located 5' or 6' from the target gene.

[0048] In some instances, the polynucleic acid molecule comprises exons 2, 3, 4, 5, 6, 7 of the DMD gene. , 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, 4 4, 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, At least one upstream of 71, 72, 73, 74, 75, 76, 77, and 78 (a In some examples, the polynucleic acid molecule hybridizes to a target region located at Exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, and 2 of the DMD gene 4, 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 Hybridizes to at least one upstream (or 5') of the target region. In an example, the polynucleic acid molecule is selected from exons 8, 23, 35, 43, 44, 45, 60, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, A target located upstream (or 5') of at least one of 50, 51, 52, 53, or 55 In some examples, the polynucleic acid molecule hybridizes to a region of the exonucleic acid sequence of the DMD gene. 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, 5 5, 56, 57, 58, 59, 60, 61, 62, and 63. , 10, 15, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') to a target region.

[0049] In some examples, the polynucleic acid molecules described herein comprise exons in the DMD gene. 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2 7, 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, 6 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 At most 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt t, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10 nt, or 5 nt downstream (or 3' from) the region. The polynucleic acid molecules described herein are selected from exons 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 1, 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, At least 1000 nucleotides (nt 58, 59, 60, 61, 62, or 63 ), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60n t, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or In some instances, the polynucleic acid molecules described herein target a region 3' to the The child has exons 8, 23, 35, 43, 44, 45, 50, 51, 52, and 6 of the DMD gene. 53, or 55 at least 1000 nucleotides (nt), 500 nt, 400 nt , 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 3 Targeting the region 0nt, 20nt, 10nt, or 5nt downstream (or 3') In some examples, the polynucleic acid molecules described herein comprise exons of the DMD gene. At least 1000 nucleotides (nt), 500 nt, 400 nt, 300 nt of sequence 8 , 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of the target region. In some examples, the polynucleic acid molecules described herein comprise at least one sequence encoding exon 23 of the DMD gene. At most 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt t, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10 nt or 5 nt downstream (or 3'). The polynucleic acid molecules described herein comprise at least 10 sequences of exon 35 of the DMD gene. 00 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100 nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, In some instances, the target region is a region 5 nt downstream (or 3') of the target gene. The polynucleic acid molecule described in is a polynucleotide that confers at least 1000 nucleotides of exon 43 of the DMD gene. Otid (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80 nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt In some instances, the downstream (or 3') region of the target gene can be targeted to a region of the target gene. The polynucleic acid molecule comprises at least 1000 nucleotides (n) of exon 44 of the DMD gene. t), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60 nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (some In some instances, the polynucleic acid described herein targets a region of the nucleic acid sequence (e.g., 3' to 3'). The molecule comprises at least 1000 nucleotides (nt), 50 nucleotides (nt) of exon 45 of the DMD gene. 0nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50 nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3' In some examples, the polynucleic acid molecules described herein target a region of the D At least 1000 nucleotides (nt), 500 nt, or 4 nt of exon 48 of the MD gene 00nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40 nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') In some examples, the polynucleic acid molecules described herein target the DMD gene. At least 1000 nucleotides (nt), 500 nt, 400 nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30 Targeting regions nt, 20nt, 10nt, or 5nt downstream (or 3') In some examples, the polynucleic acid molecules described herein are selected from the group consisting of exons of the DMD gene. 50 of at least 1000 nucleotides (nt), 500nt, 400nt, 300nt , 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20 nt, 10 nt, or 5 nt downstream (or 3') of the target region. In some examples, the polynucleic acid molecules described herein comprise at least one sequence encoding exon 51 of the DMD gene. At most 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt t, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10 nt or 5 nt downstream (or 3'). The polynucleic acid molecules described herein comprise at least 10 sequences of exon 52 of the DMD gene. 00 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100 nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, In some instances, the target region is a region 5 nt downstream (or 3') of the target gene. The polynucleic acid molecule described in is a polynucleotide comprising at least 1000 nucleotides of exon 53 of the DMD gene. Otid (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80 nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt In some instances, the downstream (or 3') region of the target gene can be targeted to a region of the target gene. The polynucleic acid molecule comprises at least 1000 nucleotides (n) of exon 55 of the DMD gene. t), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60 nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (some The target region is the 3' or 4' end of the ribosome.

[0050] In some instances, the polynucleic acid molecule comprises exons 2, 3, 4, 5, 6, 7 of the DMD gene. , 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, 4 4, 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, At least one downstream of 71, 72, 73, 74, 75, 76, 77, and 78 (a In some instances, the polynucleic acid molecule hybridizes to a target region 3' or 3' of the DNA. Exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24 of the D gene 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 3 8, 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 In some instances, the target region hybridizes to at least one downstream (or 3') target region. The polynucleic acid molecule is selected from exons 3, 4, 5, 6, 7, 18, 19, 20, and 21 of the DMD gene. , 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, 4 8, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , and 63 at least one of about 5, 10, 15, 20, 50, 100, 200, 3 Hybridize to the target region 00, 400, or 500 bp downstream (or 5') In some examples, the polynucleic acid molecule is selected from exons 8, 23, 35, 43, and 50 of the DMD gene. , 44, 45, 50, 51, 52, 53, or 55. 5, 20, 50, 100, 200, 300, 400, or 500 bp downstream (or 3') hybridizes to the target region.

[0051] In some instances, the polynucleic acid molecule comprises exons 2, 3, 4, 5, 6, 7 of the DMD gene. , 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, 4 4, 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, Targeting internal regions within 71, 72, 73, 74, 75, 76, 77, or 78. In some examples, the polynucleic acid molecules described herein comprise exon 3 of the DMD gene. 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, 5 Targeting internal regions within 5, 56, 57, 58, 59, 60, 61, 62, or 63 In some examples, the polynucleic acid molecules described herein comprise a nucleic acid sequence encoding exon 8 of the DMD gene. , 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 In some examples, the polynucleic acid molecules described herein target the DMD gene. Targeting an internal region within exon 8. In some instances, the polynuclear The acid molecule targets an internal region within exon 23 of the DMD gene. In some instances, 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 comprise an exon of the DMD gene. In some instances, the polynucleic acid described herein targets an internal region within Son 43. The molecule targets an internal region within exon 44 of the DMD gene. The polynucleic acid molecule described herein targets an internal region within exon 45 of the DMD gene. In some examples, the polynucleic acid molecules described herein comprise exons of the DMD gene. In some instances, the polynucleic acid molecules described herein target an internal region within region 48. The molecule targets an internal region within exon 49 of the DMD gene. 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 are selected from the group consisting of exons of the DMD gene. In some examples, the polynucleic acid molecule described herein targets an internal region within 51. In some instances, the present invention targets an internal region within exon 52 of the DMD gene. The described polynucleic acid molecule targets an internal region within exon 53 of the DMD gene. In some examples, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to exon 5 of the DMD gene. Targeting the internal area within 5.

[0052] In some instances, the polynucleic acid molecule comprises exons 2, 3, 4, 5, 6, 7 of the DMD gene. , 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, 4 4, 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 at least one of the target regions In some examples, the polynucleic acid molecule hybridizes to exon 3 of the DMD gene. 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, 5 6, 57, 58, 59, 60, 61, 62, and / or 63 In some examples, the polynucleic acid molecule hybridizes to the target region of the DMD gene. 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 Each hybridization sequence hybridizes to at least one internal target region.

[0053] In some embodiments, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to that of the DMD gene. Targets partially spliced ​​mRNA sequences that contain exon 44. In this example, the polynucleic acid molecule is a nucleic acid sequence that is nucleotide sequence specific for the target region upstream (or 5') to exon 44. In some examples, the polynucleic acid molecule hybridizes to about 5, 10, 1 5, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of the target region. In some examples, the polynucleic acid molecule 44. In some instances, , the polynucleic acid molecule is about 5, 10, 15, 20, 50, 100, 200, 3 Hybridize to the target region 00, 400, or 500 bp downstream (or 3') do.

[0054] In some instances, the polynucleic acid molecule is a nucleic acid sequence that is specific to a target region within exon 44 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to the 5' intron-exon 4 Hybridizes to target regions at the 4 junction or the 3' exon 44-intron junction do.

[0055] In some embodiments, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to that of the DMD gene. Targets partially spliced ​​mRNA sequences that contain exon 45. In this example, the polynucleic acid molecule is a nucleic acid sequence that is nucleotide sequence specific for the target region upstream (or 5') to exon 45. In some examples, the polynucleic acid molecule hybridizes to about 5, 10, 1 5, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of the target region. In some examples, the polynucleic acid molecule 45. In some instances, , the polynucleic acid molecule is about 5, 10, 15, 20, 50, 100, 200, 3 Hybridize to the target region 00, 400, or 500 bp downstream (or 3') do.

[0056] In some instances, the polynucleic acid molecule is a nucleic acid sequence that is specific to a target region within exon 45 of the DMD gene. In some examples, the polynucleic acid molecule hybridizes to the 5' intron-exon 4 Hybridizes to target regions at the 5' junction or the 3' exon 45-intron junction do.

[0057] In some embodiments, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to that of the DMD gene. Targets partially spliced ​​mRNA sequences that contain exon 51. In this example, the polynucleic acid molecule is a nucleic acid sequence that is nucleotide sequence specific for the target region upstream (or 5') to exon 51. In some examples, the polynucleic acid molecule hybridizes to about 5, 10, 1 5, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of the target region. In some examples, the polynucleic acid molecule 51. In some instances, , the polynucleic acid molecule is about 5, 10, 15, 20, 50, 100, 200, 3 Hybridize to the target region 00, 400, or 500 bp downstream (or 3') do.

[0058] In some instances, the polynucleic acid molecule is a nucleic acid sequence that is specific to a target region within exon 51 of the DMD gene. Optionally, the polynucleic acid molecule hybridizes to the 5' intron-exon 51 Hybridizes to a target region at the junction or 3' exon 51-intron junction do.

[0059] In some embodiments, the polynucleic acid molecules described herein comprise a nucleic acid sequence similar to that of the DMD gene. Targets partially spliced ​​mRNA sequences that contain exon 53. In this example, the polynucleic acid molecule is a nucleic acid sequence that is nucleotide sequence specific for the target region upstream (or 5') to exon 53. In some examples, the polynucleic acid molecule hybridizes to about 5, 10, 1 5, 20, 50, 100, 200, 300, 400, or 500 bp upstream (or 5') of the target region. In some examples, the polynucleic acid molecule 53. In some instances, , the polynucleic acid molecule is about 5, 10, 15, 20, 50, 100, 200, 3 Hybridize to the target region 00, 400, or 500 bp downstream (or 3') do.

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

[0061] In some embodiments, the polynucleic acid molecule has at least 5'-nucleotide sequence identity to a target sequence of interest. 0%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 9 The sequences may share 6%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the polynucleic acid molecule is at least 50% identical to the target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to the target In some embodiments, the sequence has at least 60% sequence identity to the target sequence. In one embodiment, the polynucleic acid molecule has at least 70% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having a target sequence of interest. In some embodiments, the sequence has at least 75% sequence identity to The polynucleic acid molecule is a sequence having at least 80% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises at least one nucleic acid sequence that is complementary to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having 85% sequence identity with the The fragment contains a sequence having at least 90% sequence identity to the target sequence of interest. In some embodiments, the polynucleic acid molecule has at least 95% homology to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to the target nucleic acid. The target sequence includes a sequence having at least 96% sequence identity to the target sequence. In some embodiments, the polynucleic acid molecule has at least 97% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence that is complementary to a target sequence of interest. In some embodiments, the sequence of the present invention includes a sequence having at least 98% sequence identity to the The polynucleic acid molecule comprises a sequence having at least 99% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule consists of a 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 nucleotide sequence that matches a target sequence of interest. and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9 0%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some examples, the second polynucleotide comprises a sequence that is complementary to a target sequence of interest. At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some instances, the polynucleic acid molecule comprises at least 50 nucleotides to a target sequence of interest. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99%, or 100% sequence identity to a first polynucleotide. The octide and the target sequence of interest are at least 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or comprises a second polynucleotide with 100% sequence identity.

[0063] In some embodiments, the polynucleic acid molecule is SEQ ID NO:964-128 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% for 5 %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N O:964-1285. In some embodiments, the polynucleic acid molecule is a nucleic acid sequence similar to SEQ ID NO:964-1285. In some embodiments, the sequence of the present invention includes a sequence having at least 60% sequence identity to the The polynucleic acid molecule has at least 70% sequence identity to SEQ ID NO:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Contains sequences having at least 75% sequence identity to ID NO:964-1285. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NO:964-12 In some embodiments, the sequence has at least 80% sequence identity to . In the present invention, the polynucleic acid molecule has at least 8 sequences corresponding to SEQ ID NO:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having 5% sequence identity to the Has at least 90% sequence identity to SEQ ID NO:964-1285 In some embodiments, the polynucleic acid molecule comprises the sequence SEQ ID NO:96 4-1285. In an embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:964-1285. In some embodiments, the polynucleic acid comprises a sequence having at least 96% sequence identity. The molecule has at least 97% sequence identity to SEQ ID NO:964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N O:964-1285. In some embodiments, the polynucleic acid molecule is a nucleic acid sequence similar to SEQ ID NO:964-1285. In some embodiments, the polynucleotide comprises a sequence having at least 99% sequence identity to the polypeptide. The nucleic acid molecule consists of SEQ ID NO:964-1285.

[0064] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1056-10 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8% of 94 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. Contains sequences having at least 50% sequence identity to NO:1056-1094. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1056-109 In some embodiments, the sequence has at least 60% sequence identity to 4. In this case, the polynucleic acid molecule has at least 7 sequences corresponding to SEQ ID NOs: 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1056-1094 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. Contains sequences with at least 80% sequence identity to 056-1094. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1056-1094. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO:1056-1094 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO: 1056- 1094. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NO:1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1056-1094. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1056-1094. become.

[0065] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1147-11 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 for 62 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. It includes sequences having at least 50% sequence identity to NO:1147-1162. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1147-116 In some embodiments, the sequence has at least 60% sequence identity to In this case, the polynucleic acid molecule has at least 7 sequences identical to SEQ ID NO:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1147-1162 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. 147-1162. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1147-1162. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO:1147-1162 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO:1147- 1162. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NOs: 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1147-1162. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1147-1162. become.

[0066] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1173-12 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 for 11 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. Contains sequences having at least 50% sequence identity to NO:1173-1211. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1173-121 In some embodiments, the sequence of the present invention includes a sequence having at least 60% sequence identity to . In this case, the polynucleic acid molecule has at least 7 sequences corresponding to SEQ ID NOs: 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1173-1211 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. 173-1211. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1173-1211. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO:1173-1211 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO:1173- 1211. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NOs: 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1173-1211. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1173-1211. become.

[0067] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1056-10 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 for 76 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. Contains sequences having at least 50% sequence identity to NO:1056-1076. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1056-107 In some embodiments, the sequence has at least 60% sequence identity to 6. In this case, the polynucleic acid molecule has at least 7 sequences corresponding to SEQ ID NOs: 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1056-1076 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. Contains sequences with at least 80% sequence identity to 056-1076. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1056-1076. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO:1056-1076 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO: 1056- 1076. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NO:1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1056-1076. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1056-1076. become.

[0068] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1077-10 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8% of 94 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. Contains sequences having at least 50% sequence identity to NO:1077-1094. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1077-109 In some embodiments, the sequence has at least 60% sequence identity to 4. In this case, the polynucleic acid molecule has at least 7 sequences corresponding to SEQ ID NO:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1077-1094 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. 077-1094. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1077-1094. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO:1077-1094 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO: 1077- 1094. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NO:1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1077-1094. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1077-1094. become.

[0069] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1056-10 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 8 for 58 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. It includes sequences having at least 50% sequence identity to NO:1056-1058. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1056-105 8. In some embodiments, the sequence of the present invention is a sequence having at least 60% sequence identity to In this case, the polynucleic acid molecule has at least 7 sequences corresponding to SEQ ID NOs: 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1056-1058 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. 056-1058. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1056-1058. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO: 1056-1058 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO: 1056- 1058. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NO:1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1056-1058. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1056-1058. become.

[0070] In some embodiments, the polynucleic acid molecule is SEQ ID NO:1087-10 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 89% 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some embodiments, the polynucleic acid molecule comprises a sequence having a sequence ID. Contains sequences having at least 50% sequence identity to NO:1087-1089. In some embodiments, the polynucleic acid molecule is SEQ ID NO:1087-108 In some embodiments, the sequence has at least 60% sequence identity to 9. In this case, the polynucleic acid molecule has at least 7 sequences corresponding to SEQ ID NO:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having 0% sequence identity to the Has at least 75% sequence identity to SEQ ID NO:1087-1089 In some embodiments, the polynucleic acid molecule comprises a sequence comprising SEQ ID NO:1. Contains sequences with at least 80% sequence identity to 087-1089. In one embodiment, the polynucleic acid molecule has the sequence corresponding to SEQ ID NO:1087-1089. In some embodiments, the polynucleotide comprises a sequence having at least 85% sequence identity with the polynucleotide. The nucleic acid molecule has at least 90% sequence identity to SEQ ID NO:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having sequence identity to SEQ ID NO: Sequences having at least 95% sequence identity to ID NO:1087-1089 In some embodiments, the polynucleic acid molecule comprises SEQ ID NO: 1087- 1089. In one embodiment, the polynucleic acid molecule has a sequence similar to SEQ ID NO:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence that has 97% sequence identity with the The offspring have at least 98% sequence identity to SEQ ID NO:1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having SEQ ID N Contains a sequence having at least 99% sequence identity to O:1087-1089. In some embodiments, the polynucleic acid molecule is selected from the group consisting of SEQ ID NOs:1087-1089. become.

[0071] In some embodiments, the polynucleic acid molecule is SEQ ID NO:964-128 At least 10, 15, 16, 17, 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, Contains 1, 22, 23, 24, 25, 26, or more contiguous bases. In examples, the polynucleic acid molecules include SEQ ID NOs: 1056-1094, 1147-116 2, or at least 10, 15, or 16 of a sequence selected from 1173-1211 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more neighbors In some examples, the polynucleic acid molecule comprises SEQ ID NO:1056. At least 10, 15, 16, 17, 18, 19, or 20 of a sequence selected from -1076, Contains 20, 21, 22, 23, 24, 25, 26 or more contiguous bases. In some examples, the polynucleic acid molecule is selected from SEQ ID NOs:1077-1094. At least 10, 15, 16, 17, 18, 19, 20, 21, 22, or 24 of the sequence In some instances, the polynucleotide may contain 3, 24, 25, 26, or more contiguous bases. The nucleic acid molecule comprises at least one of a sequence selected from SEQ ID NOs:1147-1162. 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 In some instances, the polynucleic acid molecule comprises SEQ ID NO: 1 or more contiguous bases. At least 10, 15, 16 or 20 of a nucleotide sequence selected from ID NO:1173-1211 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more neighbors In some examples, the polynucleic acid molecule comprises SEQ ID NO:1056. At least 10, 15, 16, 17, 18, 19, or 20 of a sequence selected from -1058, Contains 20, 21, 22, 23, 24, 25, 26 or more contiguous bases. In some examples, the polynucleic acid molecule is selected from SEQ ID NOs: 1087-1089. At least 10, 15, 16, 17, 18, 19, 20, 21, 22, or 24 of the sequence Contains 3, 24, 25, 26, or more contiguous bases. In some cases, polynuclear The acid molecule further contains one, two, three or four mismatches.

[0072] In some embodiments, the polynucleic acid molecule comprises a guide strand and a passenger strand. In some examples, the guide strand is at least Also 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , 96%, 97%, 98%, 99%, or 100% sequence identity. Optionally, the guide strand is selected from SEQ ID NOs:964-1285. Contains arrays.

[0073] In some embodiments, the polynucleic acid molecules described herein are RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some examples, the RNA is Small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA NA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal In some instances, the nucleobase is a ribosomal RNA (rRNA), a nucleobase is a ribosomal RNA (RNP), or a heterogeneous nuclear RNA (hnRNA). In some instances, the RNA comprises an shRNA. In some instances, the RNA comprises an miRNA. In some examples, the RNA comprises dsRNA. In some examples, the RNA comprises tRNA. In some instances, the RNA comprises rRNA. In some instances, the RNA comprises hnRNA. In some examples, the RNA comprises a siRNA. In some examples, the polynucleic acid molecule is In some instances, the polynucleic acid molecule comprises 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 comprises from about 10 to about 30, from about 15 to about 30, from about 18 to about 30 to about 30, about 18 to about 25, form about 18 to about 24, about 19 to about 23, about 1 9 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 2 It is 2 nucleotides in length.

[0075] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some examples, the polynucleic acid molecule is about 45 nucleotides in length. In some examples, the polynucleic acid molecule is about 40 nucleotides in length. In some examples, the polynucleic acid molecule is about 30 nucleotides in length. In some examples, the polynucleic acid molecule is about 25 nucleotides in length. In some examples, the polynucleic acid molecule is about 20 nucleotides in length. In some examples, the polynucleic acid molecule is about 19 nucleotides in length. In some examples, the polynucleic acid molecule is about 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 instances, the polynucleic acid molecule is about 14 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. The nucleic 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 instances, the polynucleic acid molecule is from about 10 to about 40 nucleotides in length. In some examples, the polynucleic acid molecule is from about 10 to about 35 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. The nucleic acid molecule is about 20 to about 25 nucleotides in length.

[0076] In some embodiments, the polynucleic acid molecule comprises at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 4 In some embodiments, the polynucleic acid molecule is 0, 45, or 50 nucleotides in length. At least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, or 30 nucleotides in length. In some instances, the polynucleic acid molecule is at least 15 nucleotides in length. In some instances, the polynucleic acid molecule is at least 18 nucleotides in length. In some instances, the polynucleic acid molecule is at least 20 nucleotides in length. In some instances, the polynucleic acid molecule is at least 21 nucleotides in length. In some instances, the polynucleic acid molecule is at least 22 nucleotides in length. In some instances, the polynucleic acid molecule is at least 23 nucleotides in length. In some instances, the polynucleic acid molecule is at least 24 nucleotides in length. In some instances, the polynucleic acid molecule is at least 25 nucleotides in length. It is at least 30 nucleotides in length.

[0077] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some examples, the polynucleic acid molecule is about 45 nucleotides in length. In some examples, the polynucleic acid molecule is about 40 nucleotides in length. In some examples, the polynucleic acid molecule is about 30 nucleotides in length. In some examples, the polynucleic acid molecule is about 29 nucleotides in length. In some examples, the polynucleic acid molecule is about 28 nucleotides in length. In some examples, the polynucleic acid molecule is about 27 nucleotides in length. In some examples, the polynucleic acid molecule is about 25 nucleotides in length. In some examples, the polynucleic acid molecule is about 24 nucleotides in length. In some examples, the polynucleic acid molecule is about 23 nucleotides in length. In some examples, the polynucleic acid molecule is about 22 nucleotides in length. In some examples, the polynucleic acid molecule is about 20 nucleotides in length. In some examples, the polynucleic acid molecule is about 19 nucleotides in length. In some examples, the polynucleic acid molecule is about 18 nucleotides in length. In some examples, the polynucleic acid molecule is about 17 nucleotides in length. In some examples, the polynucleic acid molecule is about 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 instances, the polynucleic acid molecule is about 12 nucleotides in length. In some examples, the polynucleic acid molecule is about 10 nucleotides in length. It is.

[0078] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide. In some instances, the polynucleic acid molecule comprises a second polynucleotide. The acid molecule comprises a first polynucleotide and a second polynucleotide. The first polynucleotide is a sense strand or a passenger strand. The second polynucleotide is the antisense strand or guide strand.

[0079] In some embodiments, the polynucleic acid molecule is the 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 3 0, about 18 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 1 9 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 2 It is 2 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 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. The nucleotide is about 25 nucleotides in length. In some examples, the first polynucleotide In some examples, the first polynucleotide is about 20 nucleotides in length. In some examples, the first polynucleotide is about 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 instances, the first polynucleotide is about 10 nucleotides in length. A polynucleotide of one is about 10 to about 50 nucleotides in length. In some embodiments, the first polynucleotide is about 10 to about 45 nucleotides in length. In some embodiments, 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 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. The length of the leotide.

[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 3 0, about 18 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 1 9 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 2 It is 2 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 examples, the second polynucleotide is about 40 nucleotides in length. In some instances, the second polynucleotide is about 35 nucleotides in length. In some instances, the second polynucleotide is about 30 nucleotides in length. The nucleotide is about 25 nucleotides in length. In some examples, the second polynucleotide In some instances, the second polynucleotide is about 20 nucleotides in length. In some examples, the second polynucleotide is about 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 instances, the second polynucleotide is about 10 nucleotides in length. The two polynucleotides are about 10 to about 50 nucleotides in length. In some embodiments, the second polynucleotide is about 10 to about 45 nucleotides in length. In some embodiments, 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 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. The length of the leotide.

[0083] In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the polynucleic acid molecule further comprises blunt ended, overhanging oligonucleotides. In some instances, the blunt ends include 5' blunt ends, 3' blunt ends, or combinations thereof. In some cases, the overhangs are 5' overhangs, blunt ends, or both. 3' overhang, 3' overhang, or both. contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base paired nucleotides. In some cases, the overhang may consist of 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, or 4 non-base paired nucleotides. In some cases, the overhang contains one non-base paired nucleotide. In some cases, the overhang contains two non-base paired nucleotides. In some cases, the overhang contains three non-base paired nucleotides. The cDNA contains four non-base paired nucleotides.

[0084] In some embodiments, the sequence of the polynucleic acid molecule is a target sequence described herein. At least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80% %, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary. In some embodiments, the sequence of the polynucleic acid molecule is at least equal to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is In some embodiments, the target sequence is at least 60% complementary to the target sequence set forth in The sequence of the polynucleic acid molecule is at least 70% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least partially similar to a target sequence described herein. In some embodiments, the sequences of the polynucleic acid molecules are at least 80% complementary to each other. At least 90% complementary to the target sequence described herein. In some embodiments, Thus, the sequence of the polynucleic acid molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule is at least 99% complementary to the sequence of the It is 100% complementary to the target sequence shown.

[0085] In some embodiments, the sequence of the polynucleic acid molecule corresponds to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule has five or fewer mismatches. The sequence has no more than four mismatches to the target sequence described herein. In this case, the sequence of the polynucleic acid molecule has no more than three mismatches with the target sequence described herein. In some cases, the sequence of the polynucleic acid molecule corresponds to a target sequence described herein. Optionally, the sequence of the polynucleic acid molecule may be any of the sequences described herein. It has no more than one mismatch to the target sequence listed.

[0086] In some embodiments, a polypeptide that hybridizes to a target sequence described herein is The specificity of the polynucleic acid molecule for the target sequence is 95%, 98%, 99%, or 99%. 0.5%, or 100% sequence complementarity. In some instances, the hybridization sequence These are 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 a substance that is The target polynucleic acid polymer is a nucleic acid that binds to another mRNA sequence, a DNA sequence, or a cellular protein. Direct or indirect interaction with the quality or other parts of the In some cases, other transcripts and polynucleotides are involved. by partial homology or complementarity between the sense and / or antisense strands of "Off-target effects" occur when there is simultaneous degradation of other transcripts.

[0088] In some embodiments, the polynucleic acid molecule may be a mixture of natural or synthetic or artificial nucleotides. In some cases, the polynucleic acid molecule may be DNA, RNA, or and / or combinations of nucleotide analogs. In some instances, synthetic or human The nucleotide analog or base of the nucleotide is a ribose moiety, a phosphate moiety, a nucleoside moiety, The compound may include modifications in one or more of the following:

[0089] In some embodiments, the nucleotide analog or artificial nucleotide base is , including nucleic acids having modifications at the 2' hydroxyl group of the ribose moiety. In some instances, the modifications are H, including OR, R, halo, SH, SR, NH2, NHR, NR2, 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), Amides, ethers, esters, alcohols, and oxygen. The alkyl moiety further comprises a modification. In some instances, the modification is an azo group, a keto group, an aldehyde group, or an aldehyde group. A carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocycle (e.g., imidazo hydroxyl, hydrazino, or hydroxylamino groups, isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxide, sulfone, sulfide, or disulfide); In some instances, the alkyl moiety further comprises heterosubstitution. In some cases, a carbon in the heterocyclic group is replaced by nitrogen, oxygen, or sulfur. In examples of the heterocyclic substituents, include, but are not limited to, morpholino, imidazole, and pyrrolo. Including Lisino.

[0090] In some cases, the modification of the 2' hydroxyl group is a 2'-O-methyl modification or a 2' 2'-O-Methoxyethyl (2'-O-MOE) modification. The methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2'O-methionine modification adds a methyl group to the 2' hydroxyl group of the ribose moiety. The methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. Representative examples of 2'-O-methyl modifications of adenine molecules and 2'O-methoxyethyl modifications of uridine Typical chemical structures are illustrated below.

[0091] [ka]

[0092] In some embodiments, the modification of the 2' hydroxyl group is an extension comprising a propyl linker. The modified amine group is a 2'-O-aminopropyl modification that attaches the amine group to the 2' oxygen. In some instances, this modification involves the introduction of one positive charge from an amine group per sugar. neutralizing the overall negative charge from the phosphates of the oligonucleotide molecule, thereby Its zwitterionic properties improve its cellular uptake properties. A typical chemical structure of a leucoside phosphoramidite is illustrated below.

[0093] [ka]

[0094] In some instances, the 2' hydroxyl modification is a locked or bridged ribose modification (e.g., nucleotides, or LNAs, in which the oxygen molecule attached at the 2' carbon is a methylene group. is attached to the 4' carbon by, thus, a 2'-C, 4'-C-oxy-methylene bond It forms a bicyclic ribonucleotide monomer. A representative example of the chemical structure of LNA is illustrated below. The representative example shown on the left highlights the chemical bond nature of the LNA monomer. The example shown is a locked 3'-endo( 3 E) emphasize structure. There are.

[0095] [ka]

[0096] In some embodiments, the modification at the 2' hydroxyl group converts the sugar structure to a 3'-endo sugar. to the sugar puckering conformation Ethylene nucleic acids (ENAs), e.g., 2'-4'-ethylene bridged nucleic acids, ENAs are part of the bridged nucleic acid class of modified nucleic acids that also includes LNAs. Exemplary chemical structures of ENAs and cross-linked nucleic acids are illustrated below.

[0097] [ka]

[0098] In some embodiments, the additional modification at the 2' hydroxyl group is 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-DM AEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0099] In some embodiments, the nucleotide analog includes, but is not limited to, 5-propionyl Niluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N ,N,-Dimethyladenine, 2-Propyladenine, 2-Propylguanine, 2-Amino Adenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyl Uridine and other nucleotides with modifications at the 5-position, 5-(2-amino)propyl Luuridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyl Adenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methyl 7-methylguanosine, 2,2-dimethylguanosine, 5-methylamino Deazanidine such as 5-ethyluridine, 5-methoxyuridine, and 7-deaza-adenosine 6-Azouridine, 6-Azocytidine, 6-Azothymidine, 5-methyl-2-thio Auridine, 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 N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine , 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl Nyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, G-clan Modified cytosines, 8-substituted adenines and guanidines that act as p-nucleotides 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyapatidine alkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkyl Modified nucleotides include modified bases such as polycarbonyl alkylated nucleotides. In addition, the sugar moiety may be a nucleotide that does not have a ribosyl sugar or its analogues, as well as a nucleotide that does not have a ribosyl sugar or its analogues. For example, in some cases, the sugar moiety is , arabinose, glucopyranose, galactopyranose, 4'-thioribose, and , other sugars, heterocycles, or carbocycles, or based on these. The term nucleotide further includes those known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindolyltransferase, Contains dhole, or nebularine.

[0100] In some embodiments, the nucleotide analogs are morpholinos, peptide nucleic acids (PN A), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'- Fluoro-N3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (H NA), or combinations thereof. Promorpholino oligos (PMOs) are synthetic molecules whose structures consist of normal sugars and phosphates. It mimics the natural nucleic acid structure by deviating from the structure. In this example, the five-membered ribose ring contains four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is replaced with a six-membered morpholino ring. In some cases, modifications of the backbone are made by phosphorodiamidate groups instead of the aryl groups. is a cellular delivery agent, such as that used by charged oligonucleotides. They can cross cell membranes without the aid of delivery agents. Remove all positive and negative charges making the morpholino neutral molecule.

[0101] [ka]

[0102] In some embodiments, peptide nucleic acids (PNAs) contain phosphate linkages in the sugar backbone ring. Instead, the bases are linked and appropriately spaced by molecules such as oligoglycines, so In addition, the backbone charges are removed.

[0103] [ka]

[0104] In some embodiments, one or more modifications optionally occur at an internucleotide linkage. In some examples, the modified internucleotide linkage can be, but is not limited to, a phosphorothioate. phosphate, phosphorodithioate, methylphosphonate, 5'-alkylenephosphonate, 5 '-Methylphosphonate, 3'-alkylenephosphonate, boron trifluoride trifluoridates), 3'-5' or 2'-5' boranoline Acid esters and selenophosphates, phosphotriesters, thionoalkylphosphotriesters Esters, hydrogen phosphonate bonds, alkyl phosphonates, alkyl phosphonothioates , aryl phosphorothioates, phosphoroselenoates, phosphorodiselenoates, phosphatides Phosphoramidates, 3'-alkyl phosphoramidates, aminoalkyl Phosphoramidate, thionophosphoramidate, phosphoropiperazidate, phosphoroa Nitrothioates, phosphoranilidates, ketones, sulfones, sulfonamides, carbonyls esters, carbamates, methylenehydrazo, methylenedimethyldimethylhydrazo, formaldehyde Cetyl, thioformyl acetal, oxime, methyleneimino, methylenemethylimino, thio Amidates, bonds with riboacetyl groups, aminoethylglycines, silyl, or siloxy groups hydroxyl groups, e.g., saturated or unsaturated and / or substituted and / or heterocyclic groups. Alkyl or cycloalkyl with or without heteroatoms of 1 to 10 carbons, including heteroatoms alkyl bond, bond with morpholino structure, amide, base is directly bonded to the aza nitrogen of the backbone or indirectly linked polyamides, or combinations thereof. PS ASO contains phosphorothioate linkages Exemplary PS ASOs are described below.

[0105] [ka]

[0106] In some instances, the modification is a methylphosphonate or thiolphosphonate modification. Which methyl or thiol modification. A typical thiol phosphonate nucleotide (left) ) and methylphosphonate nucleotides (right) are illustrated below.

[0107] [ka]

[0108] In some examples, modified nucleotides include, but are not limited to, the following: The 2'-fluoro N3-P5'-phosphoramidites include:

[0109] [ka]

[0110] In some examples, modified nucleotides include, but are not limited to, the following: Hexitol nucleic acid (also called 1',5'-anhydrohexitol nucleic acid (HNA) ) including:

[0111] [ka]

[0112] In some embodiments, the nucleotide analog or artificial nucleotide base is 5'-vinylphosphonate modified nucleotides having modifications at the 5' hydroxyl group of the ribose moiety In some embodiments, the 5'-vinylphosphonate modified nucleic acid The nucleotide is selected from the nucleotides provided below, where 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 an extension comprising a propyl linker. The modified amine group is a 2'-O-aminopropyl modification, which attaches an amine group to the 2' oxygen. In some instances, this modification involves the introduction of one positive charge from the amine group per sugar. This neutralizes the overall negative charge of the oligonucleotide molecule resulting from the phosphate groups, thereby , improving cellular uptake properties due to its zwitterionic properties.

[0115] In some instances, 5'-vinylphosphonates are used in combination with locked or bridged ribose modifications (e.g. For example, a nucleic acid such as a locked nucleic acid or LNA, in which the 2'-carbon linked The molecule is linked to the 4' carbon by a methylene group, thus forming a 2'-C, 4'-C-oxygen bond. Forms a 5'-vinylphosphonate bicyclic ribonucleotide monomer. Exemplary representations of the chemical structures of modified LNAs are illustrated below, where X is O or S. B is a heterocyclic base moiety; and J is an adjacent nucleotide of the polynucleotide. It is an internucleotide linker that binds to a nucleotide.

[0116] [ka]

[0117] In some embodiments, the additional modification at the 2' hydroxyl group is 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-DM AEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0118] In some embodiments, the nucleotide analogue is a modified base, e.g., However, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6- Methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine adenine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylsilicate uridine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-( 2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine Cytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyl Uridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine uridine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides (7 -Deaza-adenosine, 6-azolidine, 6-azocytidine, or 6-azocytidine 5-methyl-2-thiouridine, other thio bases (2-thiouridine, 4-thio uridine, 2-thiocytidine, etc.), dihydrouridine, pseudouridine, -osine, alkaeosine, naphthyl and substituted naphthyl groups, O- and N-alkanes Chlorinated purines and pyrimidines, e.g., N6-methyladenosine, 5-methylcarbonyl dimethyluridine, uridine, 5-oxyacetic acid, pyridin-4-one, pyridin-2-one phenyl and modified phenyl groups, e.g., aminophenol or 2,4,6-triphenylphenol; Methoxybenzene, a modified cytosine that acts as a G-clamp nucleotide, 8-substituted substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidins nucleotides, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides nucleotides and alkylcarbonyl alkylated nucleotides. Phosphonate-modified nucleotides may also contain non-ribosyl sugars or their analogs. The sugar moiety is modified in the same manner as the 5'-vinylphosphonate-modified nucleotides with For example, in some cases, the sugar moiety is mannose, alanine, or glyceryl. Rabinose, glucopyranose, galactopyranose, 4'-thioribose, and It may be, or be based on, other sugars, heterocycles, or carbocycles. The term nucleotide further includes those known in the art as universal bases. By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, Contains ru, or nebularine.

[0119] In some embodiments, the 5'-vinylphosphonate modified nucleotide analyte Logs are further classified into morpholino, peptide nucleic acid (PNA), and methylphosphonate nucleotides. , thiol phosphonate nucleotides, 2'-fluoro N3-P5' phosphoramidites, or 1',5'-anhydrohexitol nucleic acid (HNA). Phosphorodiamidate morpholino oligos (PMOs) comprise synthetic molecules whose structures are similar to those of natural They mimic the structure of nucleic acids but deviate from the normal sugar and phosphate structures. In this example, the five-membered ribose ring contains four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is replaced with a six-membered morpholino ring. In such cases, the backbone is linked by a phosphorodiamidate group instead of a cyclic group. The alteration can be achieved by using cell delivery agents such as those used by charged oligonucleotides. They can cross cell membranes without the aid of r delivery agents. It is possible to remove all positive and negative charges making a morpholino neutral molecule. Non-limiting examples of sulfonate-modified morpholino oligonucleotides are illustrated, wherein: X is O or S; and B is a heterocyclic base moiety.

[0120] [ka]

[0121] In some embodiments, the 5'-vinylphosphonate modified monoclonal antibody described above is Phosphorinos or PMOs are PMOs that contain a positive or cationic charge. Some examples So, PMO is PMOplus (Sarepta). PMOplus is an arbitrary number (1-piperazino)phosphinylideneox y), 1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinyli deoxy bond (e.g., those described in PCT Publication WO2008 / 036127) In some instances, the term refers to a phosphorodiamidate morpholino oligomer that includes The PMO is a PMO described in US Pat. No. 7,943,762.

[0122] In some embodiments, the morpholino or PMO described above is PMO-X( In some cases, PMO-X is a compound disclosed in PCT Publication WO2011 No. 2012 / 0065169 and other related art-disclosed At least one bond or at least one of the terminal modifications This refers to morpholino oligomers.

[0123] In some embodiments, the morpholinos or PMOs described above are those disclosed in U.S. Patent Publication No. 2005 / 0133636. The PMO is as described in Table 5 of Jpn. Jpn. Cit. No. 2014 / 0296321.

[0124] An exemplary representation of the chemical structure of a 5'-vinylphosphonate modified nucleic acid is illustrated below: wherein 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 phosphate linkages in the sugar backbone ring. The bases are linked and properly spaced by oligoglycine-like molecules, and thus the bone Remove the negative charge.

[0127] [ka]

[0128] In some embodiments, the 5'-vinylphosphonate modified oligonucleotide One or more modifications of the nucleotides are optionally made at the internucleotide linkages. The internucleotide linkages include, but are not limited to, phosphorothioate; phosphorodithioate; 5'-Methylphosphonate;5'-Alkylenephosphonate;5'-Methylphosphonate; 3'-Alkylenephosphonate; Boron trifluoride; 3'-5' or 2'-5' bond Boranophosphates and selenophosphates; Phosphotriesters; Thionoalkyl Phosphotriester; Hydrogen phosphonate bond; Alkyl phosphonate; Alkyl phosphono Thioate;Arylphosphonothioate;Phosphoroselenoate;Phosphorodiselenoate Phosphinates;Phosphinates;Phosphoramidates;3'-Alkylphosphoramidates;Amino Noalkylphosphoramidates;Thionophosphoramidates;Phosphoropiperazidates; Phosphoroanilothioates; Phosphoroanilidates; Ketones; Sulfones; Sulfonamides ;Carbonate;Carbamate;Methylenehydrazo;Methylenedimethyldimethylhydrazo;Form Muacetal;Thioformacetal;Oxime;Methyleneimino;Methylenemethylimino; Thioamidate; Riboacetyl group bond; Aminoethylglycine; Silyl or silyl xanthane bonds; e.g., saturated or unsaturated and / or substituted and / or Heteroatom-containing alkyl or silyl groups of 1 to 10 carbons with or without heteroatoms chloroalkyl bond; bond with morpholino structure; amide; base is directly bonded to the aza nitrogen of the backbone or indirectly bonded to a polyamide, or a combination thereof.

[0129] In some instances, the modification is a methylphosphonate or thiolphosphonate modification. Which methyl or thiol modification. A typical thiol phosphonate nucleotide (left) ), phosphorodithioate (middle), and methylphosphonate nucleotides (right) are Illustrated below.

[0130] [ka]

[0131] In some instances, the 5'-vinylphosphonate modified nucleotide can be, but is not limited to, However, examples of phosphoramidites include those exemplified as follows:

[0132] [ka]

[0133] In some instances, the modified internucleotide linkage is a phosphorodiamidate linkage. A non-limiting example of a phosphorodiamidate linkage using the morpholino system is shown below.

[0134] [ka]

[0135] In some instances, the modified internucleotide linkage is a methylphosphonate linkage. A non-limiting example of a methylphosphonate linkage is shown below.

[0136] [ka]

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

[0138] [ka]

[0139] In some instances, the 5'-vinylphosphonate modified nucleotide can be, but is not limited to, However, it includes modified nucleic acids, examples of which are given below.

[0140] In some embodiments, one or more modifications are such that the modifications result in a neutral or uncharged backbone. In some examples, the phosphate backbone comprises a modified phosphate backbone comprising: It is modified by alkylation to produce an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, the alkyl group, as used herein in the context of alkylation, is It refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms. In this example, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, butyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n- Pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyric acid butyl, 2,2-dimethylbutyl, 3.3-dimethylbutyl, and 2-ethylbutyl groups In some cases, the modified phosphates are described in U.S. Pat. No. 5,399,623.

[0141] In some embodiments, the additional modified phosphate backbone is a methylphosphonate. phosphonate, ethyl phosphonate, methylthiophosphonate, or methoxyphosphonate Optionally, the modified phosphate is a methylphosphonate. In some cases, the modified phosphate is an ethyl phosphonate. The modified phosphate is a methylthiophosphonate. The phosphonate is a methoxyphosphonate.

[0142] In some embodiments, the one or more modifications further include a ribose moiety, a phosphate Backbone and nucleoside modifications, or 3' or 5' terminal nucleotide analogs For example, the 3' terminus optionally includes a 3' cationic group, or the 3'-3' bond In another alternative, the 3'-terminus is optionally , conjugated to an aminoalkyl group, e.g., 3'C5-aminoalkyl dT. In some embodiments, the 3'-terminus is optionally terminated with an abasic site, e.g., an apurinic or apyrimidinic acid moiety. In some instances, the 5'-terminus is conjugated to an aminoalkyl group, e.g., Optionally, the 5'-end is conjugated with an abasic site, For example, it is conjugated to an apurinic acid or apyrimidinic acid moiety.

[0143] In some embodiments, the polynucleic acid molecule comprises an artificial nucleotide sequence as described herein. In some examples, the polynucleic acid molecules described herein include one or more of the following analogs: , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, It includes 17, 18, 20, 25 or more artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analog 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), TO-Dimethylaminoethyloxyethyl (2'-O-DM AEOE), or modified 2'-ON-methylacetamide (2'-O-NM A) LNA, ENA, PNA, HNA, morpholino, and methylphosphonate nucleotides , thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites In some embodiments, the polynucleic acid molecule comprises a 2' -O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Aminopropanol Pyr, 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-Dimethylaminoethyloxy diethyl (2'-O-DMAEOE) or modified 2'-ON-methylacetate amide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methyl Phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3- P5'-phosphoramidites, or combinations of these , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20 In some embodiments, the polynucleic acid molecule comprises 25 or more artificial nucleotide analogs. is 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 embodiments, the polynucleic acid molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 2, 13, 14, 15, 16, 17, 18, 20, 25 or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some instances, the nucleotides described herein include The polynucleic acid molecule is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 Contains 4, 15, 16, 17, 18, 20, 25 or more thiol phosphonate nucleotides nothing.

[0144] In some embodiments, the polynucleic acid molecule comprises a plurality of phosphorodiamidate morpholinosides. A peptide nucleic acid oligomer or a plurality of peptide nucleic acids, including modified non-natural nucleotides, and and optionally at least one inverted abasic portion. 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- In some instances, the polynucleic acid molecule is 100% phosphorodiaminodiphenyl phosphate. Midate morpholino oligomers contain 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 modifications In some examples, the polynucleic acid molecule comprises 100% peptide. The nucleic acids contain modified and non-natural nucleotides.

[0146] In some embodiments, the polynucleic acid molecule is a nucleic acid molecule in which each nucleotide analog is stereochemically In such an example, the polynucleic acid molecule contains one or more nucleotide analogs that are isomeric to the nucleotide sequence. The nucleotides are chiral molecules. In some cases, the nucleotide analogs contain backbone stereochemistry. In further instances, the nucleotide analogs are described in U.S. Pat. ,211, or chiral analogs such as those described in 9,605,019.

[0147] In some examples, the polynucleic acid molecule comprises at least one of the following: about 5% to about 10 0% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% from about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification , about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 10 0% modification, and from about 90% to about 100% modification.

[0148] Optionally, the polynucleic acid molecule comprises at least one of the following: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 9 0% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to About 90% modified, and about 80% to about 100% modified.

[0149] Optionally, the polynucleic acid molecule comprises at least one of the following: from about 10% to about 80% about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 8 0% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% modification. 0% to approximately 80% modification.

[0150] In some examples, the polynucleic acid molecule comprises at least one of the following: about 10% to about 7 0% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modified, about 50% to about 70% modified, and about 60% to about 70% modified.

[0151] In some examples, the polynucleic acid molecule comprises at least one of the following: about 10% to about 6 0% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to About 60% modified, and about 50% to about 60% modified.

[0152] Optionally, the polynucleic acid molecule comprises at least one of the following: about 10% to about 50% about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about Approximately 50% modified.

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

[0154] Optionally, the polynucleic acid molecule comprises at least one of the following: about 10% to about 30% modification, and from about 20% to about 30% modification.

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

[0156] In some cases, the polynucleic acid molecules are about 15% to about 90%, about 20% to about 80%, about 30% to about 50%, about 60% to about 70%, about 80% to about 90%, about 10% to about 15%, about 20% to about 30%, about 30% to about 50%, about 10% to about 15%, about 20% to about 30%, about 30% It includes from 0% to about 70%, or from about 40% to about 60% modifications.

[0157] In further instances, the polynucleic acid molecule is at least about 15%, 20%, 30%, 40%, 50%, Includes 0%, 60%, 70%, 80%, 90%, 95%, or 99% qualification.

[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 1 7, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides. Includes do.

[0160] In some instances, about 5 to about 100% of the polynucleic acid molecules are selected from the group consisting of the artificial polynucleic acid molecules described herein. In some instances, about 5%, 10%, 15%, or 20% of the polynucleic acid molecule comprises a nucleotide analog. %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, or 100% are herein defined as In some instances, about 5% of the polynucleic acid molecule comprises an artificial nucleotide analog as described. In some examples, the polynuclear nucleotide analog comprises an artificial nucleotide analog as described herein. Approximately 10% of the acid molecules contain the artificial nucleotide analogs described herein. In one example, about 15% of the polynucleic acid molecules contain an artificial nucleotide analog as described herein. In some examples, about 20% of the polynucleic acid molecules comprise an artificial nucleic acid as described herein. In some examples, about 25% of the polynucleic acid molecules comprise a nucleic acid analog as described herein. In some instances, about 30% of the polynucleic acid molecule comprises an artificial nucleotide analog. In some examples, the polynuclear nucleotide analog comprises an artificial nucleotide analog as described herein. Approximately 35% of the acid molecules contain the artificial nucleotide analogs described herein. In one embodiment, about 40% of the polynucleic acid molecules are comprised of the artificial nucleotide analogs described herein. In some examples, about 45% of the polynucleic acid molecules comprise an artificial nucleic acid as described herein. In some examples, about 50% of the polynucleic acid molecules comprise a nucleic acid analog as described herein. In some instances, about 55% of the polynucleic acid molecule comprises an artificial nucleotide analog. In some examples, the polynuclear nucleotide analog comprises an artificial nucleotide analog as described herein. Approximately 60% of the acid molecules contain the artificial nucleotide analogs described herein. In one example, about 65% of the polynucleic acid molecules are comprised of the artificial nucleotide analogs described herein. In some examples, about 70% of the polynucleic acid molecules comprise an artificial nucleic acid as described herein. In some examples, about 75% of the polynucleic acid molecules comprise a nucleic acid analog as described herein. In some instances, about 80% of the polynucleic acid molecule comprises an artificial nucleotide analogue. In some examples, the polynuclear nucleotide analog comprises an artificial nucleotide analog as described herein. Approximately 85% of the acid molecules contain the artificial nucleotide analogs described herein. In one embodiment, about 90% of the polynucleic acid molecule comprises an artificial nucleotide analog as described herein. In some examples, about 95% of the polynucleic acid molecules comprise the artificial nucleic acid sequences described herein. In some examples, about 96% of the polynucleic acid molecules comprise a nucleic acid analog as described herein. In some instances, about 97% of the polynucleic acid molecule comprises an artificial nucleotide analogue. In some examples, the polynuclear nucleotide analog comprises an artificial nucleotide analog as described herein. Approximately 98% of the acid molecules contain the artificial nucleotide analogs described herein. In one embodiment, about 99% of the polynucleic acid molecule comprises an artificial nucleotide analog as described herein. In some examples, about 100% of the polynucleic acid molecules are comprised of the artificial nucleic acid sequences described herein. In some embodiments, the artificial nucleotide analog comprises 2 '-O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Aminopeptide propyl, 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-Dimethylaminoethyl 2'-O-DMAEOE, or modified 2'-ON-methyl Setoamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methyl 2'-Fluoro-N3 -P5'-phosphoramidites, or combinations thereof.

[0161] In some embodiments, the polynucleic acid molecule comprises from about 1 to about 25 modifications, wherein the modifications The decoration comprises an artificial nucleotide analog described herein. In some embodiments, In accordance with the present invention, the polynucleic acid molecule comprises about one modification, wherein the modification is an artificial nucleotide as described herein. In some embodiments, the polynucleic acid molecule comprises about two modifications. , where the modification comprises an artificial nucleotide analog as described herein. In embodiments, the polynucleic acid molecule comprises about three modifications, where the modifications are as described herein. In some embodiments, the polynucleic acid molecule comprises an artificial nucleotide analogue having about 4 wherein the modification includes an artificial nucleotide analog as described herein. In some embodiments, the polynucleic acid molecule comprises about 5 modifications, wherein the modifications are In some embodiments, the polynuclear nucleotide analogs include those described in the above. The acid molecule contains about six modifications, where the modifications are selected from the group consisting of the artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 7 modifications, wherein Modifications include artificial nucleotide analogs as described herein. In one embodiment, the polynucleic acid molecule comprises about 8 modifications, wherein the modifications are selected from the group consisting of artificial nucleotides described herein. In some embodiments, the polynucleic acid molecule comprises about 9 modifications. wherein the modification includes an artificial nucleotide analog as described herein. In an embodiment, the polynucleic acid molecule comprises about 10 modifications, wherein the modifications are as described herein. In some embodiments, the polynucleic acid molecule comprises an artificial nucleotide analogue. and about 11 modifications, wherein the modifications are selected from the group consisting of artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, wherein the modifications comprises an artificial nucleotide analog as described herein. In some embodiments, The polynucleic acid molecule comprises about 13 modifications, where the modifications are artificial nucleotides as described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications. wherein the modification includes an artificial nucleotide analog as described herein. In an embodiment, the polynucleic acid molecule comprises about 15 modifications, wherein the modifications are as described herein. In some embodiments, the polynucleic acid molecule comprises an artificial nucleotide analogue. and about 16 modifications, wherein the modifications are selected from the group consisting of artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 17 modifications, wherein the modifications comprises an artificial nucleotide analog as described herein. In some embodiments, The polynucleic acid molecule comprises about 18 modifications, where the modifications are artificial nucleotides as described herein. In some embodiments, the polynucleic acid molecule comprises about 19 modifications. wherein the modification includes an artificial nucleotide analog as described herein. In an embodiment, the polynucleic acid molecule comprises about 20 modifications, wherein the modifications are as described herein. In some embodiments, the polynucleic acid molecule comprises an artificial nucleotide analogue. The modified nucleotides include about 21 modifications, where the modifications are the artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 22 modifications, wherein the modifications comprises an artificial nucleotide analog as described herein. In some embodiments, The polynucleic acid molecule comprises about 23 modifications, where the modifications are artificial nucleotides as described herein. In some embodiments, the polynucleic acid molecule comprises about 24 modifications. wherein the modification includes an artificial nucleotide analog as described herein. In an embodiment, the polynucleic acid molecule comprises about 25 modifications, wherein the modifications are as described herein. The artificial nucleotide analogues include

[0162] In some embodiments, the polynucleic acid molecule is composed of two separate polynucleotides. wherein one polynucleotide comprises a sense strand and the second polynucleotide comprises a sense strand. In another embodiment, the sense strand is linked to a linker molecule. Thus, the antisense strand is connected to the polynucleotide linker, which in some instances is - or a non-nucleotidic linker.

[0163] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, The pyrimidine nucleotides in the sense strand are 2'-O-methylpyrimidine nucleotides. The purine nucleotides in the sense strand include 2'-deoxy purine 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 are 2'-deoxy-2'-fluoropyrimidines. The purine nucleotides present in the sense strand are 2'-deoxy purine nucleotides. Contains phosphorus nucleotides.

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

[0165] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy 2'-fluoropyrimidine nucleotides, and purine nucleotides are When present in the source strand, it contains 2'-deoxy-purine nucleotides.

[0166] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, The sense strand can be the 5'-end, the 3'-end, or both the 5' and 3' ends of the sense strand. In another embodiment, the terminal cap moiety comprises an inverted deoxy abasic moiety. minutes.

[0167] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, In the antisense strand, a phosphate backbone modification is included at the 3' end of the antisense strand. In an example, the phosphate backbone modification is phosphorothioate. The guide strand contains more phosphorothioate modifications than the guide strand. The passenger strand contains more phosphorothioate modifications than the passenger strand. The passenger strand may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more In additional cases, the guide strand may comprise about 2, 3, 4, 5, 6, Contains 7, 8, 9, 10 or more phosphorothioate modifications.

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

[0169] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand has one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phospho thioate 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) and / or Universal base-modified nucleotides, and optionally 3'-terminus, 5'-terminus, or or containing terminal cap molecules at both the 3'- and 5'-ends; and is from 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 phosphoro thioate 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 the 3'-terminus, 5'-terminus, or 3'- and 5'-terminus of the antisense strand. In other embodiments, the amine comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the sense strand and / or The pyrimidine nucleotides in the antisense strand are 2'-deoxy, 2'-O-methyl, and and / or 2'-deoxy-2'-fluoro nucleotides, or For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioates. oate internucleotide linkages and / or 3'- with end-capping molecules at the termini, the 5'-terminus, or both the 3'- and 5'-terminus, Alternatively, it may be chemically modified without using any of the above.

[0170] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand is about 1 to about 25, for example, 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 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of universal base-modified nucleotides nucleotide, and optionally the 3'-terminus, 5'-terminus, or 3'- and 5'-terminus of the sense strand. and the antisense strand comprises about 1 to about 25 , for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5, 16, 17, 18, 19, 20 or more phosphorothioate nucleotides and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of 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 capsids may be added to the 3'-terminus, 5'-terminus, or both 3'- and 5'-terminus of the sense strand. In other embodiments, the peptide comprises one or more, e.g., about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10 or more sense and / or antisense strand peptides Amino acid nucleotides can be 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy. Using 2'-fluorooxy nucleotides, from 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, 1 8, 19, 20 or more phosphorothioate internucleotide linkages, and / or or 3'-end, 5'-end, or 3'- and 5'-ends present on the same or different strands. -terminus is chemically modified with or without an end cap molecule.

[0171] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand is one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 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. Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the universal base modification nucleic acids nucleotide, and optionally the 3'-terminus, 5'-terminus, or 3'- and 5'-terminus of the sense strand. and the antisense strand comprises an end cap molecule at both ends of the antisense strand and an end cap molecule at both ends of the antisense strand. 0, especially about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hosts Holothioate internucleotide linkages, and / or one or more (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10 or more) of 2'-deoxy, 2'-O-methyl 1, 2'-difluoro, 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 modification nucleoside tide, and optionally the 3'-terminus, 5'-terminus, or 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, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20 or more, sense and / or antisense strands The pyrimidine nucleotides are 2'-deoxy, 2'-O-methyl, and / or 2' 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide bonds and / or 3'-terminus, 5'-terminus or Chemically modified with or without end capping molecules at both the 3' and 5' ends. will be done.

[0172] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand is 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 one 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) of 2'-deoxy, 2 '-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g. For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more universal bases Modified nucleotides, and optionally, the 3'-terminus, 5'-terminus, or 3' and the antisense strand comprises a terminal cap molecule at both the - and 5'-ends, and to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleic acids Internucleotide bonds and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10 or more) of 2'-deoxy, 2'-O-methyl, 2'-deoxy 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, the 3'-terminus, the 5'-terminus, or both the 3'- and 5'-terminus of the antisense strand. In other embodiments, the end capping molecule comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more of the sense and / or antisense strands Pyrimidine nucleotides can be 2'-deoxy, 2'-O-methyl, and / or 2'- Using deoxy-2'-fluoro nucleotides, the , 4, 5 or more phosphorothioate internucleotide linkages, and / or the same or 3'-terminus, 5'-terminus, or both 3'- and 5'-terminus on different strands It is chemically modified with or without end-capping molecules.

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

[0174] In some embodiments, the double-stranded polynucleic acid molecule has one or more of the modifications described above. and a guide strand (e.g., antisense strand) having a plurality of phosphorodiamidate molecules. Polynucleotide oligomers or peptide nucleic acids containing modified non-natural nucleotides A passenger strand (eg, a sense strand) is included.

[0175] In some embodiments, the polynucleic acid molecules described herein are In each chain, about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more hosts Chemically modified short interfering nucleic acid molecules having holothioate internucleotide linkages.

[0176] In another embodiment, the polynucleic acid molecules described herein have a 2'-5' internucleotide linkage. In some instances, the 2'-5' internucleotide linkage is At the 3'-end, 5'-end, or both the 3'-end and 5'-end of the strand. Additional examples In the case of 2'-5' internucleotide linkages, the 2'-5' internucleotide linkages can be located at various other positions within one or both strands of the sequence. For example, all of the pyrimidine nucleotides in one or both strands of a polynucleic acid molecule Approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of all internucleotide bonds The above may contain 2'-5' internucleotide linkages or may be present at one or both ends of a polynucleic acid molecule. In one strand, approximately 1, 2, 3, 4, 5, or 6 of all internucleotide bonds of purine nucleotides are 6, 7, 8, 9, 10, or more contain 2'-5' internucleotide linkages.

[0177] In some embodiments, the polynucleic acid molecule mediates RNAi activity in a cell or A single stranded polynucleic acid molecule reconstituted in an in vitro system, wherein the polynucleic acid molecule is a target nucleic acid molecule. The nucleic acid sequence of the nucleic acid is a single stranded polynucleotide having a complementarity to the nucleic acid sequence of the nucleic acid. At least one of the pyrimidine nucleotides is a 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., where all pyrimidine nucleotides are 2'-deoxy or, alternatively, a plurality of pyrimidine nucleotides. The pyrimidine nucleotide is a 2'-deoxy-2'-fluoropyrimidine nucleotide), and Any purine nucleotide present in the polynucleic acid is converted to a 2'-deoxypurine nucleotide. (e.g., all purine nucleotides are 2'-deoxypurine nucleotides) or alternatively, the plurality of purine nucleotides are 2'-deoxypurine nucleotides. The terminal cap modification is at the 3'-end of the antisense sequence, 5'-end of the antisense sequence, and A polynucleic acid molecule may optionally be present at either the 3'-end or at both the 3'- and 5'-ends. The 3' end of the acid molecule has from about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxy Optionally, the terminal nucleotide may further comprise one or more silibonucleotides. containing the above (e.g., 1, 2, 3, or 4) phosphorothioate internucleotide linkages , and, optionally, the polynucleic acid molecule 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 naturally occurring. In comparison with the polynucleic acid molecules of, for example, ribonucleases such as RNase H, DNase Deoxyribonucleases such as 5'-3' exonucleases and 3'-5' Resistant to nucleases such as exonucleases In some examples, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MO E), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2 '-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'- O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO -Dimethylaminoethyloxyethyl (2'-O-DMAEOE), or modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleosides tides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof Artificial nucleotide analogs containing ribonucleases such as RNase H and DNase Deoxyribonucleases such as 5'-3' exonucleases and 3'-5' Resistant to nucleases such as exonucleases In some instances, the 2'-O methyl modified polynucleic acid molecule is nuclease resistant (e.g., For example, RNaseH, DNase, 5'-3' exonuclease, or 3'-5' endonuclease. xonuclease resistant). In some instances, 2'O-methoxyethyl (2'- O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNa se, 5'-3' exonuclease, or 3'-5' exonuclease resistant) In some examples, the 2'-O-aminopropyl modified polynucleic acid molecule is a nuclease Resistance to enzymes (e.g., RNase H, DNase, 5'-3' exonuclease, or In some instances, the 2'-deoxy modified nucleotide is 3'-5' exonuclease resistant. The modified polynucleotide molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3 'Exonuclease, or 3'-5' exonuclease resistant). In one example, the T-deoxy-2'-O-fluoro modified polynucleic acid molecule is nuclease resistant. activity (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3' In some instances, 2'-O-aminopropyl The 2'-O-AP modified polynucleic acid molecules are nuclease resistant (e.g., RNase H). , DNase, 5'-3' exonuclease, or 3'-5' exonuclease In some cases, 2'-O-dimethylaminoethyl (2'-O-DM The AOE (Analyzed Enzyme-Enhanced Exonucleic Acid) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase , 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some instances, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified The polynucleic acid molecule may be nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In the example, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modification The polynucleic acid molecule may be nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In the example, the 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecule is , nuclease resistance (e.g., RNaseH, DNase, 5'-3' exonuclease In some cases, LN A modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5' -3' exonuclease or 3'-5' exonuclease resistant. In some examples, the ENA modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease In some instances, the HNA modified polynucleic acid molecule is nuclease resistant. (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'- In some instances, the morpholino is a nuclease resistant (5' exonuclease resistant). Resistance to enzymes (e.g., RNase H, DNase, 5'-3' exonuclease, or In some instances, PNA modified polynuclear fragments are used. The acid molecule is resistant to nucleases (e.g., RNase H, DNase, 5 '-3' exonuclease or 3'-5' exonuclease resistant). In some instances, the methylphosphonate nucleotide modified polynucleic acid molecules are nuclease resistant. (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'- In some instances, thiolphosphonate nucleic acids are The nucleic acid modified polynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase , 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, polynucleic acids containing 2'-fluoro N3-P5'-phosphoramidites. The molecule is nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, In some cases, Therefore, the 5' conjugates described herein inhibit 5'-3' exonucleolytic cleavage. In some examples, the 3' conjugates described herein are capable of 3'-5' exonuclease cleavage. Inhibits.

[0179] In some embodiments, one of the artificial nucleotide analogs described herein These have been shown to increase the binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'- O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-amino 2'-O-Dimethylaminoethyl (2'-O-DMAO E), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminopropyl Aminoethyloxyethyl (2'-O-DMAEOE) or modified 2'-O- N-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, mol phosphonate nucleotides, methylphosphonate nucleotides, thiolphosphonate nucleotides, or An artificial nucleotide analogue containing 2'-fluoro N3-P5'-phosphoramidite The polynucleic acid molecule comprises a polynucleic acid molecule that is more selective for its mRNA target than a comparable naturally occurring polynucleic acid molecule. In some instances, the 2'-O-methyl modified polynucleic acid molecule has increased binding affinity to the nucleic acid. , which has increased binding affinity for its mRNA target compared to a comparable naturally occurring polynucleic acid molecule. In some instances, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are The molecule has increased binding affinity for its mRNA target compared to a comparable natural polynucleic acid molecule. In some instances, the 2'-O-aminopropyl modified polynucleic acid molecule is a polynucleotide having a similar structure to the natural equivalent. The polynucleic acid molecule has increased binding affinity to its mRNA target compared to other polynucleic acid molecules. In this example, the 2'-deoxy modified polynucleic acid molecule has a higher affinity for its nucleic acid than the equivalent natural polynucleic acid molecule. In some cases, T-deoxy-2 '-Fluoro modified polynucleic acid molecules have a lower mRNA targeting activity than comparable natural polynucleic acid molecules. In some instances, 2'-O-aminopropyl (2 The modified polynucleic acid molecule has a lower mRNA content than the equivalent natural polynucleic acid molecule. In some instances, 2'-O-dimethylaminoethyl esters were used to increase the binding affinity to the target. The 2'-O-DMAOE modified polynucleic acid molecules have a higher cleavage efficiency compared to the equivalent natural polynucleic acid molecules. In some instances, the 2'-O-diaminophenyl 2'-amino acid residues were used to increase the binding affinity for their mRNA targets. Methylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are similar to the natural polynucleic acid molecules. In some cases, the α-terminal end of the nucleoside analog had increased binding affinity to its mRNA target compared to the α-terminal end of the nucleoside analog. , 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynuclear The acid molecule exhibits a higher binding affinity for its mRNA target than a comparable natural polynucleic acid molecule. In some instances, 2'-ON-methylacetamide (2'-O-NMA ) modified polynucleic acid molecules have a higher affinity for their mRNA targets than comparable natural polynucleic acid molecules. In some instances, the LNA modified polynucleic acid molecule has increased binding affinity compared to the equivalent natural Compared to polynucleic acid molecules, they have increased binding affinity to their mRNA targets. In an example, an ENA-modified polynucleic acid molecule has a reduced mRNA expression level compared to a comparable natural polynucleic acid molecule. Increased binding affinity to the target. In some examples, the PNA modified polynucleic acid molecule is Increased binding affinity to its mRNA target compared to a comparable natural polynucleic acid molecule In some instances, HNA modified polynucleic acid molecules have a higher affinity for nucleotides than comparable native polynucleic acid molecules. In some instances, the morpholino modification increased the binding affinity to its mRNA target. A polynucleic acid molecule has a higher binding affinity to its mRNA target than a comparable naturally occurring polynucleic acid molecule. In some instances, methylphosphonate nucleotide modified polynucleic acid molecules The molecule has increased binding affinity for its mRNA target compared to a comparable natural polynucleic acid molecule. In some instances, the thiol phosphonate nucleotide modified polynucleic acid molecule is and the like, have increased binding affinity to their mRNA targets. In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites. has increased binding affinity for its mRNA target compared to a comparable natural polynucleic acid molecule. In some cases, the increased affinity may be due to a lower Kd, a higher melting temperature (Tm), or other Illustrated in combination.

[0180] In some embodiments, the polynucleic acid molecules described herein are chirally pure. or stereopure polynucleic acid molecules, or polynucleic acid molecules containing a single enantiomer. In some instances, the polynucleic acid molecule comprises L-nucleotides. In some instances, the polynucleic acid molecule comprises D-nucleotides. The product is 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, or 2%, 1%, or less. In some cases, the polynucleic acid molecule composition contains a racemic mixture. 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or In some examples, the polynucleic acid molecule is a nucleic acid molecule as described in U.S. Patent Application Publication No. 2014-0136624. / 194610 and 2015 / 211006; described in WO2015107425 A polynucleic acid molecule.

[0181] In some embodiments, the polynucleic acid molecules described herein are aptamer-conjugated. In some instances, the aptamer-conjugated moiety is further modified to include a DNA In some examples, the aptamer-conjugated moiety is an Alphame r (Centauri Therapeutics), which targets specific cell surface and a portion that displays a specific epitope for binding to circulating antibodies. In some instances, the polynucleic acid molecules described herein include those disclosed in U.S. Pat. As described in Nos. 8,604,184, 8,591,910, and 7,850,975 The compound may be further modified to include an aptamer-conjugated moiety such as:

[0182] In additional embodiments, the polynucleic acid molecules described herein may be modified to increase their stability. In some embodiments, the polynucleic acid molecule is an RNA (e.g., an siRNA). In some instances, the polynucleic acid molecule is a nucleic acid molecule having a structure as described above in order to increase its stability. In some cases, the polynucleic acid molecule is modified by one or more of the modifications described above. '-O-Methyl, 2'-O-Methoxyethyl (2'-O-MOE), 2'-O-Aminopeptide propyl, 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-Dimethylaminoethyl 2'-O-DMAEOE, or 2'-O-Methylacetamide ( 2'-O-NMA) modifications, or locked or bridged ribose structures (e.g., LNA In some cases, the polyamine is modified at the 2 hydroxyl position, for example with phenylalanine or phenylalanine. The nucleic acid molecules are linked by 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule is further modified with a nucleotide sequence to increase its stability. , morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate and / or 2'-fluoro N3-P5'-phosphoramidites In some instances, the polynucleic acid molecule is a chirally pure (or stereopure) polynucleic acid. In some instances, chirally pure (or stereopure) polynucleic acid molecules. has been modified to increase its stability. Suitable modifications of A will be apparent to those skilled in the art.

[0183] In some embodiments, the polynucleic acid molecules described herein include, but are not limited to, , DMD, DUX4, DYSF, EMD, or LMNA muscular dystrophies It has RNAi activity that regulates the expression of RNA encoded by the gene that it is targeting. In some examples, the polynucleic acid molecules described herein include DMD, DUX4, DYSF, E A double-stranded siRNA that downregulates the expression of at least one of MD and LMNA. NA molecule, wherein one strand of the double-stranded siRNA molecule is selected from the group consisting of DMD, DUX4, DY SF, EMD, or LMNA, or DMD, DUX4, DYSF, EMD, or or at least one RNA encoded by LMNA, or a portion thereof wherein the double-stranded siRNA comprises a nucleotide sequence complementary to another nucleotide sequence. The second strand of the A molecule is a small molecule of DMD, DUX4, DYSF, EMD, or LMNA. At least one of DMD, DUX4, DYSF, EMD, or LMNA substantially similar to the nucleotide sequence of an RNA encoded by at least one of the In some cases, the polynucleic acid molecules described herein contain similar nucleotide sequences. phenotype, and expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA. down-regulating double-stranded siRNA molecules, wherein each of the siRNA molecules The strand contains about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and and each strand has at least about 14, 17, or 18 nucleotides complementary to the other strand. Optionally, the polynucleic acid molecules described herein comprise 19 nucleotides. , DMD, DUX4, DYSF, EMD, or LMNA. wherein each of the siRNA molecules is a double-stranded siRNA molecule that downregulates Each strand contains about 19 to about 23 nucleotides, and each strand is a nucleotide sequence of the other strand. In some instances, the RNAi The activity occurs intracellularly. In another example, the RNAi activity occurs in a reconstituted in vitro system. .

[0184] In some embodiments, the polynucleic acid molecules described herein are directed to the DMD gene. Thus, the gene has RNAi activity that regulates the expression of the encoded RNA. In some examples, The polynucleic acid molecules described herein are single stranded siRNAs that downregulate the expression of DMD. A single stranded siRNA molecule, wherein the single stranded siRNA molecule is a DMD or a DMD-encoded siRNA molecule. It contains a nucleotide sequence complementary to the nucleotide sequence of the RNA or a part thereof. In some cases, the polynucleic acid molecules described herein downregulate the expression of DMD. wherein the siRNA molecule is a single stranded siRNA molecule that 24, or 19 to about 23 nucleotides. the polynucleic acid molecule is a single stranded siRNA molecule that downregulates the expression of DMD, Here, the siRNA molecule comprises about 19 to about 23 nucleotides. In another example, RNAi activity can be achieved in a reconstituted in vitro system. arises.

[0185] In some instances, the polynucleic acid molecule comprises self-complementary sense and antisense regions. The antisense region is a double-stranded polynucleotide molecule, including a target nucleic acid molecule. or a part thereof, and the sense region is a nucleotide sequence complementary to a nucleotide sequence in the target In some instances, the polypeptide has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. A nucleic acid molecule is assembled from two separate polynucleotides, one of which is the sense strand. and the other strand is the antisense strand, where the antisense strand and the sense strand are self-correlated. complementary (e.g., each strand contains nucleotides that are complementary to the nucleotide sequence of the other strand). When the antisense and sense strands form a duplex or double-stranded structure, etc. For example, the double-stranded region may be about 19, 20, 21, 22, 23, or more base pairs. The antisense strand is a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof. 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, The self-complementary sense and antisense regions of the nucleic acid molecule may be nucleic acid-based or non-nucleic acid-based. The linkers are connected to the base.

[0186] In some cases, the polynucleic acid molecule has self-complementary sense and antisense regions. duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structures wherein the antisense region is a polynucleotide having the structure The sense region contains a nucleotide sequence complementary to a part of the nucleotide sequence of the target nucleic acid. In other cases, the nucleic acid molecule has a nucleotide sequence corresponding to the nucleic acid sequence or a portion thereof. The fragment has two or more loop structures and a base that contains self-complementary sense and antisense regions. wherein the antisense region is a circular single-stranded polynucleotide having a target nucleic acid sequence. A nucleotide sequence complementary to the nucleotide sequence of the nucleic acid molecule or a portion thereof, The region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, The tides are then transfected in vivo to generate active polynucleic acid molecules capable of mediating RNAi. In a further instance, the polynucleic acid molecule is further treated with a target nucleic acid sequence. A single-stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid fragment or a portion thereof. Such polynucleic acid molecules include nucleic acid fragments that are directed to a target nucleic acid sequence or a portion thereof. (It is not necessary that the corresponding nucleotide sequence is present within a polynucleic acid molecule) Nucleotides can also be converted to 5'-phosphates (e.g., Martinez et al., 2 002, Cell., 110, 563-574 and Schwarz et al. , 2002, Molecular Cell, 10, 537-568) or contains a terminal phosphate group, such as 5',3'-diphosphate.

[0187] In some instances, the antisense region and the nucleotide or non-nucleotide A linear polynucleic acid molecule comprising a loop portion and a sense region is asymmetric, and the sense region is sufficient to base-pair with the antisense region and form a looped duplex. The region may have fewer nucleotides than the antisense region so that it has sufficient complementary nucleotides. For example, asymmetric hairpin-shaped polynucleic acid molecules can be used to generate R sequences in cells or in vitro systems. has a length sufficient to mediate NAi and contains about 4 to about 8 nucleotides An antisense region having a loop region (e.g., about 19 to about 22 nucleotides) and an antisense region having an antisense loop region (e.g., about 19 to about 22 nucleotides). and a sense region having about 3 to about 18 nucleotides complementary to the sense region. Therefore, the asymmetric hairpin polynucleic acid molecule further comprises a chemically modified 5'-terminal linker. In a further embodiment, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises , nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0188] In some embodiments, the asymmetric duplex comprises a sense region and an antisense region. A polynucleic acid molecule having two separate strands containing a sense region and an antisense region. has sufficient complementary nucleotides to base pair with and form a duplex , which contains fewer nucleotides than the antisense region. For example, an asymmetric double polynucleotide The acid molecule is an amplicon of sufficient length to mediate RNAi in cells or in vitro systems. a sense region (e.g., about 19 to about 22 nucleotides) and a region complementary to the antisense region and a sense region having a target sequence of about 3 to about 18 nucleotides.

[0189] In some cases, natural DNA / RNA bases that are barely distinguishable from universal bases The term "nucleotidic base analogues" refers to nucleotide base analogues that form base pairs with each of the universal bases. Examples include C-phenyl, C-naphthyl, and other aromatic derivatives as known in the art. Conductors, inosine, azole carboxamide, and 3-nitropyrrole, 4-nitro Nitroazoles such as indole, 5-nitroindole, and 6-nitroindole Derivatives (e.g., Loakes, 2001, Nucleic Acids R (see Research, 29, 2437-2447).

[0190] Polynucleic acid molecule synthesis In some embodiments, the polynucleic acid molecules described herein are Constructed using chemical synthesis and / or enzymatic ligation reactions using known procedures. For example, polynucleic acid molecules can be constructed using naturally occurring nucleotides or by synthesis. To increase the biostability of the nucleic acid molecule, or to enhance the affinity of the nucleic acid molecule formed between the polynucleic acid molecule and the target nucleic acid. A variety of modified nucleotides designed to increase the physical stability of the resulting duplex were used. Exemplary methods are described in U.S. Patents 5,142,047; 5,185 ,444; No. 5,889,136; No. 6,008,400; and No. 6,111 ,086; PCT Publication No. WO2009099942; or European Publication No. 1579015. Additional exemplary methods are described below. Griffey et al., “2'-O-aminopropyl r ibonucleotides: a zwitterionic modificat ion that enhances the exonuclease resist ance and biological activity of antisensors e oligonucleotides,” J. Med. Chem. 39(26 ):5100-5109 (1997)); Obika, et al. hesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides ha ving a fixed C3, -endo sugar puckering”. Tetrahedron Letters 38 (50): 8735 (1997 ); Koizumi, M. “ENA oligonucleotides as therapeutics”.Current opinion in molecu lar therapeutics 8 (2): 144-149 (2006); and Abramova et al., “Novel oligonucleot ide analogues based on morpholino nucleo side subunits-antisense technologies: ne w chemical possibilities,” Indian Journa l of Chemistry 48B:1721-1726 (2009). alternatively The polynucleic acid molecule may be an expression vector into which the polynucleic acid molecule has been subcloned in an antisense orientation. is biologically produced using the is in the antisense orientation relative to a desired target polynucleic acid molecule).

[0191] In some embodiments, the polynucleic acid molecule is synthesized by a tandem synthesis method, Here, both strands are bound to a single adjacent oligonucleotide fragment separated by a cleavable linker. The fragments or strands are then cleaved to form the hybridization duplex. This results in separate fragments or strands which allow for purification of the duplex.

[0192] In some instances, a polynucleic acid molecule may also be composed of two distinct nucleic acid strands or fragments. A fragment containing the sense region and a second fragment containing the split region are constructed. The antisense region of the gene is included.

[0193] Further modification methods, for example to incorporate sugar, base, and phosphate modifications, include the following: Includes: Eckstein et al., International Publ ication PCT No. WO 92 / 07065; al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; n and Cedergren, Trends in Biochem. Sci. , 1992, 17, 334-339; Usman et al. ational Publication PCT No. WO 93 / 15187; Sproat, US Pat. No. 5,334,711 and Bei gelman et al., 1995, J. Biol. Chem., 270 , 25702; Beigelman et al., International PCT publication No. WO 97 / 26270; Beigel man et al., US Pat. No. 5,716,824; an et al., US Pat. No. 5,627,053; f et al., International PCT Publication No. WO 98 / 13526; Thompson et al., US S er. No. 60 / 082,404 which was filed on Ap r. 20, 1998; Karpeisky et al., 1998, Tet rahedron Lett., 39, 1131; Earnshaw and G. ait, 1998, Biopolymers (Nucleic Acid Sci ences), 48, 39-55; Verma and Eckstein, 1 998, Annu. Rev. Biochem., 67, 99-134; and , Burlina et al., 1997, Bioorg. Med. Chem ., 5, 1999-2010. The above publication discloses a method for the synthesis of a nucleic acid molecule without regulating the catalytic activity. Methods for determining locations for incorporating sugar, base, and / or phosphate modifications into a It describes tactics.

[0194] In some instances, phosphorothioate, phosphorodithioate, and / or 5' Chemical modification of the internucleotide bonds of polynucleic acid molecules with -methylphosphonate bonds increases their stability. While this improves the overall activity of the protein, excessive modification often leads to toxicity or reduced activity. However, when designing nucleic acid molecules, the amount of these internucleotide bonds may be minimized. In such cases, decreasing the concentration of these bonds reduces the toxicity of these molecules. This increases efficacy and high specificity.

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

[0196] In some instances, at least one polypeptide A is conjugated to at least one B. In some examples, at least one polypeptide A is In some embodiments, at least one B is conjugated to A is covalently linked 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 linked to at least two B. In some examples, at least one polypeptide A is conjugated to at least 2, 3 , 4, 5, 6, 7, 8 or more B's may be conjugated.

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

[0198] In some examples, at least one B and / or at least one C, and Optionally, at least one D is conjugated to at least one polypeptide A. In the above example, at least one B is attached to at least one polypeptide A at a terminal (e.g., 5 at least one polypeptide A is conjugated to the polypeptide A at its 3' or 3' terminus or internally to the polypeptide A In some cases, at least one C is conjugated via at least one poly The peptide is conjugated directly to peptide A or indirectly via at least one B. Indirectly, by at least one B, at least one C at the same end as polypeptide A and at the opposite end from at least one polypeptide A, Alternatively, they are independently conjugated at internal sites. In some instances, at least one additional Polypeptide A is further conjugated to at least one of polypeptides A, B, or C. In a further example, at least one D is optionally linked to at least one polypeptide A, Directly or indirectly conjugated to at least one B or at least one C When directly conjugated to at least one polypeptide A, at least one D is also Optionally conjugated to at least one B to form an ADB conjugate, or -Optionally, at least one B and at least one C to form a DBC conjugate. In some instances, at least one The D is directly linked to at least one polypeptide A and at least one B. Indirectly conjugated to at least one C. Indirectly conjugated to at least one polypeptide A. When at least one D is used to form an ABD conjugate, at least one or at least one aryl group to form an ABDC conjugate. Optionally conjugated to one B and at least one C. In some examples, at least one The additional D is further conjugated to at least one of the polypeptides 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] are for illustrative purposes only and include humanized antibodies or binding fragments thereof, chimeric antibodies or a monoclonal antibody or a binding fragment thereof, a monovalent Fab ', bivalent Fab 2 , single chain variable fragment (scFv), diabody, minibody ( minibody, nanobody, single domain antibody (sdAb), or camel antibody The term encompasses the agonist or binding fragment thereof.

[0226] joining part In some embodiments, the binding moiety A is a polypeptide. The polypeptide is an antibody or a fragment thereof. Optionally, the fragment is In some examples, the antibody or binding fragment thereof is a human a mouse antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, antibody or binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab', bivalent Fab 2 , F(ab)' 3 Fragment, single chain variable fragment (scF v), bis-scFv (scFv) 2 , diabodies, minibodies, nanobodies, triple special Heteroantibodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), single domain In-antibody (sdAb), Ig NAR, camelid antibody or binding fragment thereof, double It includes specific antibodies or binding fragments thereof, or chemically modified derivatives thereof.

[0227] In some examples, A is an antibody or a binding fragment thereof. A is a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof , chimeric antibodies or binding fragments thereof, monoclonal antibodies or binding fragments thereof Fab', monovalent Fab', divalent Fab' 2 , F(ab)' 3 Fragment, single chain variable fragment scFv, bis-scFv (scFv) 2 , diabody, minibody, nanobody Antibodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv "), single domain antibodies (sdAbs), Ig NARs, camelid antibodies or their binding fragments. a fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof In some examples, A is a humanized antibody or binding fragment thereof. In some embodiments, A is a mouse antibody or a binding fragment thereof. and A is a chimeric antibody or binding fragment thereof. In some examples, A is a monoclonal antibody. In some instances, A is a monovalent Fab' antibody or binding fragment thereof. In some instances, A is a bivalent Fab 2 In some embodiments, A is a single chain variable fragment. It is a fragment (scFv).

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

[0229] In some cases, the bispecific antibody is a bispecific miniantibody. Bispecific miniantibodies consist of bivalent Fab 2 , F(ab)' 3 Fragment, bis-scFv(s cFv) 2 , diabodies, minibodies, triabodies, tetrabodies, or bispecific antibodies. In some embodiments, the bispecific T cell engager (BiTE) is The specific T cell engager is composed of two scFvs targeting epitopes on two different antigens. It is a fusion protein containing two single-chain variable fragments (scFvs) of interest.

[0230] In some embodiments, binding moiety A is a bispecific miniantibody. So, A is bispecific Fab 2 In some examples, A is a bispecific F(ab)' 3 Optionally, A is a bispecific bis-scFv fragment. Optionally, A is a bispecific bis-scFv fragment. Thus, in some embodiments where A is a bispecific (scFv), A is a bispecific In some embodiments, A is a bispecific minibody. In some embodiments, A is a bispecific triabody. In other embodiments, A is In another embodiment, A is a bispecific T cell antigen binding antibody. The device is called BiTE.

[0231] In some embodiments, binding moiety A is a trispecific antibody. , and trispecific antibodies are F(ab)' 3 Fragment or trispecific antibody. In one example, A is a trispecific F(ab)' 3 A is a fragment. In some cases, A is In some embodiments, A is a trispecific antibody. , “Development of a trispecific antibody designed to simultaneously and efficient ly target three different antigens on tu mor cells,” Mol. Pharmaceutics, 12(9): 34 90-3501(2015).

[0232] In some embodiments, binding moiety A is an antibody or is a binding fragment thereof. In some instances, binding moiety A is a cell surface The antibody or binding fragment thereof recognizes the protein. Exemplary cell surface proteins recognized by the Sca- 1, CD34, Myo-D, Myogenin, MRF4, NCAM, CD43, and CD9 Includes 5(Fas).

[0233] In some instances, the cell surface protein is 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, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD3 D4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, C D16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD4 5RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD 49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD5 3, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD 61, 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. nothing.

[0234] In some instances, binding moiety A is an antibody or a polypeptide that recognizes a CD cell surface marker. In some examples, binding moiety A is a binding fragment of CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b , CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23 , CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31 , CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39 , CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD 45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, C D49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD 53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, C D61, CD62E, CD62L (L-selectin), CD62P, CD63, CD6 4, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD7 9 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD10 3, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD27 9 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and are antibodies or binding fragments thereof that recognize these combinations.

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

[0236] In some instances, binding moiety A is an anti-myosin antibody. The antibody is a humanized antibody. In other cases, the anti-myosin antibody is a chimeric antibody. In some cases, the anti-myosin antibody is a monovalent, divalent, or polyvalent antibody.

[0237] In some instances, binding moiety A is an anti-transferrin (anti-CD71) antibody. In some cases, the anti-transferrin antibody is a humanized antibody. In a further instance, the anti-transferrin antibody is a chimeric antibody. In some embodiments, an exemplary anti-transferase antibody is a bivalent or polyvalent antibody. The IL-1 antibody was MAB5746 from R&D Systems and MAB5746 from Bio-Rad Laboratories. atories AHP858, Bethyl Laboratories, Inc. A80-128A from MilliporeSigma, and T2027 from MilliporeSigma.

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

[0239] In some embodiments, the binding moiety A is non-specifically conjugated to the polynucleic acid molecule (B). In some instances, the binding moiety A is linked to a lysine residue or a sphingotropic residue in a non-site-specific manner. The binding moiety A is conjugated to the polynucleic acid molecule (B) via a stain residue. is conjugated to the polynucleic acid molecule (B) via a lysine residue in a non-site-specific manner. In some cases, the binding moiety A is linked to the polynuclear ligand via a cysteine ​​residue in a non-site specific manner. It is conjugated to an acid molecule (B).

[0240] In some embodiments, the binding moiety A binds to the polynucleic acid molecule in a non-site specific manner. (B). In some examples, the binding moiety A is conjugated to lysine in a site-specific manner. at the 5'-terminus, at the 3'-terminus, via a cysteine ​​residue, a non-natural amino acid, or is an enzyme-modified or enzyme-catalyzed residue that is conjugated to a polynucleic acid molecule (B). In the example of FIG. 1, the binding moiety A is attached in a site-specific manner to a polynucleic acid molecule (B In some instances, the binding moiety A is conjugated to a cysteine ​​in a site-specific manner. In some instances, the binding moiety A is conjugated to the polynucleic acid molecule (B) via a residue. In some instances, the nucleic acid molecule (B) is conjugated at its 5' end in a specific manner. The binding moiety A is conjugated to the polynucleic acid molecule (B) at its 3' end in a site-specific manner. In some examples, the binding moiety A is linked to the polynucleic acid via the unnatural amino acid in a site-specific manner. In some instances, the binding moiety A is conjugated to an enzyme in a site-specific manner. It is conjugated to a polynucleic acid molecule (B) via a modified or enzyme-catalyzed residue.

[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, 1 3, 14, 15, 16 or more polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about one polynucleic acid molecule is conjugated to one binding moiety A. In some examples, about 2 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 3 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 4 polynucleic acids The molecule is conjugated to one binding moiety A. In some examples, about 5 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 6 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 7 polynucleic acid molecules are conjugated to one binding moiety A. In some examples, about 8 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, about 9 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, 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. The molecule is conjugated to one binding moiety A. In some examples, about 12 polynucleic acid molecules are conjugated to one 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 other instances, one or more of the polynucleic acid molecules are different. .

[0242] In some embodiments, the number of polynucleic acid molecules (B) conjugated to the binding moiety A is In some instances, the ratio is referred to as the DAR (drug-to-antibody) ratio, and is used in the present invention. The drug as referred to in the specification is a polynucleic acid molecule (B). In some instances, the binding moiety The DAR ratio of the polynucleic acid molecule (B) to A is about 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, or more. In some cases, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1 or more. In this example, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 2 or more. In some embodiments, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 3 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 4 or more. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is: The DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 5 or more. In some embodiments, the D of the polynucleic acid molecule (B) to the binding moiety A is about 6 or more. The AR ratio is greater than or equal to about 7. In some examples, the ratio of the polynucleic acid molecule (B) to the binding moiety A is In some instances, the DAR ratio of the polynucleic acid moiety A to the binding moiety B is about 8 or greater. The DAR ratio of the molecule (B) to the binding moiety A is about 9 or greater. The DAR ratio of the nucleic acid molecule (B) is about 10 or more. The DAR ratio of the polynucleic acid molecule (B) that binds the nucleotide sequence is about 11 or more. The DAR ratio of polynucleic acid molecule (B) to component A is about 12 or greater.

[0243] In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 1. In some instances, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 2 In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is: In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is about 4. , about 5. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is is about 6. In some examples, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety A is In some examples, the DAR of the polynucleic acid molecule (B) to the binding moiety A is about 7. The ratio is about 8. In some examples, the DA of the polynucleic acid molecule (B) to the binding moiety A is The R ratio is about 9. In some examples, the D of the polynucleic acid molecule (B) to the binding moiety A The AR ratio is about 10. In some examples, the ratio of the polynucleic acid molecule (B) to the binding moiety A is about 10. The DAR ratio of the polynucleic acid molecule ( B) has a DAR ratio of about 12. The DAR ratio of the polynuclear molecule (B) to the binding moiety A is about 13. The DAR ratio of the acid molecule (B) is about 14. In some examples, the DAR ratio of the poly(A) to the binding moiety (A) is about 14. The DAR ratio of the nucleic acid molecule (B) to the binding moiety A is about 15. The DAR ratio of the polynucleic acid molecule (B) is about 16.

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

[0245] In some instances, compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some cases, conjugates comprising a polynucleic acid molecule (B) and a binding moiety (A) have improved activity. The improved activity may be an enhancement of a biologically relevant function, for example, the treatment of a disease state or This results in improved stability, affinity, binding, functional activity, and efficacy in prophylaxis. In some instances, the disease state is the result of one or more mutated exons of a gene. In some instances, the polynucleic acid molecule (B) is more specific than the conjugate (B) without the binding moiety A. A conjugate comprising a nucleic acid molecule (B) and a binding moiety (A) binds one or more mutated exons. In some instances, the polynucleic acid molecule without the binding moiety A may be Exon skipping is a process that involves the synthesis of a conjugate that contains a polynucleotide (B) and a nucleic acid molecule (C). In conjugates containing moiety A, at least or about 5%, 10%, 20%, 25%, 30%, %, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% Increase.

[0246] In some embodiments, the antibodies or binding fragments thereof, alone or in combination, In combination with conventional techniques known in the art, e.g., amino acid deletions By using deletions, insertions, substitutions, additions, and / or recombinations, and / or or other modifications known in the art (e.g., translational modifications such as glycosylation and phosphorylation). In some instances, the modification is by F c Receptor. In some instances, one or more The modifications include, for example, those described in International Publication No. WO97 / 34631, discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Such a nucleic acid sequence underlying the amino acid sequence of the antibody or binding fragment thereof may be Methods for introducing modifications are well known to those of skill in the art.

[0247] In some instances, the antibody binding fragment further includes derivatives thereof, and may include at least one The polypeptide sequence comprises one CDR.

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

[0249] In some instances, the bispecific single chain antibody construct comprises binding domains from two antibodies. In such an embodiment, the bispecific single chain antibody construct comprises a tandem b In some embodiments, the scFv is a linker peptide. In some embodiments, the linker comprises a VH domain and a VL domain connected by a peptide. Each of the first and second domains retains its differential binding specificity independently of the other. The length and sequence are sufficient to support the

[0250] In some embodiments, as used herein, the term "conjugate" refers to a bond with or by Interaction defines the binding / interaction of at least two antigen interaction sites with each other. In some instances, the antigen interaction site is identified as a specific antigen or a specific group of antigens. Define motifs of polypeptides that exhibit the ability to interact with one another. In some cases, binding / interaction The interaction is also understood to define a specific recognition. In such a case, the specific recognition is determined by an antibody or or the binding fragment is specific for at least two amino acids of each of the target molecules. It refers to the ability to interact and / or bind. For example, a particular recognition , which refers to the specificity of an antibody molecule, or its ability to distinguish a particular range of target molecules. In this example, the specificity of the antigen interaction site and the specific interaction with the antigen can be determined, for example, by the positioning of the antigen. This leads to signal initiation by inducing conformational changes and oligomerization of antigens. In one embodiment, the binding is based on the "key-lock principle." Thus, in some instances, the antigen interaction site and Certain motifs in the amino acid sequence of the antigen and the nucleotide sequence of ... , which bind to each other as a result of their primary, secondary, or tertiary structure. In such cases, antigens The specific interaction of an interaction site with its specific antigen allows for easy binding of the site to the antigen. bring about.

[0251] In some instances, the specific interaction further results in a reduction in the antibody or binding fragment thereof. This refers to less cross-reactivity or reduced off-target effects. peptide / protein but does not bind essentially to any other polypeptides The antibody or binding fragment thereof is specific for the desired polypeptide / protein. Specificity of the antigen interaction site Examples of specific interactions with antigens include ligands The specificity of the antibody with its receptor, e.g., the antigen-binding site of an antibody This includes interactions with the position.

[0252] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. Plasma proteins include albumin. In some instances, binding moiety A is albumin. In some instances, albumin is conjugated to the albumin complex by one or more of the conjugation chemistries described herein. , is attached to a polynucleic acid molecule. In some instances, albumin is a natural ligation In some instances, albumin is linked to a polynucleic acid molecule by lysine binding. is attached to the polynucleic acid molecule by

[0253] In some instances, the conjugated moiety is a steroid. Exemplary steroids are cholesterol steroids. glycerols, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons They can be elementary (saturated, unsaturated, and substituted), or a combination of these. In some examples, the steroid is cholesterol. In some instances, cholesterol is a conjugation chemistry described herein. In some instances, cholesterol is attached to the polynucleic acid molecule by one or more In some instances, the nucleic acid is linked to a polynucleic acid molecule by natural ligation chemistry. The terol is attached to the polynucleic acid molecule by a lysine bond.

[0254] In some instances, the binding moiety may bind to, but is not limited to, a specific surface marker on a cell. In this example, the binding moiety is a polymer that contains a polynucleic acid molecule aptamer that binds to a target gene. It does not hybridize to the DNA or mRNA but instead identifies a cell surface marker that is specific for the Similar to antibodies that bind to epitopes on the surface of cells, It is a polynucleic acid.

[0255] In some cases, the binding moiety is a peptide. In some cases, the peptide is about 1 to about 3 In some cases, the peptides may be from about 1.2 to about 2.8 kDa, from about 1.5 to about In some embodiments, the peptide comprises a bicyclic peptide having a molecular weight of about 2.5 kDa, or about 1.5 to about 2 kDa. In some cases, the bicyclic peptide is a constrained bicyclic peptide. (constrained bicyclic peptide). Some examples In the case of the peptide moiety, the peptide is a bicyclic peptide (e.g., Bicycle Therapeutic cs bicycles).

[0256] In further instances, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. antibody-recruiting small molecule In some cases, the antibody-mobilizing small molecule comprises a target-binding end and an antibody-binding end, The binding end can then recognize and interact with cell surface receptors. For example, In some cases, targets containing glutamate urea compounds The binding terminus allows interaction with PSMA, thereby binding to cells expressing PSMA. In some instances, the binding moiety enhances antibody interactions as described by Zhang et al. , “A remote arene-binding site on prosta specific membrane antigen revealed by antibody-recruiting small molecules,” J Am Chem Soc. 132(36): 12711-12716 (2010 ); or McEnaney, et al., “Antibody-recruitment iting molecules: an emerging paradigm fo r engaging immune function in treating h ACS Chem Biol. 7(7): 1139 -1151 (2012).

[0257] bond chemistry In some embodiments, polynucleic acid molecule B is linked to a binding moiety. So, the binding moiety can be an amino acid, peptide, polypeptide, protein, antibody, antigen, or toxin. , hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polyethylene glycosides Polymers such as glycols and polypropylene glycols, as well as all of these classes of materials Additional examples of binding moieties include cholesterol, Phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g. For example, saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and In some instances, the binding moiety includes an antibody or a binding moiety thereof. In some instances, the polynucleic acid molecule is further linked to a polymer, Optionally, it is conjugated to an endosomolytic moiety.

[0258] In some embodiments, the polynucleic acid molecule is synthesized by a chemical ligation process. In some instances, the polynucleic acid molecule is linked to the binding moiety by natural ligation. In some instances, the linkage is as described below. Dawson, et al. “Synthesis of proteins by native chemical ligation,” Science 19 94, 266, 776-779; Dawson, et al. tion of Reactivity in Native Chemical Li gation through the Use of Thiol Additive s,” J. Am. Chem. Soc. 1997, 119, 4325-43 29; Hackeng, et al. “Protein synthesis b y native chemical ligation:Expanded scop e by using straightforward methodology. ” Proc. Natl. Acad. Sci. USA 1999, 96, 1 0068-10073; or Wu, et al. “Building com plex glycopeptides: Development of a cys teine-free native chemical ligation prot ocol,” Angew. Chem. Int. Ed. 2006, 45, 4 116-4125. In some instances, the conjugation is as described in U.S. Pat. No. 8,936,910. In some embodiments, the polynucleic acid molecule is a naturally occurring ligation The antibodies can be linked to binding moieties site-specifically or non-specifically via ligand chemistry.

[0259] In some instances, the polynucleic acid molecule may be a "traceless" capsule. Attached to the binding moiety in a site-directed manner using RING technology (PhiloChem) In some instances, the "traceless" coupling technique involves the use of a carboxylate that contains an aldehyde group. The polynucleic acid molecule is then linked to a 1,2-aminothiol group at the N-terminus of the linking moiety. Ru. (see Casi et al., “Site-specific trace less coupling of potent cytotoxic drugs to recombinant antibodies for pharmacode livery,” JACS 134(13): 5887-5892 (2012) reference)

[0260] In some instances, the polynucleic acid molecule utilizes unnatural amino acids incorporated into the binding moiety. In some instances, the non-natural amino acid is attached to the binding moiety in a site-directed manner. The acid includes p-acetylphenylalanine (pAcPhe). The keto group of Phe can be linked to an alkoxy-amine derived linker to form an oxime bond. (Axup et al., “Synthesis of s ite-specific antibody-drug conjugates us ing unnatural amino acids,” PNAS 109(40) : 16101-16106 (2012)).

[0261] In some instances, the polynucleic acid molecules are synthesized in a site-directed manner utilizing an enzyme-catalyzed process. In some instances, the site-directed method is SMARTag ( In some instances, SMARTag™ technology is used. The technique uses the formylglycine-producing enzyme ( generation of formylglycine (FGly) residues from cysteine ​​by FGE; and Alkyl cation via hydrazino-Pictet-Spengler (HIPS) ligation The subsequent conjugation of FGly to a chiral hydrazine-functionalized polynucleic acid molecule (Wu et al. al., “Site-specific chemical modification n of recombinant proteins produced in ma mmalian cells by using the genetically e ncoded aldehyde tag,” PNAS 106(9): 3000- 3005 (2009); Agarwal, et al., “A Pictet- Spengler ligation for protein chemical m odification,” PNAS 110(1): 46-51 (2013) reference)

[0262] In some instances, the enzyme-catalyzed process uses microbial transglutaminase (mTG). In some instances, the polynucleic acid molecule is selected from the group consisting of a microbial transglutaminase catalyzed process. In some instances, mTG is attached to the binding moiety using a glutamine in the recognition sequence. and catalyzing the formation of a covalent bond between the amide side chain of a functionalized polynucleic acid molecule. In some cases, mTG is expressed in Streptomyces mobaraensis. myces mobarensis (Strop et al., “ Location matters: site of conjugation mo dulates stability and pharmacokinetics o f antibody drug conjugates,”Chemistry an d Biology 20(2) 161-167 (2013)

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

[0264] In some examples, the polynucleic acid molecule is a nucleic acid molecule as described in U.S. Patent Publication No. 2015 / 0105539 and U.S. Patent Publication No. 2015 / 0105539. and attached to the binding moiety by the methods described in US Pat. No. 2015 / 0105540.

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

[0266] In some instances, the antibody or binding fragment thereof is recombinantly expressed, and The nucleic acid encoding the antibody or binding fragment thereof may be a chemically synthesized oligonucleotide. Reotide (e.g., Kutmeier et al., 1994, BioTechn iques 17:242), which encodes an antibody. Synthesis of overlapping oligonucleotides containing portions of the sequence, Annealing and ligation of nucleotides, followed by synthesis of the ligated oligonucleotides. It is involved in PCR amplification of nucleotides.

[0267] Alternatively, the nucleic acid molecule encoding the antibody may be a nucleic acid sequence that hybridizes to the 3' and 5' ends of the sequence. by PCR amplification using synthetic primers that can be used to identify specific gene sequences By cloning using oligonucleotide probes specific for the sequence, Any suitable source (e.g., antibody cDNA library or any nucleic acid expressing immunoglobulin) may be used. The cDNA may be optionally generated from a cDNA library generated from tissues or cells.

[0268] In some instances, the antibody or conjugate thereof is cultured in rabbit serum to generate polyclonal antibodies. or more preferably, by immunizing an animal such as a hamster, e.g., as described in the Kohler and Milstein (1975, Nature 256:495-497) or as described by Kozbor et al. (1983, Imm Uniology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Ther apy, Alan R. Liss, Inc., pp. 77-96) Alternatively, the antibody may be produced by producing a monoclonal antibody as described above. In addition, clones encoding at least the Fab portion of an antibody are generated that produce FAbs that bind to specific antigens. To clone fragments, use a Fab expression library (e.g., Huse et al. ., 1989, Science 246:1275-1281) By screening or antibody libraries (Clackson et al. , 1991, Nature 352:624; Hane et al., 199 7 Proc. Natl. Acad. Sci. USA 94:4937) can be optionally obtained by screening.

[0269] In some embodiments, together with genes for suitable biologically active human antibody molecules, By splicing genes from mouse antibody molecules of appropriate antigen specificity, mela antibody" (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) Chimeric antibodies are antibodies in which different parts are derived from a mouse monoclonal antibody. and human immunoglobulin constant regions (e.g., humanized antibodies). These molecules are derived from various animal species, including

[0270] In some embodiments, single chain antibodies (U.S. Pat. No. 4,694,778; Birth d, 1988, Science 242:423-42; Huston et a. l., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; and Ward et al., 1989, Nature 3 34:544-54) are suitable for producing single chain antibodies. Single chain antibodies are composed of heavy or light chain fragments of the Fv region bounded by an amino acid bridge. The resulting polypeptide chain is a single chain. Optionally, techniques for the assembly of functional Fv fragments such as those described in Skerra et al. are also used. et al., 1988, Science 242:1038-1041).

[0271] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or an antibody The nucleotide sequence of the vector can be introduced into the host cell using conventional techniques (e.g., electroporation, liposome transfer, etc.). The vector is introduced into the host cell by a method such as transfection, calcium phosphate precipitation, etc. The transfected cells are then cultured by conventional techniques to produce antibodies. In certain embodiments, expression of the antibody is constitutive, inducible, or tissue-specific. It is regulated by a promoter.

[0272] In some embodiments, various host-expression vector systems can be used to express the antibodies described herein. Such host expression systems are utilized to express the polypeptides or binding fragments thereof. Not only do they represent the vehicle in which the coding sequence of the antibody is generated and subsequently purified, but they also represent the appropriate nucleic acid. When modified or transfected with a nucleic acid coding sequence, the antibody or also refers to cells expressing the binding fragments in situ. These include, but are not limited to, However, recombinant bacteriophage DNA, plasmid DNA, or antibodies or Bacteria transformed with cosmid DNA expression vectors containing the ligation fragment coding sequences ( Microorganisms such as Escherichia coli and Bacillus subtilis; antibody or binding fragment coding sequences Yeast (e.g., Saccharomyces pichia) transformed with a recombinant yeast expression vector containing ); into a recombinant viral expression vector containing the antibody or binding fragment thereof coding sequence. Infected insect cell systems (e.g., baculovirus); recombinant viral expression vectors (e.g., , Cauliflower mosaic virus (CaMV) and Tobacco mosaic virus (TMV) ) or a recombinant plasmid containing the antibody or its binding fragment coding sequence. a plant cell line transformed with a smid expression vector (e.g., a Ti plasmid); or , the genome of mammalian cells (e.g., metallothionein promoter), or Animal viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K A mammalian cell line containing a recombinant expression construct comprising a promoter derived from Cellular lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells).

[0273] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express the antibodies are engineered. Rather than using expression vectors containing an origin, the host cell must contain appropriate expression control elements (e.g. For example, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc. The exogenous DNA is transformed into a host cell by a selectable marker. After induction of A, cells were engineered to grow in enriched medium for 1-2 days and then transferred to selective medium. The selectable marker on the recombinant plasmid confers resistance to the selection. The cells stably integrate the plasmid into their chromosomes, grow, and are cloned to produce cells. This method allows the formation of foci that expand into the cell line. It can be advantageously used to engineer cell lines which express the fragment.

[0274] In some examples, but not limited to, tk cells, hgprt cells, or a Herpes simplex virus thymidine kinase (HSK) is recruited to prt cells (Wigler et al. l., 1977, Cell 11:223), hypoxanthine-guanine phosphoribonuclease Silyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and Ad. Nin phosphoribosyltransferase (Lowy et al., 1980, Ce Many selection systems are available, including the use of the genotype gene (Ill 22:817). Resistance is used as a selection criterion for genes that confer resistance to methotrexate. dhfr (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 19 81, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1 981, Proc. Natl. Acad. Sci. USA 78:2072) ;neo(Clinical Pha racy 12:488-505; Wu and Wu, 1991, Bioth erapy 3:87-95; Tolstoshev, 1993, Ann. Re v. Pharmacol. Toxicol. 32:573-596; Mulli gan, 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 (Santer re et al., 1984, Gene 30:147). Known methods in the art for NA technology are generally described by Ausubel et al. l. (eds., 1993, Current Protocols in Mol etcular Biology, John Wiley & Sons, NY; riegler, 1990, Gene Transfer and Express ion, A Laboratory Manual, Stockton Press , NY; and in Chapters 12 and 13, Dracopo li et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, N. Y.; Colberre-Garapin et al., 1981, J. Mo l. Biol. 150:1).

[0275] In some instances, antibody expression levels are increased by vector amplification (see for review). For this purpose, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammals ian cells in DNA cloning, Vol. 3. (Acade (See Microbiol Press, New York, 1987.) Antibody-expressing vectors If the marker in the gene expression system is amplifiable, the level of the inhibitor present in the host cell culture can be determined. As the level increases, the number of copies of the marker gene increases. The amplified region is then inserted into the nucleotide sequence of the antibody. The increased production of antibodies due to its association with the tid sequence (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0276] In some instances, methods known in the art for purifying or analyzing antibodies or antibody conjugates are well known in the art. Any known method may be used, for example, chromatography (e.g., ion exchange, affinity chromatography, etc. The affinity to specific antigens after protein A and sizing column cleavage are important. chromatography), centrifugation, differential solubility, or other methods for the purification of proteins. Standard technology methods are used. Exemplary chromatographic methods include limited Although not used, strong anion exchange chromatography, hydrophobic interaction chromatography These include chromatography, size exclusion chromatography, and fast protein liquid chromatography. Can be obtained.

[0277] Polymer Binding Moiety In some embodiments, the polymer moiety C is a polynucleic acid molecule described herein. , or a binding moiety described herein, or a combination thereof. In some instances, the polymer moiety C is attached to a polynucleic acid molecule. In other cases, the polymer moiety C is linked to a polynucleic acid molecule binding moiety. In a further case, the polymer moiety C is bonded as illustrated above. can be.

[0278] In some examples, the polymer portion C may be a branched or unbranched monomer, and and / or natural or synthetic, consisting of long chains of cross-linked networks of two- or three-dimensional monomers. In some examples, the polymer moiety C is a polysaccharide, lignin, rubber, , or polyalkylene oxides (e.g., polyethylene glycol). In some examples, the at least one polymer moiety C can include, but is not limited to, alpha- Dihydroxyl polyethylene glycol, omega-dihydroxyl polyethylene glycol , biodegradable lactone-based polymers, e.g., polyacrylic acid, polylactide acid ( PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin Fin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (PET, PETG), Polyethylene terephthalate (PETE), Polytetramethyl Polyethylene glycol (PTG), polyurethane, and mixtures of these. As used herein, a mixture is similar to the reference to a block copolymer. In some cases, block copolymers refer to the use of different polymers within the same compound. is a polymer in which at least one portion of the polymer is constructed from monomers of another polymer. In some examples, the polymer moiety C comprises a polyalkylene oxide. In examples, the polymer moiety C comprises PEG. In some examples, the polymer moiety C comprises polyethylene. These include polyethylenimide (PEI) or hydroxyethyl starch (HES).

[0279] In some instances, C is a PEG moiety. In some instances, the PEG moiety is a polynucleic acid. The binding moiety is attached at the 3' end of the polynucleic acid molecule, while the binding moiety is attached at the 5' end of the molecule. In some instances, the PEG moiety is attached at the 3' end of the polynucleic acid molecule; In some instances, the PEG moiety is attached at an internal site of the polynucleic acid molecule. In some examples, the PEG moiety, the linking moiety, or a combination thereof is In some instances, the conjugate is a direct conjugate. In some instances, the attachment is via native ligation.

[0280] In some embodiments, the polyalkylene oxide (e.g., PEG) is polydisperse or In some instances, polydisperse materials are compounds that are monodisperse or have an average weight (weight average) size. It includes a dispersed distribution of materials of different molecular weights, characterized by size and dispersity. In examples, monodisperse PEG contains molecules of one size. is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), The molecular weight represents the average molecular weight of the polyalkylene oxide (eg, 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, 1 100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1 800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2 600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3 750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6 000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20 ,000, 35,000, 40,000, 50,000, 60,000, or 100 ,000Da.

[0282] In some embodiments, C is a polyalkylene oxide (e.g., PEG). , about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1 800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2 600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3 750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6 000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20 ,000, 35,000, 40,000, 50,000, 60,000, or 100 In some embodiments, C is PEG and has a molecular weight of about 2,000 Da. 00, 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, 20,00 0, 35,000, 40,000, 50,000, 60,000, or 100,00 In some instances, C has a molecular weight of about 200 Da. In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 40 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, C has a molecular weight of about 600 Da. In some instances, C has a molecular weight of about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about In some instances, the molecular weight of C is about 1300 Da. In some examples, the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some examples, the molecular weight of C is about 2100 Da. In some instances, C has a molecular weight of about 2200 Da. In some instances, C has a molecular weight of about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, 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 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about In some instances, the molecular weight of C is about 3350 Da. In some examples, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some examples, the molecular weight of C is about 5500 Da. In some instances, C has a molecular weight of about 6000 Da. In some instances, C has a molecular weight of about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da. In some instances, C has a molecular weight of about 20,000 Da. In some instances, C has a molecular weight of about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,0 In some instances, the molecular weight of C is about 60,000 Da. In this example, C has a molecular weight of about 100,000 Da.

[0283] In some embodiments, the polyalkylene oxide (e.g., PEG) is dispersed in a dispersion P EG, and the dispersed PEG is a polymeric PEG containing one or more repeating ethylene oxide units. In some instances, the dispersed PEG (dPEG) may be 2-60, 2-50, or 2 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 In some instances, the dPEG contains about 2 or more repeating ethylene oxide units. In some instances, dPEG contains about 3 or more repeating ethylene oxide units. In some instances, dPEG contains about 4 or more repeating ethylene oxide units. In some examples, the dPEG contains about 5 or more repeating ethylene oxide units. dPEG contains about 6 or more repeating ethylene oxide units. In some examples, dPEG In some instances, the dPEG contains about 8 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 9 or more repeating ethylene oxide units. In some instances, dPEG contains about 10 or more repeating ethylene oxide units. In some instances, dPEG contains about 11 or more repeating ethylene oxide units. In some embodiments, dPEG contains about 12 or more repeating ethylene oxide units. In some instances, dPEG contains about 13 or more repeating ethylene oxide units. In some instances, the dPEG contains about 14 or more repeating ethylene oxide units. In some instances, the 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, the dPEG contains about 17 or more repeating ethylene oxide units. dPEG contains about 18 or more repeating ethylene oxide units. In some examples, dPE G comprises about 19 or more repeating ethylene oxide units. In some embodiments, dPEG comprises about In some instances, the dPEG contains about 22 or more repeating ethylene oxide units. In some instances, the dPEG contains about 24 or more repeating ethylene oxide units. In some embodiments, the dPEG contains about 26 or more repeating ethylene oxide units. In some instances, the dPEG contains about 28 or more repeating ethylene oxide units. In some instances, 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 instances, dPEG contains about 40 or more repeating ethylene oxide units. In some instances, the dPEG contains about 42 or more repeating ethylene oxide units. In some examples, dPEG contains about 48 or more repeating ethylene oxide units. In an example, the dPEG contains about 50 or more repeating ethylene oxide units. The dPEG may be a pure (e.g., about 95%, 98%, 99%, or even 99.5%) starting material. In some cases, dPEG is synthesized stepwise from the matrix as a single molecular weight compound. In some cases, the dPE described herein has a specific molecular weight, rather than a specific molecular weight. G is dPEG from Quanta Biodesign, LMD.

[0284] In some embodiments, the polymer moiety C is a cationic mucic acid-based polymer. In some instances, the cMAP comprises at least one repeat subunit. The subunit structure is represented by formula (V):

[0285] [ka]

[0286] wherein m at each occurrence is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Preferably, n is 4-6, or 5; and n, at each occurrence, is independently 1, 2, In some embodiments, m and n are, for example, about 10. do.

[0287] In some instances, cMAP can be further conjugated to a PEG moiety to form a cMAP-PEG copolymer. mer, mPEG-cMAP-PEG triblock polymer, or cMAP-PEG In some instances, the PEG moiety is about 500 In some examples, the PEG moiety ranges from about 500 Da to about 50,000 Da. a to about 1000 Da, more than 1000 Da to about 5000 Da, more than 5000 Da ~ approx. 10,000 Da, > 10,000 ~ approx. 25,000 Da, 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges It is a combination.

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

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

[0290] Endosomolytic moiety In some embodiments, the molecule of formula (I): AXBYC may further include an additional conjugated moiety. In some examples, the additional binding moiety is an endosomolytic moiety. In some cases, the endosomolytic moiety may be a cellular compartment releasing component, e.g., an endosome, Lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or cells The cells are released from any of the compartments of the cell known in the art, such as the endoplasmic reticulum or other In some cases, the endosomolytic moiety is a compound that can a lytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or In some cases, the endosomolytic moiety is an endosomolytic moiety. In other cases, the endosomolytic moiety comprises an endosomolytic polypeptide. Contains a soluble polymer.

[0291] Endosomolytic Polypeptides In some embodiments, the molecule of formula (I): AXBYC is an endosomolytic In some embodiments, the compound of formula (V): A-( X 1 -B) n Or formula (II): AX 1 -(BX 2 -C) n The molecule is an endosome In some cases, the endosomolytic polypeptide further binds to the Tides are pH-dependent membrane active peptides. In some cases, endosomolytic polypeptides In additional cases, endosomolytic polypeptides are In some instances, the endosomolytic polypeptide is an IN F, melittin, mucin, or their respective derivatives. In some examples, the endosomolytic polypeptide comprises INF or a derivative thereof. In some cases, the endosomolytic polypeptide comprises melittin or a derivative thereof. In some cases, the endosomolytic polypeptide comprises a mucin or a derivative thereof.

[0292] In some instances, INF7 is a 24-residue polypeptide, and these sequences include the CG IFGEIEELIEEGLENLIDWGNA (SEQ ID NO:1), or Contains GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO:2) In some instances, INF7 or a derivative thereof comprises the following sequence: GLFEAIE GFIENGWEGMIWDYGSGSCG (SEQ ID NO: 3), GLFE AIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 4), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K (GalNAc)2 (SEQ ID NO: 5).

[0293] In some cases, melittin is a 26 residue polypeptide, the sequence of which is LKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 6), or G Contains IGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:7) In some instances, melittin may be prepared using the polysaccharides described in U.S. Pat. No. 8,501,930. Contains a peptide sequence.

[0294] In some instances, mucins are derived from the venom of the scorpion Mesobuthus eupeus. In some cases, mucins are mucin-13, an antimicrobial peptide (AMP) derived from the mucin gland. (These sequences are: IFGAIAGLLKNIF-NH2 (SEQ ID NO:8) ) and mucin-18 (these sequences are SEQ ID NO:9)).

[0295] In some instances, the endosomolytic polypeptide has a sequence similar to that of INF7 or its derivatives. Derivatives, melittin or its derivatives, or mucin or its derivatives At least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity In some examples, the endosomolytic moiety comprises a polypeptide that is soluble in INF7. or derivatives thereof, melittin or derivatives thereof, or mucin or derivatives thereof .

[0296] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% %, 96%, 97%, 98%, 99%, or 100% sequence identity In some examples, the endosomolytic moiety comprises a nucleotide sequence similar to that of SEQ ID NO:1. At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some examples, the endosomolytic moiety comprises a polypeptide having SEQ ID NO: At least 50%, 55%, 60%, 65%, 70%, 75% for 2-5, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Optionally, the endosomolytic moiety comprises a polypeptide having sequence identity to Optionally, the endosomolytic moiety comprises SEQ ID NO:1. D NO:2-5. O:1. Optionally, the endosomolytic moiety comprises SEQ ID NO:2- It consists of 5.

[0297] In some instances, the endosomolytic moiety is melittin or a derivative thereof. In some examples, the endosomolytic moiety has a low affinity to SEQ ID NO:6 or 7. At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, Polypeptides with 95%, 96%, 97%, 98%, 99% or 100% sequence identity In some examples, the endosomolytic moiety comprises a peptide having SEQ ID NO: At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% for 6 %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity In some examples, the endosomolytic moiety includes a polypeptide having SEQ ID NO: At least 50%, 55%, 60%, 65%, 70%, 75% for ID NO:7, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Optionally, the endosomolytic moiety comprises a polypeptide having sequence identity to Optionally, the endosomolytic moiety comprises SEQ ID NO:6. Optionally, the endosomolytic moiety comprises SEQ ID NO: 6. Optionally, the endosomolytic moiety consists of SEQ ID NO:7. do.

[0298] In some instances, the endosomolytic moiety is a mucin or a derivative thereof. In some examples, the endosomolytic moiety has at least one amino acid sequence similar to SEQ ID NO:8 or 9. At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9 Polypeptides with 5%, 96%, 97%, 98%, 99%, or 100% sequence identity In some examples, the endosomolytic moiety comprises a peptide of SEQ ID NO:8 At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some examples, the endosomolytic moiety includes a polypeptide having SEQ ID NO:1. D NO:9 at least 50%, 55%, 60%, 65%, 70%, 75%, 8 0%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Optionally, the endosomolytic moiety comprises a polypeptide having sequence identity to S Optionally, the endosomolytic moiety comprises SEQ ID NO:8. 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 is selected from the group consisting of Bcl-2 and / or Bcl-x L Bak BH3 polypeptide induces apoptosis through antagonism of inhibitory gene targets such as In some instances, the endosomolytic moiety includes a cleavage site as described in Albarran, et al. al., “Efficient intracellular delivery o fa pro-apoptotic peptide with a pH-resp onsive carrier,” Reactive & Functional P The Bak BH3 polypeptide described in Oligonucleotides 71: 261-265 (2011) Contains lipids.

[0303] In some examples, the endosomolytic moiety is a compound described in PCT Publication No. WO2013 / 16 6155 and WO2015 / 069587. , cell-penetrating polypeptides).

[0304] Endosomolytic polymers In some embodiments, the compound of formula (V): A-(X 1 -B) n Or formula (VI): A -X 1 -(BX 2 -C) n The molecule is further conjugated with an endosomolytic polymer. As used herein, endosomolytic polymers include linear, branched networks. In some cases, endosomes are used. Mulubricating polymers are homopolymers or copolymers that contain two or more different types of monomers. In some cases, the endosomolytic polymer is a polycationic polymer. In other cases, the endosomolytic polymer is a polyanionic polymer.

[0305] In some instances, the polycationic polymer may be positively, neutrally, or negatively charged. In other cases, polycations contain monomeric units that are charged, resulting in a net positive charge. Polymers include non-polymeric molecules that contain two or more positive charges. Examples of the mer 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 (DMAE MA), or N,N-diethylaminoethyl methacrylate (DEAEMA). Can be obtained.

[0306] In some cases, the polyanionic polymer may be positively, neutrally, or negatively charged. In other cases, the polyanion polymer comprises a monomer unit having a net charge of 0.01 to 0.01, and the net charge is negative. Polymers include non-polymeric molecules that contain two or more negative charges. The mer is a p(alkyl acrylate) (e.g., poly(propyl acrylic acid) (PPAA ) or poly(N-isopropylacrylamide) (NIPAM). Examples of this are PP75, Khormaee, et al., “Edosomolytic anionic polymer for the cytoplasmic del ivery of siRNAs in localized in vivo app lications,” Advanced Functional Material s 23: 565-574 (2013) Contains L-lysine isophthalamide polymer.

[0307] In some embodiments, the endosomolytic polymers described herein are A pH-responsive endosomolytic polymer. pH-responsive polymers respond to changes in the pH of the environment. It contains a polymer that either increases in size (swells) or collapses depending on the and chitosan are examples of pH-responsive polymers.

[0308] In some instances, the endosomolytic moieties described herein are membrane disruptive polymers. (membrane-disruptive polymer). The membrane disruptive polymer may be a cationic polymer, a neutral or hydrophobic polymer, or an anionic polymer. In some instances, the membrane disruptive polymer is a hydrophilic polymer.

[0309] In some instances, the endosomolytic moieties described herein are capable of binding to pH-responsive membrane An exemplary pH-responsive membrane disruptive polymer is p(alkylacryloyl) poly(N-isopropylacrylamide) (NIPAM) copolymer, succinyl These include dimethylated p(glycidol), and p(β-malic acid) polymers.

[0310] In some instances, the p(alkyl acrylic acid) may be poly(propyl acrylic acid) (poly PAA), poly(methacrylic acid) (PMAA), poly(ethyl acrylic acid) (PEAA), and poly(propylacrylic acid) (PPAA). In some examples, p(alkyl (acrylic acid) was synthesized by Jones, et al., Biochemistry Jou p(alkylacrylic acid) described in Journal of Polymer Science 372: 65-75 (2003) Includes.

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

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

[0313] In some embodiments, the pH responsive membrane disruptive polymer is a pyridyl disulfide pyridyldisulfide acrylate (PDSA) polymers, such as Poly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-c o-PDSA), poly(MAA-co-BA-co-PDSA), poly(EAA-co- Poly(PAA-co-BA-co-PDSA) or poly(PAA-co-BA-co-PDSA) polymer (El-Sayed, et al., “Rational design of composition and activity correlation s for pH-responsive and glutathione-reac tive polymer therapeutics,” Journal of C ontrolled Release 104: 417-427 (2005); For more information, see Flanary et al., “Antigen delivery with poly(propylacrylic acid) conjugation en hanced MHC-1 presentation and T-cell act ivation,” Bioconjugate Chem. 20: 241-248 (2009)

[0314] In some embodiments, the pH-responsive membrane disruptive polymer comprises the basic structure Cytolytic polymers include:

[0315] [ka]

[0316] In some examples, the endosomolytic moiety described herein can be coupled to an additional conjugate, For example, a polymer (e.g., PEG) or a modified polymer (e.g., cholesterol The polymer is further attached to a polymer modified with a carboxyl group.

[0317] In some instances, the additional conjugate includes a detergent (e.g., Triton X-100). In some examples, the endosomolytic moieties described herein are surfactants (e.g., e.g., Triton X-100) and a polymer (e.g., poly(amidoamine)) In some examples, the endosomolytic moieties described herein include poly(amino acids). Amidoamine)-Triton X-100 conjugate (Duncan, et al., “A p olymer-Triton X-100 conjugate capable of pH-dependent red blood cell lysis: a mo del system illustrating the possibility of drug delivery within acidic intracell ular compartments,” Journal of Drug Targ eting 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 compound of formula (V): A-(X 1 -B) n Or the formula (V I):AX 1 -(BX 2 -C) n The molecules are endosomolytic lipids (e.g., fusogenic An exemplary fusogenic lipid is 1,2-dileoyl. l)-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z, 9Z, ​​28Z, 31Z)-Heptatriaconta-6, 9, 28, 31-tetraene-19 -ol (Di-Lin), and N-methyl(2,2-di(9Z,12Z)-octadecanol DLin(9,12-dienyl)-1,3-dioxolan-4-yl)methanamine -k-DMA) and N-methyl-2-(2,2-di(9Z,12Z)-octadeca- 9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC) nothing.

[0319] In some examples, the endosomolytic moiety is a compound described in PCT Publication No. WO09 / 126, 933 (eg, fusogenic lipids).

[0320] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In this embodiment, the compound represented by formula (I): A-(X 1 -B) n Or formula (II): AX 1 -(BX 2 -C) n The molecule is further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as moieties include, but are not limited to, quinine, chloroquine, water Chloroquine oxide, amodiaquine (carnoquines), amoxicilloquine, primaquine , mefloquine, nivaquines, halofantrine, quinoneimines, or In some examples, the quinoline endosomolytic moiety includes a combination of: Including but 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; 7-Hydroxy-4-(4-diethyl- 7-Chloro-4-(4-diethylamino- 1-Butylamino)quinoline (desmethylchloroquine); 7-Fluoro-4-(4-diphenyl 4-(4-diethylamino-1-butylamino)quinoline; 7-Hydroxy-4-(4-diethylamino-1-butylamino)quinoline Phosphorus;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)chloride Nolin; 7-Fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamine 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-methylbutyl 7-Fluoro-4-(1-carboxy-4-diethyl-amino-1- 4-(1-carboxy-4-diethylamino-1-methyl)quinoline 7-Hydroxy-4-(1-carboxy-4-diethylamino)quinoline; 7-Fluoro-4-(4-ethyl-(2-hydro) 4-(4-ethyl-(2-methyl-1-methoxyethyl)amino-1-methylbutylamino)quinoline; -Hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline;7-Hydroxy C-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino) Quinoline; Hydroxychloroquine phosphate; 7-Chloro-4-(4-ethyl-(2- Hydroxyethyl-1)-amino-1-butylamino)quinoline (desmethylhydroxy Chloroquine; 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) 7-Chloro-4-(1-carboxy-4 -Ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-fluro O-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butanol 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-methyl butylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyphenyl) 4-(1-Carboxy)-amino-1-methylbutylamino)quinoline; -4-Ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline 7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-a 8-[(4-aminopentyl)amino-6- Methoxydihydrochloridequinoline; 1-Acetyl-1,2,3,4-tetrahydroquinoline Phosphorus;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride 1-Butyryl-1,2,3,4-tetrahydroquinoline;3-chloro-4-(4-hydro Roxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinol 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxy- Nolin; 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidone) Lysinyl)-2,5-xylidinoquinoline(4-[(4-diethylamino)-1-methyl butyl-amino]-6-methoxyquinoline; 4-(4-hydroxy-α,α'-bis(2 -Methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-[(4-diethyl amino)-1-methylbutyl-amino]-6-methoxyquinoline;3,4-dihydro-1- (2H)-Quinolinecarboxaldehyde; 1,1'-Pentamethylenequinolinium iodide 8-Quinolinol sulfate and amino, aldehyde, carboxylic acid, hydroxyl, halide aryl, keto, sulfhydryl, and vinyl derivatives or analogs thereof. In some instances, the endosomolytic moiety is a cytosolic moiety as described by Naisbitt et al. 997, J Pharmacol Exp Therapy 280:884-893 ) and small molecules described in U.S. Pat. No. 5,736,557. In certain embodiments, the endosomolytic moiety is nigericin or a conjugate thereof, e.g., folic acid -Ester conjugate of nigericin, amide conjugate of folic acid-nigericin, or amide conjugate of folic acid-nigericin In some instances, the endosomolytic moiety is a carbamate bond of R angasamy, et. al., “New mechanism for re lease of endosomal contents: osmotic lys is via nigericin-mediated K+ / H+ exchange ,” Bioconjugate Chem. 29:1047-1059 (2018 ) is nigericin.

[0321] Linker In some embodiments, the linkers described herein are cleavable linkers. or a non-cleavable linker. In some instances, 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 is a polymeric Refers to a linker that does not contain repeating units of the monomers produced by the process. Examples of the rema linker include, but are not limited to, C 1 -C 6 Alkyl groups (e.g., C 5 , C 4 , C 3 , C 2 , or C 1 Alkyl groups), homobifunctional cross-linkers, heterobifunctional Cross-linker, peptide linker, traceless linker, self-immolative linker, maleimide In some cases, the linker may be a non-poly Marlin Carr is C 1 -C6 Alkyl groups (e.g., C 5 , C 4 , C 3 , C 2 , or C 1 alkyl group), homobifunctional cross-linker, heterobifunctional cross-linker, peptide linker linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or includes combinations thereof. In further cases, the non-polymeric linker may include more than two Linkers of the same type, e.g., more than two homobifunctional cross-linkers, or In further instances, the non-polymeric linker is optionally Contains one or more reactive functional groups.

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

[0324] In some examples, the linker comprises a homobifunctional linker. Examples of anchors include, but are not limited to, Lomant's reagent dithiobis(succinimidyl phosphate propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate Disuccinimidyl suberate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl Disuccinimidyl suberate (BS), Disuccinimidyl tartrate (DST), Disulfosuccinimidyl Succinimidyl tartrate (Sulfo-DST), Ethylene glycobis(succinimidyl Succinimidyl glutarate (EGS), N,N'-di Succinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethy Dimethylsuberimidate (DMP), Dimethylsuberimidate (DMS), Dimethyl Thiol-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-( 2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimide hexa (BMH), e.g., 1,5-difluoro-2,4-dinitrobenzene, 1,3-di Halogenated aryl-containing compounds such as fluoro-4,6-dinitrobenzene (DFDNB ), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), β-(4-azidosalicylamido)ethyl disulfide (BASED), formamide Aldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, azide Pinic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfone Acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexame Examples include ethylene bis(iodoacetamide).

[0325] In some embodiments, the linker comprises a heterobifunctional linker. Bifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-bridged linkers. anchors, such as N-succinimidyl 3-(2-pyridyldithio)propionate, (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate ,(LC-sPDP), a 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) m-maleimide benzyl cyclohexane-1-carboxylate (sulfo-sMCC) Benzoyl-N-hydroxysuccinimide esters (MBs), m-maleimidobenzoyl Sulfo-MB, N-hydroxysulfosuccinimide ester Di(4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacteyl) aminobenzoate (sulfo-sIAB), succinimide Dimethyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl N-(γ-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB) thyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6 -[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sI AXX), Succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane San-1-carboxylate (sIAC), succinimidyl 6-((((4-iodo Acetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), 4-(4-N-ma N-Maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclo Hexane-1-carboxyl-hydrazide-8 (M 2 C 2 H), 3-(2-pyridyldithio e) Carbonyl-reactive and sulfhydryl compounds such as propionyl hydrazide (PDPH) Reactive cross-linkers, amine reactive and photoreactive cross-linkers, e.g., N-hydroxyl Roxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysul Fosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl Imidyl-(4-azidosalicylamide)hexanoate (sulfo-NH-LC-AsA ), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3'-di Thiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzo HsAB, N-hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HsAB), N-Succinimidyl-6-(4'-azido-2'-nitrophenyl) 4'-nylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'- Azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N- 5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), Sulfos Chuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-di Thiopropionate (sAND), N-succinimidyl-4(4-azidophenyl) 1 , 3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azide phenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimide 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinic acid Imidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3 '-Dithiopropionate (sAED), Sulfosuccinimidyl 7-azido-4-methyl Chillcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyridine Rubato (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoro Propionate (PNP-DTP), a sulfhydryl-reactive and photoreactive cross-linker For example, 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane ( AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridin a) propionamide (APDP), benzophenone-4-iodoacetamide, Benzophenone-4-maleimidocarbonyl reactive and photoreactive cross-linkers, e.g. For example, ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive Cross-linkers, such as 4-(ρ-azidosalicylamido)butylamine (AsBA); and arginine-reactive and photoreactive cross-linkers, e.g., ρ-azidophenyl Examples include glyoxal (APG).

[0326] In some instances, the linker comprises a reactive functional group. In some instances, the reactive functional group is , which contains a nucleophilic group reactive to an electrophilic group present in the binding moiety. Exemplary electrophilic groups are aldehydes, amides, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides In some embodiments, the reactive functional group comprises an aldehyde, Exemplary nucleophilic groups are hydrazide, oxime, amino, hydrazine, thiosemicarbamates, These include arylhydrazides, hydrazine carboxylates, and aryl hydrazides.

[0327] In some embodiments, the linker comprises a maleimide group. The maleimide group is also called a maleimide spacer. In some instances, the maleimide group is a caproic acid spacer. Optionally, phosphoric acid may be further included to form maleimidocaproyl (mc). The linker comprises maleimidocaproyl (mc). In another embodiment, the maleimide group is a succinimidyl group as described above. 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) , or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- Contains a maleimidomethyl group, such as 1-carboxylate (sulfo-sMCC).

[0328] In some embodiments, the maleimide group is a self-stabilizing maleimide. In the example, the self-stabilizing maleimide is involved in the intramolecular catalysis of thiosuccinimide ring hydrolysis. To provide this, diaminopropyl ether was added to incorporate a basic amino group adjacent to the maleimide. The maleimide is eliminated by the retro-Michael reaction using diphenyl ether (DPR). In some instances, the self-stabilizing maleimide is L yon, et al., “Self-hydrolyzing maleimide s improve the stability and pharmacology cal properties of antibody-drug conjugat es,” Nat. Biotechnol. 32(10):1059-1062 ( In some instances, the linker is a self-stabilizing maleimide group as described in Maleimide. In some instances, the linker is a self-stabilizing maleimide.

[0329] In some embodiments, the linker comprises a peptide moiety. The do portion comprises at least 1, 2, 3, 4, 5, or more than 6 amino acid residues. In some instances, the peptide moiety may be at most 1, 2, 3, 4, 5, 6, 7, or 8. In some examples, the peptide portion comprises about 2, about 3, about 4, about 5, Alternatively, the peptide moiety comprises about 6 amino acid residues. In some instances, the peptide moiety is a peptide moiety (e.g., enzymatically or chemically). In some examples, the peptidic moiety is a 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- In some examples, the linker comprises Val-Cit (Val-Cit Lulin), 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 cases, the linker comprises a peptide moiety such as Val-Cit. Thus, 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 its derivatives include para-aminobenzoic acid (PABA). In some instances, the benzoic acid group or its derivatives include γ-aminobutyric acid (GABA). nothing.

[0331] In some embodiments, the linker comprises, in any combination, a maleimide group, a peptide In some embodiments, the aryl group comprises one or more phenylmethylsulfonyl groups, a phenylmethylsulfonyl group ... and / or a benzoic acid group. , the linker is 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 instances, the peptide group is val-cit. In some instances, the benzoic acid group is P In some instances, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. Contains the mc-val-cit-PABA group.

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

[0333] In some embodiments, the linker is a dendritic linker. 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 is cleaved and then A linker moiety (e.g., an atom or a linker group) is connected to a linking moiety A, a polynucleotide B, The linker does not leave the endosomolytic moiety C or the endosomolytic moiety D. The sulfur linker includes, but is not limited to, a germanium linker, a silicon linker (s elenium linker), sulfur linker, selenium linker, nitrogen linker, phosphorus linker linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. In some cases, the linker may be a cyclic amine, as described in Hejesen, et al., “A raceless aryl-triazene linker for DNA-di rected chemistry,” Org Biomol Chem 11(15 ): 2493-2497 (2013) In some examples, the linker is a cyclic 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. Pat. ,783.

[0335] In some embodiments, the linker is a cyclic amine as described in U.S. Pat. Nos. 6,884,869; 7,498, ,298; No. 8,288,352; No. 8,609,105; or No. 8,69 No. 7,688; U.S. Patent Publication No. 2014 / 0127239; No. 2013 / 028919 No.2014 / 286970;No.2013 / 0309256;No.2015 / 037 360; or 2014 / 0294851; or PCT Publication WO20 15057699;WO2014080251;WO2014197854;WO201 4145090; or a linker described in WO2014177042.

[0336] In some embodiments, X, Y, and L are independently a bond or a linker. In some examples, X, Y, and L are independently a single bond. , Y, and L are independently linkers.

[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 C 1 - C 6 In some cases, X is, for example, C 5 , C 4 , C 3 , C 2 ,be IwaC 1 C such as alkyl group 1 -C6 It is an alkyl group. In some cases, C 1 -C 6 a The alkyl group is an unsubstituted C 1 -C 6 In the context of the linker, inter alia: When used in the context of X, alkyl means a saturated linear alkyl group containing up to 6 carbon atoms. In some instances, X is a non-polymeric phosphorus radical. In some embodiments, X is a homobifunctional linker or a heterobifunctional linker as described above. Optionally, X comprises a heterobifunctional linker. In some instances, X comprises sMCC. In other instances, X comprises C 1 -C 6 Optionally bonded to an alkyl group In another embodiment, X is a heterobifunctional linker having a C 1 -C 6 The alkyl group may optionally be In some additional examples, X comprises a homobifunctional sMCC linked to The linker does not include a heterobifunctional linker or a heterobifunctional linker.

[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 C 1 - C 6 In some embodiments, Y is an alkyl group. In some embodiments, Y is a homobifunctional linker as described above. In some examples, Y is a heterobifunctional linker as described above. In some examples, Y is a maleimidocaproyl (mc), such as described above. In some embodiments, Y comprises a Val- In some instances, Y contains a benzoic acid group, such as PABA. In further examples, Y is a maleimide group, a peptide moiety, and / or a benzoic acid moiety. In an additional example, Y includes a combination of mc groups. In an additional example, Y includes a combination of mc-v groups. In a further example, Y comprises a val-cit-PABA group. In an example, Y comprises a mc-val-cit-PABA group.

[0339] In some examples, L is a single bond or a linker. In some embodiments, L is a linker. 1 -C 6 In some examples, L is a homobifunctional linker or In some embodiments, L is a homobifunctional linker as described above. In some examples, L is a heterobifunctional linker as described above. In some examples, L is a maleimidocaproyl (mc), such as described above. In some instances, L contains a Val-Ci In some instances, L contains a peptide moiety, such as PABA. In further examples, L is a maleimide group, a peptide moiety, and / or a benzoic ...

Claims

1. A pharmaceutical composition for treating a disease or disorder characterized by defective DMD mRNA in a subject in need thereof, comprising a polynucleic acid conjugate, the polynucleic acid conjugate comprises a full-length anti-human transferrin receptor antibody and at least one polynucleic acid molecule; The at least one polynucleic acid molecule hybridizes to a target region within exon 44 or exon 45 or at an exon-intron junction of exon 44 or exon 45 of a pre-mRNA transcript of the DMD gene, the at least one polynucleic acid molecule comprises at least one phosphorodiamidate morpholino oligomer (PMO) modified non-naturally occurring nucleotide, the at least one polynucleic acid molecule induces splicing out of exon 44 or exon 45 from the pre-mRNA transcript in human muscle cells to generate an mRNA transcript encoding a functional dystrophin protein, and the at least one polynucleic acid molecule comprises at least 26 consecutive nucleotides from a nucleic acid sequence selected from SEQ ID NOs: 1056-1067, 1079-1082 and 1084-1089. Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the functional dystrophin protein is a truncated form of the dystrophin protein.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the target region is at the exon-intron junction of exon 44 or exon 45, or the target region is an intron region upstream or downstream of the exon-intron junction of exon 44 or exon 45.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein said at least one polynucleic acid molecule induces the splicing out of exon 44 from said pre-mRNA transcript of the DMD gene.

5. The polynucleic acid conjugate is A-(X １ -B)n Formula (V) wherein A comprises the full length anti-human transferrin receptor antibody; B consists of one of said at least one polynucleic acid molecule; X １ comprises a single bond or a first non-polymeric linker; and The pharmaceutical composition according to any one of claims 1 to 4, wherein n is an average value selected from 1 to 12.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein said at least one polynucleic acid molecule is a single-stranded oligonucleotide.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the at least one polynucleic acid molecule comprises at least one modified internucleotide linkage, at least one inverted abasic moiety, at least one 5'-vinylphosphonate modified non-natural nucleotide, or a combination thereof.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the disease or condition is Duchenne muscular dystrophy.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the subject is a human.

Images