Compositions and methods for treating facioscapulohumeral muscular dystrophy

Abstract

Disclosed herein are polynucleic acid molecules, pharmaceutical compositions, and methods for treating muscular dystrophy.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 001,211, filed Mar. 27, 2020, which is incorporated herein by reference in its entirety.

Background Art

[0002] Background of the Invention Gene silencing induced by RNA results in gene suppression that provides multiple levels of control, namely transcriptional inactivation, mRNA degradation induced by small interfering RNA (siRNA), and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) provides a long-lasting effect over multiple cell divisions. Thus, RNAi represents a viable method useful for drug target validation, gene function analysis, pathway analysis, and disease treatment.

[0003] Incorporation by Reference I All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Summary of the Invention

[0004] In certain embodiments, polynucleotide molecules and pharmaceutical compositions for modulating genes associated with muscular dystrophy, particularly facioscapulohumeral muscular dystrophy (e.g., DM1), are disclosed herein. In some embodiments, methods of treating muscular dystrophy, particularly FSHD, with the polynucleotide molecules or polynucleotide molecule conjugates disclosed herein are also described herein.

[0005] Disclosed herein is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK4 in a particular embodiment. The polynucleic acid molecule has a sense strand having at least 80% identical sequence to SEQ ID NO: 1 and an antisense strand having at least 80% identical sequence to SEQ ID NO: 2. The polynucleic acid molecule conjugate mediates RNA interference with DMPK.

[0006] In some embodiments, the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence EINPIX1GRSNYAX2KFQG where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence including SEQ ID NO: 19. In some embodiments, the VH region comprises an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence including one of SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 21, and an HCDR3 sequence including SEQ ID NO: 19. In some embodiments, the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence QHFWGTPLTX6 where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, and if present, is F. In some embodiments, the VL region includes the LCDR1 sequence containing sequence number 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 is present or absent, if present it is F. In some embodiments, the VL region includes the LCDR1 sequence containing sequence number 22 or sequence number 27, the LCDR2 sequence containing sequence number 23, sequence number 25, or sequence number 28, and the LCDR3 sequence containing sequence number 24 or sequence number 26. In some embodiments, the VH region includes the HCDR1 sequence containing sequence number 17, the HCDR2 sequence containing sequence number 18, and the HCDR3 sequence containing sequence number 19, and the VL region includes the LCDR1 sequence containing sequence number 22, the LCDR2 sequence containing sequence number 23, and the LCDR3 sequence containing sequence number 24. In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 17, an HCDR2 sequence containing sequence number 20, and an HCDR3 sequence containing sequence number 19, and the VL region includes an LCDR1 sequence containing sequence number 22, an LCDR2 sequence containing sequence number 23, and an LCDR3 sequence containing sequence number 24.In some embodiments, the VH region includes the HCDR1 sequence containing sequence number 17, the HCDR2 sequence containing sequence number 21, and the HCDR3 sequence containing sequence number 19, and the VL region includes the LCDR1 sequence containing sequence number 22, the LCDR2 sequence containing sequence number 25, and the LCDR3 sequence containing sequence number 26. In some embodiments, the VH region includes the HCDR1 sequence containing sequence number 17, the HCDR2 sequence containing sequence number 20, and the HCDR3 sequence containing sequence number 19, and the VL region includes the LCDR1 sequence containing sequence number 27, the LCDR2 sequence containing sequence number 28, and the LCDR3 sequence containing sequence number 26. In some embodiments, the VH region includes at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to sequences selected from sequence numbers 29-33. In some embodiments, the VL region includes at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to sequences selected from sequence numbers 34-38. In some embodiments, the VH region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to sequence number 30, and the VL region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to sequence number 34.

[0007] In some embodiments, the anti-transferrin receptor antibody includes a humanized antibody or its antigen-binding fragment, or a chimeric antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment. In some embodiments, the anti-transferrin receptor antibody includes IgG-scFv, nanobody, BiTE, diabody, DART, TandAb, scdiabody, scdiabody-CH3, triplebody, mini-antibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, or intrabody. In some embodiments, the anti-transferrin receptor antibody includes an IgG1 framework. Alternatively, in some embodiments, the anti-transferrin receptor antibody comprises an IgG2 framework. In some cases, the IgG2 framework is an IgG2b framework. Alternatively, in some embodiments, the anti-transferrin receptor antibody comprises an IgG4 framework.

[0008] In some embodiments, the anti-transferrin receptor antibody further includes at least one mutation in the Fc region. In some embodiments, at least one mutation modulates effector function or attenuates or eliminates Fc-γ receptor binding. In some embodiments, at least one mutation is located at residue positions D265, N297, K322, L328, or P329, with the residue position being relative to IgG1. In some embodiments, the Fc region includes two or more, three or more, or four or more mutations. In some embodiments, the Fc region includes mutations at L233 and L234, with the residues corresponding to positions 233 and 234 of SEQ ID NO: 39. In some embodiments, the Fc region includes mutations at D265 and N297. In some embodiments, the Fc region includes mutations at D265 and N297. In some embodiments, the anti-transferrin receptor antibody includes a heavy chain (HC) sequence selected from SEQ ID NOs: 39-62 and a light chain (LC) sequence selected from SEQ ID NOs: 63-66. In some embodiments, the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

[0009] In some embodiments, the sense strand and the antisense strand each independently contain at least one 2'-modified nucleotide, at least one modified nucleotide bond, or at least one reverse debase moiety. In some embodiments, the sense strand contains a 2'-O-methyl modified nucleotide at its 5' end. Alternatively and / or further, the sense strand contains at least two consecutive 2'-O-methyl modified nucleotides at its 5' end. Alternatively and / or further, the sense strand contains at least three, four, five, or six consecutive 2'-O-methyl modified nucleotides at its 5' end. Alternatively and / or further, the sense strand contains six consecutive 2'-O-methyl modified nucleotides at its 5' end. Alternatively and / or further, the sense strand contains at least one 2'-F modified nucleotide. Alternatively and / or further, the sense strand contains at least two and at least three 2'-F modified nucleotides. Alternatively and / or further, the sense strand contains at least two and at least three consecutive 2'-F modified nucleotides. Alternatively and / or further, the sense strand contains a 2'-O-methyl-modified nucleotide at its 3' end. Alternatively and / or further, the sense strand contains at least two consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or further, the sense strand contains at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or further, the sense strand contains 10 consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or further, the sense strand contains at least two phosphorothioate nucleotide interlinks. Alternatively and / or further, the sense strand has the sequence of SEQ ID NOs: 3, 5, 7, 9, 11, 13, or 15.

[0010] In some embodiments, the antisense strand contains a 2'-O-methyl modified nucleotide at its 5' end. Alternatively and / or further, the antisense strand contains a 2'-O-methyl modified nucleotide at its 3' end. Alternatively and / or further, the antisense strand contains at least two, at least three, at least four, or at least five consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or further, the antisense strand contains five consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or further, the antisense strand contains at least one, at least two, at least three, or at least four 2'-F modified nucleotides. Alternatively and / or further, the antisense strand contains four 2'-F modified nucleotides, any two of which are not consecutive. Alternatively and / or further, the antisense strand contains two overhanging nucleotides at its 3' end. Alternatively and / or further, the antisense strand contains at least two and at least three phosphorothioate nucleotide interbonds. Alternatively and / or further, the antisense strand has the sequence of SEQ ID NOs: 4, 6, 8, 10, 12, 14, or 16.

[0011] In some embodiments, the polynucleic acid molecule conjugate includes a linker that connects an anti-transferrin receptor antibody or its antigen-binding fragment to the polynucleic acid molecule. In some embodiments, the linker is a C6 linker. In some embodiments, the C6 linker is a 6-amino-1-hexanol linker. In some embodiments, the linker is a homobifunctional or heterobifunctional linker, a maleimide group, a dipeptide moiety, a benzoic acid group, or a derivative thereof. In some embodiments, the linker includes 4-(N-maleimidomethyl)cyclohexane-1-amidate (SMCC). In some embodiments, the linker is bound to the 5' end of the sense strand. In some embodiments, the polynucleic acid molecule is conjugated to a cysteine ​​residue of the anti-transferrin receptor antibody or its antigen-binding fragment. In some embodiments, the cysteine ​​residue is located in the Fc domain of the anti-transferrin receptor antibody or its antigen-binding fragment. In some embodiments, the ratio between the polynucleic acid molecule and the anti-transferrin receptor antibody or its antigen-binding fragment is approximately 1:1, 2:1, 3:1, or 4:1.

[0012] In some embodiments, the polynucleic acid moiety mediates RNA interference with the human DMPK gene to regulate muscle atrophy in the subject. In some embodiments, the RNA interference includes reducing the expression of the DMPK gene mRNA transcript by at least 50%, at least 60%, or at least 70% compared to the amount of the DMPK gene mRNA transcript in cells affected by muscular dystrophy. In some embodiments, the muscular dystrophy is myotonic dystrophy type 1 (DM1).

[0013] Disclosed herein are polynucleic acid molecule conjugates comprising, in certain embodiments, an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. The antitransferrin receptor antibody or its antigen-binding fragment comprises a polynucleic acid molecule having a sense strand having the sequence of SEQ ID NOs: 3, 5, 7, 9, 11, 13, or 15, and an antisense strand having the sequence of SEQ ID NOs: 4, 6, 8, 10, 12, 14, or 16, wherein the antitransferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region comprising an HCDR1 sequence including SEQ ID NOs: 17, an HCDR2 sequence including SEQ ID NOs: 20, and an HCDR3 sequence including SEQ ID NOs: 19, the VL region comprising an LCDR1 sequence including SEQ ID NOs: 22, an LCDR2 sequence including SEQ ID NOs: 23, and an LCDR3 sequence including SEQ ID NOs: 24, and the antitransferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a linker comprising 4-(N-maleimidomethyl)cyclohexane-1-amidate (SMCC).

[0014] Disclosed herein are polynucleic acid molecule conjugates comprising, in certain embodiments, an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. The polynucleic acid molecule has a sense strand having the sequence of SEQ ID NOs: 3, 5, 7, 9, 11, 13, or 15, and an antisense strand having the sequence of SEQ ID NOs: 4, 6, 8, 10, 12, 14, or 16. The anti-transferrin receptor antibody or its antigen-binding fragment includes a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region having 90%, 95%, 99%, or 100% sequence identity with respect to SEQ ID NO: 30, including at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to SEQ ID NO: 34. The anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0015] Disclosed herein are polynucleic acid molecule conjugates comprising, in certain embodiments, an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. The polynucleic acid molecule has a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 2, wherein the sense strand contains at least 3, 4, 5, or 6 consecutive 2'-O-methyl-modified nucleotides and at least 2 or at least 3 2'-F-modified nucleotides at its 5' end, the anti-transferrin receptor antibody or its antigen-binding fragment contains a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0016] Disclosed herein are polynucleic acid molecule conjugates comprising, in certain embodiments, an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. The polynucleic acid molecule has a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 2, wherein the antisense strand contains at least two, at least three, at least four, and at least five consecutive 2'-O-methyl modified nucleotides at its 3' end and at least one, at least two, at least three, and at least four 2'-F modified nucleotides, and the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region contains the HCDR1 sequence including SEQ ID NO: 17, the HCDR2 sequence including SEQ ID NO: 20, and the HCDR3 sequence including SEQ ID NO: 19, and the VL region contains the LCDR1 sequence including SEQ ID NO: 22, the LCDR2 sequence including SEQ ID NO: 23, and the LCDR3 sequence including SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0017] Disclosed herein is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK in a particular embodiment. The polynucleic acid molecule has a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 2, the antisense strand comprising 2'-O-methyl-modified nucleotides at its 5' and 3' ends, the anti-transferrin receptor antibody or its antigen-binding fragment comprising a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region comprising an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 18, and an HCDR3 sequence comprising SEQ ID NO: 19, the VL region comprising an LCDR1 sequence comprising SEQ ID NO: 22, an LCDR2 sequence comprising SEQ ID NO: 23, and an LCDR3 sequence comprising SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0018] Disclosed herein are polynucleic acid molecule conjugates comprising, in certain embodiments, an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. The polynucleic acid molecule has a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 2, wherein the antisense strand contains at least five consecutive 2'-O-methyl modified nucleotides at its 3' end and four 2'-F modified nucleotides, any two of which are not consecutive; the anti-transferrin receptor antibody or its antigen-binding fragment contains a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34; and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a 6-amino-1-hexanol linker.

[0019] Disclosed herein are polynucleic acid molecule conjugates comprising, in certain embodiments, an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. The polynucleic acid molecule has a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 2, wherein the antisense strand contains at least five consecutive 2'-O-methyl-modified nucleotides at its 3' end, and contains four 2'-F-modified nucleotides, of which any two are not consecutive; the anti-transferrin receptor antibody or its antigen-binding fragment contains a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34; and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a 6-amino-1-hexanol linker.

[0020] Disclosed herein are pharmaceutical compositions comprising, in certain embodiments, the polynucleic acid molecular conjugates and pharmaceutically acceptable excipients described herein. In some embodiments, the pharmaceutical compositions are formulated as nanoparticle formulations. In some embodiments, the pharmaceutical compositions are formulated for parenteral, oral, intranasal, oral cavity, rectal, or transdermal administration.

[0021] Disclosed herein are, in certain embodiments, a method for treating muscular dystrophy in a subject of interest, comprising the steps of providing a polynucleic acid conjugate or pharmaceutical composition described herein, and administering the polynucleic acid conjugate to the subject of interest for the treatment of muscular dystrophy, wherein the polynucleic acid conjugate reduces the amount of human DMPK mRNA transcript. In some embodiments, the polynucleic acid moiety mediates RNA interference with human DMPK and modulates muscle atrophy in the subject. In some embodiments, the muscular dystrophy is myotonic dystrophy type 1 (DM1).

[0022] Disclosed herein are, in certain embodiments, the use of the polynucleic acid conjugates or pharmaceutical compositions described herein for treating subjects diagnosed with or suspected of having myotonic dystrophy type 1 (DM1), or for manufacturing pharmaceuticals for treating myotonic dystrophy type 1 (DM1). Disclosed herein are, in certain embodiments, kits comprising the polynucleic acid conjugates or pharmaceutical compositions described herein. [Brief explanation of the drawing]

[0023] Various aspects of this disclosure are specifically described in the attached claims. A better understanding of the features and merits of this disclosure can be obtained by referring to the following detailed description and attached drawings illustrating exemplary embodiments in which the principles of this disclosure are utilized. This patent application file includes at least one drawing performed in color. A copy of this publication of the patent application containing the color drawing will be provided by the Secretariat upon request and payment of the necessary fees. [Figure 1] A schematic diagram of an antibody-siRNA conjugate is illustrated. [Figure 2] This illustrates the schematic structure of DMPK siRNA. [Figure 3] The graph illustrating the binding of anti-TfR antibodies to TfR2 using ELISA is illustrated. [Figure 4] Illustrate a graph of the binding of an anti-TfR antibody to TfR1 in the presence of a cofactor. [Figure 5] Illustrate a graph of the in vivo dose-response of AOC-mediated DMPK knockdown in mouse skeletal muscle. [Figure 6] Illustrate a graph of the time course of AOC-mediated DMPK knockdown in mouse tissues (left), and the concentration of siDMPK.36 over time in mouse tissues (right). [Figure 7] Illustrate a graph showing AOC-mediated DMPK knockdown in cynomolgus monkey skeletal muscle over a 12-week time period after administration. **[Mode for Carrying Out the Invention]**

[0024] Based on clinical confirmation, DM1 is a rare, single-gene, autosomal dominant, repeat expansion disorder that affects approximately one in 8,000 people in the United States. However, recent gene-based studies have estimated the prevalence of DM1 in the United States to be one in 2,532 people. DM1 is caused by an expansion of the CTG triplet repeat in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. This expansion ranges from less than 35 repeats in healthy individuals to thousands of repeats in DM1 patients. When the mutant DMPK gene is translated into mRNA, self-complementary CUG repeats induce the formation of large hairpin loops, trapping the DMPK mRNA in the nucleus and conferring a gain-of-toxicity function. This toxicity is not due to the translation of the mRNA into toxic proteins, but rather to the presence of high concentrations of CUG repeats in the nucleus that function as traps for the important CUG-binding protein muscleblind-like protein 1 (MBNL1). Binding to the retained DMPK CUG repeats in the nucleus sequesters MBNL1, preventing it from performing its normal function of guiding mRNA processing. As a result, multiple mRNAs encoding important proteins are misprocessed. The resulting aberrant proteins translated from these mis-spliced mRNAs are the ultimate cause of the phenotypic changes characteristic of this disease.

[0025] Nucleic acid (e.g., RNAi) therapy is a targeted therapy boasting high selectivity and specificity. However, in some cases, nucleic acid therapy is also hindered by fragile intracellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications of nucleic acid compositions are being explored, such as novel linkers for better stability and / or lower toxicity, optimization of binding sites for increased target specificity and / or target delivery, and nucleic acid polymer modifications for increased stability and / or reduced off-target effects.

[0026] In some embodiments, the composition or order of the various components constituting the nucleic acid composition further results in intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, if the nucleic acid components include binding moieties, polymers, and polynucleic acid molecules (or polynucleotides), the order or composition of the binding moieties, polymers, and / or polynucleic acid molecules (or polynucleotides) (e.g., binding moieties, polynucleic acid molecules, polymers, binding moieties, polymers, or polymer-binding moieties, polynucleic acid molecules) further results in intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0027] In some embodiments, those described herein include polynucleic acid molecules and polynucleic acid molecule conjugates for the treatment of muscular dystrophy. In some examples, the polynucleic acid molecule conjugates described herein enhance intracellular uptake, stability, and / or efficacy. In some examples, the polynucleic acid molecule conjugate includes an anti-transferrin antibody or antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. In some cases, the polynucleic acid molecule includes a molecule of formula (I): A-X1-B.

[0028] Further embodiments described herein include a method for treating muscular dystrophy, comprising the step of administering a polynucleic acid molecule or polynucleic acid molecule conjugate described herein to a subject.

[0029] Polynucleic acid molecules In certain embodiments, the polynucleic acid molecule hybridizes to the target sequence of a muscular dystrophy-related gene. Preferably, the polynucleic acid molecule described herein hybridizes to the target sequence of a myotonic dystrophy protein kinase gene (DMPK, DM, DM1, DM1PK, DMK, MDPK, MT-PK, Dm15, also known as myotonic dystrophy protein kinase or DM1 protein kinase gene) among the muscular dystrophy-related genes.

[0030] In some embodiments, the polynucleic acid molecule contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NO: 1. In some embodiments, the polynucleic acid molecule contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NO: 2. In some embodiments, the polynucleic acid molecule contains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NOs. 4, 6, 8, 10, 12, 14, or 16.

[0031] In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NO: 1. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NO: 2. In some cases, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. 3, 5, 7, 9, 11, 13, or 15. In some cases, the second polynucleotide contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs. 4, 6, 8, 10, 12, 14, or 16.

[0032] In some embodiments, the polynucleic acid molecule includes a sense strand (e.g., a passenger strand) and an antisense strand (e.g., a guide strand). In some cases, the sense strand (e.g., the passenger strand) includes a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NO: 1. In some cases, the antisense strand (e.g., the guide strand) includes a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from SEQ ID NO: 2. In some embodiments, the polynucleic acid molecule includes a sense strand (e.g., a passenger strand) and an antisense strand (e.g., a guide strand). In some cases, the sense strand (e.g., passenger strand) contains a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs. 3, 5, 7, 9, 11, 13, or 15. In some cases, the antisense strand (e.g., guide strand) contains a sequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs. 96%, 97%, 98%, 99%, or 16. Table 1 presents the nucleic acid sequences and modified sequences of SEQ ID NOs. 1–16.

[0033] [Table 1]

[0034] In some embodiments, the polynucleic acid molecules described herein include RNA, DNA, or PMOs. In some cases, the polynucleic acid molecule includes RNA. In some examples, RNA includes small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heteronuclear RNA (hnRNA). In some examples, RNA includes shRNA. In some examples, RNA includes miRNA. In some examples, RNA includes dsRNA. In some examples, RNA includes tRNA. In some examples, RNA includes rRNA. In some examples, RNA includes hnRNA. In some examples, RNA includes siRNA. In some examples, the polynucleic acid molecule includes siRNA.

[0035] In some embodiments, the nucleic acid polymer is about 8 to about 50 nucleotides long. In some embodiments, the nucleic acid polymer is about 10 to about 50 nucleotides long. In some embodiments, the polynucleic acid molecule is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0036] In some embodiments, the polynucleotide molecule is about 50 nucleotides long. In some examples, the polynucleotide molecule is about 45 nucleotides long. In some examples, the polynucleotide molecule is about 40 nucleotides long. In some examples, the polynucleotide molecule is about 35 nucleotides long. In some examples, the polynucleotide molecule is about 30 nucleotides long. In some examples, the polynucleotide molecule is about 25 nucleotides long. In some examples, the polynucleotide molecule is about 20 nucleotides long. In some examples, the polynucleotide molecule is about 19 nucleotides long. In some examples, the polynucleotide molecule is about 18 nucleotides long. In some examples, the polynucleotide molecule is about 17 nucleotides long. In some examples, the polynucleotide molecule is about 16 nucleotides long. In some examples, the polynucleotide molecule is about 15 nucleotides long. In some examples, the polynucleotide molecule is about 14 nucleotides long. In some examples, the polynucleotide molecule is about 13 nucleotides long. In some examples, the polynucleotide molecule is about 12 nucleotides long. In some cases, polynucleotide molecules are approximately 11 nucleotides long. In some cases, polynucleotide molecules are approximately 10 nucleotides long. In some cases, polynucleotide molecules are approximately 8 nucleotides long. In some cases, polynucleotide molecules are approximately 8 to 50 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 50 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 45 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 40 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 35 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 30 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 25 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 20 nucleotides long. In some cases, polynucleotide molecules are approximately 15 to 25 nucleotides long. In some cases, polynucleotide molecules are approximately 15 to 30 nucleotides long.In some cases, polynucleotide molecules are approximately 12 to 30 nucleotides long.

[0037] In some embodiments, the polynucleotide molecule comprises a first polynucleotide. In some examples, the polynucleotide molecule comprises a second polynucleotide. In some examples, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide is a sense strand or passenger strand. In some examples, the second polynucleotide is an antisense strand or guide strand.

[0038] In some embodiments, the polynucleotide molecule is a first polynucleotide. In some embodiments, the first polynucleotide is about 8 to about 50 nucleotides long. The first polynucleotide is about 10 to about 50 nucleotides long. In some embodiments, the first polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0039] In some cases, the first polynucleotide is approximately 50 nucleotides long. In some cases, the first polynucleotide is approximately 45 nucleotides long. In some cases, the first polynucleotide is approximately 40 nucleotides long. In some cases, the first polynucleotide is approximately 35 nucleotides long. In some cases, the first polynucleotide is approximately 30 nucleotides long. In some cases, the first polynucleotide is approximately 25 nucleotides long. In some cases, the first polynucleotide is approximately 20 nucleotides long. In some cases, the first polynucleotide is approximately 19 nucleotides long. In some cases, the first polynucleotide is approximately 18 nucleotides long. In some cases, the first polynucleotide is approximately 17 nucleotides long. In some cases, the first polynucleotide is approximately 16 nucleotides long. In some cases, the first polynucleotide is approximately 15 nucleotides long. In some cases, the first polynucleotide is approximately 14 nucleotides long. In some cases, the first polynucleotide is approximately 13 nucleotides long. In some cases, the first polynucleotide is approximately 12 nucleotides long. In some cases, the first polynucleotide is approximately 11 nucleotides long. In some cases, the first polynucleotide is approximately 10 nucleotides long. In some cases, the first polynucleotide is approximately 8 nucleotides long. In some cases, the first polynucleotide is approximately 8 to 50 nucleotides long. In some cases, the first polynucleotide is approximately 10 to 50 nucleotides long. In some cases, the first polynucleotide is approximately 10 to 45 nucleotides long. In some cases, the first polynucleotide is approximately 10 to 40 nucleotides long. In some cases, the first polynucleotide is approximately 10 to 35 nucleotides long. In some cases, the first polynucleotide is approximately 10 to 30 nucleotides long. In some cases, the first polynucleotide is approximately 10 to 25 nucleotides long.In some examples, the first polynucleotide is approximately 10 to 20 nucleotides long. In some examples, the first polynucleotide is approximately 15 to 25 nucleotides long. In some examples, the first polynucleotide is approximately 15 to 30 nucleotides long. In some examples, the first polynucleotide is approximately 12 to 30 nucleotides long.

[0040] In some embodiments, the polynucleotide molecule is a second polynucleotide. In some embodiments, the second polynucleotide is about 8 to about 50 nucleotides long. In some embodiments, the second polynucleotide is about 10 to about 50 nucleotides long. In some embodiments, the second polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0041] In some cases, the second polynucleotide is approximately 50 nucleotides long. In some cases, the second polynucleotide is approximately 45 nucleotides long. In some cases, the second polynucleotide is approximately 40 nucleotides long. In some cases, the second polynucleotide is approximately 35 nucleotides long. In some cases, the second polynucleotide is approximately 30 nucleotides long. In some cases, the second polynucleotide is approximately 25 nucleotides long. In some cases, the second polynucleotide is approximately 20 nucleotides long. In some cases, the second polynucleotide is approximately 19 nucleotides long. In some cases, the second polynucleotide is approximately 18 nucleotides long. In some cases, the second polynucleotide is approximately 17 nucleotides long. In some cases, the second polynucleotide is approximately 16 nucleotides long. In some cases, the second polynucleotide is approximately 15 nucleotides long. In some cases, the second polynucleotide is approximately 14 nucleotides long. In some cases, the second polynucleotide is approximately 13 nucleotides long. In some cases, the second polynucleotide is approximately 12 nucleotides long. In some cases, the second polynucleotide is approximately 11 nucleotides long. In some cases, the second polynucleotide is approximately 10 nucleotides long. In some cases, the second polynucleotide is approximately 8 nucleotides long. In some cases, the second polynucleotide is approximately 8 to 50 nucleotides long. In some cases, the second polynucleotide is approximately 10 to 50 nucleotides long. In some cases, the second polynucleotide is approximately 10 to 45 nucleotides long. In some cases, the second polynucleotide is approximately 10 to 40 nucleotides long. In some cases, the second polynucleotide is approximately 10 to 35 nucleotides long. In some cases, the second polynucleotide is approximately 10 to 30 nucleotides long. In some cases, the second polynucleotide is approximately 10 to 25 nucleotides long.In some cases, the second polynucleotide is approximately 10 to 20 nucleotides long. In some cases, the second polynucleotide is approximately 15 to 25 nucleotides long. In some cases, the second polynucleotide is approximately 15 to 30 nucleotides long. In some cases, the second polynucleotide is approximately 12 to 30 nucleotides long.

[0042] In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the polynucleotide molecule further comprises blunt ends, overhangs, or a combination thereof. In some examples, the blunt ends are 5' blunt ends, 3' blunt ends, or both. In some cases, the overhangs are 5' overhangs, 3' overhangs, or both. In some cases, the overhangs contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the overhangs contain 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhangs contain 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the overhangs contain 1 non-base-paired nucleotide. In some cases, the overhangs contain 2 non-base-paired nucleotides. In some cases, the overhangs contain 3 non-base-paired nucleotides. In some cases, the overhangs contain 4 non-base-paired nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand containing two non-base-pairing nucleotides as an overhang at its 3' end, while the sense strand has no overhang. Optionally, in such embodiments, the non-base-pairing nucleotides have a TT, dTdT, or UU sequence. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand having one or more nucleotides complementary to the antisense sequence at its 5' end.

[0043] In some embodiments, the sequence of the polynucleotide molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 50% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 60% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 70% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 80% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 90% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 99% complementary to the target sequence described herein. In some cases, the sequences of the polynucleic acid molecules are 100% complementary to the target sequences described herein.

[0044] In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with respect to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has three or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has two or fewer mismatches with respect to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has one or fewer mismatches with respect to the target sequence described herein.

[0045] In some embodiments, the specificity of the polynucleic acid molecule hybridizing to the target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some examples, the hybridization is a highly stringent hybridization state.

[0046] In some embodiments, polynucleic acid molecules reduced off-target effects. In some examples, “off-target” or “off-target effect” refers to any instance in which a polynucleic acid polymer directly or indirectly interacts with another mRNA sequence, DNA sequence, or cellular protein or other part of a given target, causing an unintended effect. In some examples, “off-target effects” occur when there is simultaneous degradation of other transcripts due to partial homology or complementarity between the sense and / or antisense strands of the other transcript and the polynucleic acid molecule.

[0047] In some embodiments, polynucleic acid molecules include natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules include DNA, RNA, and / or combinations of nucleotide analogs. In some examples, the synthetic or artificial nucleotide analogs or bases include modifications with one or more ribose moieties, phosphate moieties, nucleoside moieties, or combinations thereof.

[0048] In some embodiments, nucleotide analogs or artificial nucleotide bases comprise nucleic acids having modifications to the 2' hydroxyl group of the ribose moiety. In some examples, the modifications include H, 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, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. In some examples, the alkyl moiety includes further modifications. In some examples, the modifications include azo groups, keto groups, aldehyde groups, carboxyl groups, nitro groups, nitroso groups, nitrile groups, heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) groups, isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxides, sulfones, sulfides, and disulfides). In some examples, the alkyl moiety includes further heterosubstitutions. In some cases, the carbon atoms of the heterocyclic group are substituted with nitrogen, oxygen, or sulfur. In some cases, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino compounds.

[0049] In some cases, the modification of the 2'-hydroxyl group is either 2'-O-methyl modification or 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, 2'-O-methyl modification adds a methyl group to the 2'-hydroxyl group of the ribose moiety, while 2'O-methoxyethyl modification adds a methoxyethyl group to the 2'-hydroxyl group of the ribose moiety. Typical chemical structures of 2'-O-methyl modified adenosine molecules and 2'O-methoxyethyl modified uridine are illustrated below. [ka]

[0050] In some embodiments, the modification of the 2'-hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group containing a propyl linker bonds the amine group to the 2' oxygen. In some examples, this modification neutralizes the overall negative charge derived from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving its cellular uptake properties due to its zwitterionic nature. Typical chemical structures of 2'-O-aminopropyl nucleoside phosphoramidites are illustrated below. [ka]

[0051] In some examples, the modification of the 2' hydroxyl group is locked or cross-linked ribose modification (e.g., locked nucleic acid or LNA), where the oxygen molecule bonded at the 2' carbon is bonded to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxymethylene bicyclic ribonucleotide monomer. Representative examples of the chemical structure of LNA are illustrated below. The representative example shown on the left highlights the chemical bonding properties of the LNA monomer. The representative example shown on the right shows the locked 3'-end of the furanose ring of the LNA monomer. 3 E) It emphasizes the structure. [ka]

[0052] In some embodiments, modification at the 2'-hydroxyl group locks the sugar structure into a 3'-endosugar puckering conformation, including ethylene nucleic acids (ENAs), such as 2'-4'-ethylene-bridged nucleic acids. ENAs are part of the bridged nucleic acid class of modified nucleic acids, which also includes LNAs. Typical chemical structures of ENAs and bridged nucleic acids are illustrated below. [ka]

[0053] In some embodiments, the additional modification at the 2'-hydroxyl group includes 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0054] In some embodiments, the nucleotide analog is, but is not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides having a modification at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halolysine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, 7-deaza-adenosine, deazanucleotides, 6-azouridine, 6-azocytidine, 6-azothymidine, The modified nucleotides include 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, quosin, alkaeosin, naphthyl and substituted naphthyl groups, O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine, 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. The modified nucleotides further include nucleotides modified to the sugar moiety, as well as nucleotides having a non-ribosyl sugar or its analogue. For example, in some cases, the sugar moiety may be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocyclic or carbocyclic, or based on these. The term nucleotide further includes those known in the art as universal bases.For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

[0055] In some embodiments, nucleotide analogs further include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof. Morphorino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structures mimic natural nucleic acid structures by deviating from normal sugar and phosphate structures. In some examples, a five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbon atoms, one nitrogen atom, and one oxygen atom. In some cases, the ribose monomer is bonded by a phosphorodiamidate group instead of a phosphate group. In some cases, the alteration of the skeleton removes all positive and negative charges, making a morpholino neutral molecule that can cross the cell membrane without the help of cellular delivery agents, such as those used by charged oligonucleotides. [ka]

[0056] In some embodiments, peptide nucleic acids (PNAs) do not contain phosphate bonds in the sugar skeleton ring, but the bases are bonded and appropriately spaced by oligoglycine-like molecules, thus removing the skeletal charge. [ka]

[0057] In some embodiments, one or more modifications may optionally occur at internucleotide bonds. In some examples, the modified internucleotide bonds are, but are not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, borontrifluoridates, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkylphosphonates, alkylphosphonothioates, arylphosphorothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, phosphoropiperates This includes didates, phosphoranilothioates, phosphoranilideates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazo, methylenedimethyldimethylhydrazo, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, thioamides, bonds to riboacetyl groups, aminoethylglycine, silyl, or siloxane bonds, for example saturated or unsaturated, and / or substituted and / or alkyl or cycloalkyl bonds containing or not containing 1 to 10 carbon heteroatoms, bonds to morpholino structures, amides, polyamides in which a base is directly or indirectly bonded to the aza nitrogen of the skeleton, or combinations thereof. Phosphothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate bonds. Exemplary PS ASOs are described below. [ka]

[0058] In some examples, the modifications are methyl or thiol modifications, such as methylphosphonate or thiolphosphonate modifications. Typical thiolphosphonate nucleotides (left) and methylphosphonate nucleotides (right) are illustrated below. [ka]

[0059] In some examples, the modified nucleotides include, but are not limited to, 2'-fluoroN3-P5'-phosphoamidites, as exemplified below: [ka]

[0060] In some examples, the modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNAs)) as exemplified below: [ka]

[0061] In some embodiments, one or more modifications further include modifications of the ribose moiety, phosphate backbone, and nucleoside, or modifications of nucleotide analogs at the 3' or 5' terminus. For example, the 3' terminus optionally includes a 3' cationic group, or inverts a nucleoside at the 3'-terminus containing a 3'-3' bond. In another alternative, the 3'-terminus optionally binds to an aminoalkyl group, e.g., 3'C5-aminoalkyldT. In yet another alternative, the 3'-terminus optionally binds to a debasing site, e.g., an aprinic acid or apyrimidine acid site. In some examples, the 5'-terminus binds to an aminoalkyl group, e.g., a 5'-O-aminoalkyl substituent. In some cases, the 5'-terminus binds to a debasing site, e.g., an aprinic acid or apyrimidine acid site.

[0062] In some embodiments, the polynucleic acid molecule comprises one or more artificial nucleotide analogs described herein. In some examples, the polynucleic acid molecule described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof. In some embodiments, the polynucleic acid molecule is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or This includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more artificial nucleotide analogs selected from modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof.In some embodiments, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methyl modified nucleotides. In some embodiments, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, the polynucleic acid molecule described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiol phosphonate nucleotides.

[0063] In some examples, polynucleic acid molecules contain at least one of the following: approximately 5% to approximately 100% modification, approximately 10% to approximately 100% modification, approximately 20% to approximately 100% modification, approximately 30% to approximately 100% modification, approximately 40% to approximately 100% modification, approximately 50% to approximately 100% modification, approximately 60% to approximately 100% modification, approximately 70% to approximately 100% modification, approximately 80% to approximately 100% modification, and approximately 90% to approximately 100% modification.

[0064] In some cases, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 90% modification, approximately 20% to approximately 90% modification, approximately 30% to approximately 90% modification, approximately 40% to approximately 90% modification, approximately 50% to approximately 90% modification, approximately 60% to approximately 90% modification, approximately 70% to approximately 90% modification, and approximately 80% to approximately 100% modification.

[0065] In some cases, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 80% modification, approximately 20% to approximately 80% modification, approximately 30% to approximately 80% modification, approximately 40% to approximately 80% modification, approximately 50% to approximately 80% modification, approximately 60% to approximately 80% modification, and approximately 70% to approximately 80% modification.

[0066] In some examples, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 70% modification, approximately 20% to approximately 70% modification, approximately 30% to approximately 70% modification, approximately 40% to approximately 70% modification, approximately 50% to approximately 70% modification, and approximately 60% to approximately 70% modification.

[0067] In some examples, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 60% modification, approximately 20% to approximately 60% modification, approximately 30% to approximately 60% modification, approximately 40% to approximately 60% modification, and approximately 50% to approximately 60% modification.

[0068] In some cases, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 50% modification, approximately 20% to approximately 50% modification, approximately 30% to approximately 50% modification, and approximately 40% to approximately 50% modification.

[0069] In some cases, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 40% modification, approximately 20% to approximately 40% modification, and approximately 30% to approximately 40% modification.

[0070] In some cases, polynucleic acid molecules contain at least one of the following: approximately 10% to approximately 30% modification, and approximately 20% to approximately 30% modification.

[0071] In some cases, polynucleotide molecules contain approximately 10% to 20% modifications.

[0072] In some cases, polynucleotide molecules contain approximately 15% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% modifications.

[0073] In further cases, the polynucleic acid molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification.

[0074] In some embodiments, the polynucleic acid molecule includes 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.

[0075] In some examples, polynucleic acid molecules contain 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.

[0076] In some examples, about 5 to about 100% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, about 5% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, about 10% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, about 15% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, about 20% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, about 25% of the polynucleic acid molecule contains the artificial nucleotide analogs described herein. In some examples, approximately 30% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 35% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 40% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 45% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 50% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 55% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 60% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 65% ​​of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 70% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 75% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 80% of the polynucleotide molecules contain the artificial nucleotide analogs described herein.In some examples, approximately 85% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 90% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 95% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 96% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 97% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 98% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 99% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some examples, approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof.

[0077] In some embodiments, the polynucleic acid molecule includes about 1 to about 25 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 1 modification, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 2 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 3 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 4 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 5 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 6 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 7 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 8 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule includes about 9 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 10 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, where the modifications include artificial nucleotide analogs described herein.In some embodiments, the polynucleotide molecule includes about 15 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 16 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 17 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 18 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 19 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 20 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 21 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 19 modifications, where the modifications include artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule includes about 22 modifications, where the modifications include artificial nucleotide analogs described herein.

[0078] In some embodiments, the polynucleic acid molecule is assembled from two distinct polynucleotides, where one polynucleotide comprises a sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide in the sense strand comprises a 2'-O-methylpyrimidine nucleotide and the purine nucleotide in the sense strand comprises a 2'-deoxypurine nucleotide. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide present in the sense strand comprises a 2'-deoxy-2'-fluoropyrimidine nucleotide and the purine nucleotide present in the sense strand comprises a 2'-deoxypurine nucleotide.

[0079] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide, if present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, if present in the antisense strand, is a 2'-O-methylpurine nucleotide.

[0080] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, where the pyrimidine nucleotide, if present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, if present in the antisense strand, comprises a 2'-deoxy-purine nucleotide.

[0081] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing a 2'-O-methyl modified nucleotide at its 5' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least two consecutive 2'-O-methyl modified nucleotides at its 5' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least three, four, five, or six consecutive 2'-O-methyl modified nucleotides at its 5' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing six consecutive 2'-O-methyl modified nucleotides at its 5' end.

[0082] Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least one 2'-F modified nucleotide. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least two and at least three 2'-F modified nucleotides. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least two and at least three consecutive 2'-F modified nucleotides.

[0083] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing a 2'-O-methyl modified nucleotide at its 3' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least two consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand containing 10 consecutive 2'-O-methyl modified nucleotides at its 3' end.

[0084] Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand containing a 2'-O-methyl modified nucleotide at its 5' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand containing a 2'-O-methyl modified nucleotide at its 3' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand containing at least two, at least three, at least four, or at least five consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand containing at least one, at least two, at least three, or at least four 2'-F modified nucleotides. Alternatively and / or further, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand comprising four 2'-F modified nucleotides, any two of which are not consecutive. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand comprising two overhanging nucleotides at its 3' end.

[0085] In some embodiments, the polynucleic acid molecule includes a sense strand and an antisense strand. In some embodiments, the polynucleic acid molecule includes a sense strand and an antisense strand, and the sense strand includes terminal capping portions at the 5' end, the 3' end, or both the 5' and 3' ends. In other embodiments, the terminal capping portions are reverse deoxydebase portions.

[0086] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand comprising a phosphate backbone modification at its 3' end. In some cases, the phosphate backbone modification is a phosphorothioate. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising at least two phosphorothioate nucleotide interbonds. Alternatively and / or further, the antisense strand comprising at least two, at least three phosphorothioate nucleotide interbonds.

[0087] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having a glyceryl modification at its 3' end.

[0088] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 1, and the antisense strand comprising the sequence of SEQ ID NO: 2, wherein the sense strand comprises at least three, four, five, or six consecutive 2'-O-methyl-modified nucleotides and at least two or at least three 2'-F-modified nucleotides at its 5' end.

[0089] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 1, and the antisense strand comprising the sequence of SEQ ID NO: 2, wherein the antisense strand comprises at least two, at least three, at least four, and at least five consecutive 2'-O-methyl-modified nucleotides and at least one, at least two, at least three, and at least four 2'-F-modified nucleotides at its 3' end.

[0090] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 1, the antisense strand comprising the sequence of SEQ ID NO: 2, and the antisense strand comprising 2'-O-methyl-modified nucleotides at its 5' and 3' ends.

[0091] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 1, and the antisense strand comprising the sequence of SEQ ID NO: 2, wherein the antisense strand comprises at least five consecutive 2'-O-methyl-modified nucleotides and four 2'-F-modified nucleotides at its 3' end, any two of the four 2'-F-modified nucleotides being non-consecutive.

[0092] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand comprising the sequence of SEQ ID NO: 1, and the antisense strand comprising the sequence of SEQ ID NO: 2, and the sense strand and / or antisense strand each comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% modified nucleotides of the corresponding sequences of SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0093] In some cases, one or more of the artificial nucleotide analogs described herein are resistant to nucleases such as ribonucleases like RNaseH, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonucleases and 3'-5' exonucleases, compared to natural polynucleic acid molecules. In some examples, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or modified 2'-ON-methylacetamide (2' Artificial nucleotide analogs, including 2'-O-NMA, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases such as ribonucleases like RNaseH, deoxyribonucleases like DNase, or exonucleases like 5'-3' exonucleases and 3'-5' exonucleases. In some cases, 2'-O methyl-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, 2'-deoxy-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-deoxy-2'-O-fluoro-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE)-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP)-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE)-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, LNA modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, HNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, morpholino is nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified polynucleic acid molecules are resistant to nucleases (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiol phosphonate-modified polynucleic acid molecules are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite are nuclease-resistant (e.g., RNaseH, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, the 5' conjugate described herein inhibits 5'-3' exonuclease cleavage. In some cases, the 3' conjugate described herein inhibits 3'-5' exonuclease cleavage.

[0094] In some embodiments, one or more of the artificial nucleotide analogs described herein exhibited increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. One or more artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoroN3-P5'-phosphoramidite, increased their binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In several cases, 2'-O-methyl-modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-O-aminopropyl-modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-deoxy-modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-deoxy-2'-fluoro-modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE)-modified polynucleic acid molecules exhibited increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules.In several cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, LNA modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, ENA modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, PNA modified polynucleic acid molecules showed increased binding affinity to their mRNA targets compared to equivalent naturally occurring polynucleic acid molecules. In several cases, HNA-modified polynucleotide molecules exhibited increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In several cases, morpholino-modified polynucleotide molecules exhibited increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In several cases, methylphosphonate-modified polynucleotide molecules exhibited increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In several cases, thiolphosphonate-modified polynucleotide molecules exhibited increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In several cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramidite exhibited increased binding affinity to their mRNA targets compared to equivalent native polynucleotide molecules. In some cases, the increased affinity is exemplified by a low Kd, a high melting temperature (Tm), or a combination thereof.

[0095] In some embodiments, the polynucleic acid molecules described herein are chiral pure (or stereopure) polynucleic acid molecules, or polynucleic acid molecules comprising a single enantiomer. In some examples, the polynucleic acid molecule contains an L-nucleotide. In some examples, the polynucleic acid molecule contains a D-nucleotide. In some examples, the polynucleic acid molecule composition contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomers. In some cases, the polynucleic acid molecule composition contains 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some examples, the polynucleic acid molecule is the polynucleic acid molecule described in U.S. Patent Application Publications 2014 / 194610 and 2015 / 211006; and PCT International Publication WO2015107425.

[0096] In some embodiments, the polynucleic acid molecules described herein are further modified to include an aptamer binding moiety. In some examples, the aptamer binding moiety is a DNA aptamer binding moiety. In some examples, the aptamer binding moiety is an Alphamer (Centauri Therapeutics), which includes an aptamer moiety that recognizes a specific cell surface target and a moiety that exhibits a specific epitope for binding to a circulating antibody. In some examples, the polynucleic acid molecules described herein are further modified to include an aptamer binding moiety as described in U.S. Patents 8,604,184, 8,591,910, and 7,850,975.

[0097] In additional embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA). In some examples, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2-hydroxyl position by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, or by locked or cross-linked ribose structures (e.g., LNA or ENA). In some cases, polynucleotide molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, polynucleotide molecules further contain morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphoramidite to increase their stability. In some examples, polynucleotide molecules are chiral-pure (or stereopure) polynucleotide molecules. In some examples, chiral-pure (or stereopure) polynucleotide molecules are modified to increase their stability. Appropriate modifications of RNA to increase delivery stability are obvious to those skilled in the art.

[0098] In some examples, a polynucleic acid molecule is a double-stranded polynucleotide molecule containing a self-complementary sense region and an antisense region, where the antisense region contains a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some examples, a polynucleic acid molecule is assembled from two separate polynucleotides, one strand being a sense strand and the other being an antisense strand, where the antisense and sense strands are self-complementary (e.g., each strand contains a nucleotide sequence complementary to the nucleotide sequence of the other strand; for example, when the antisense and sense strands form a duplex or double-stranded structure, e.g., the double-stranded region is approximately 19, 20, 21, 22, 23, or more base pairs); the antisense strand contains a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense strand contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, polynucleotide molecules are assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of the polynucleotide molecule are linked by nucleic acid-based or non-nucleic acid-based linkers.

[0099] In some cases, the polynucleotide molecule is a polynucleotide having a double, asymmetrical, hairpin-shaped, or asymmetrical hairpin-shaped secondary structure with a self-complementary sense region and an antisense region, where the antisense region contains a nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the polynucleotide molecule is a cyclic single-chain polynucleotide having two or more loop structures and a base containing a self-complementary sense region and an antisense region, where the antisense region contains a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the cyclic polynucleotide is processed in vivo or in vitro to produce an active polynucleotide molecule capable of mediating RNAi. In further cases, the polynucleic acid molecule further comprises a single-chain polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (for example, such a polynucleic acid molecule does not need to be present in the polynucleic acid molecule of the nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-chain polynucleotide further comprises a terminal phosphate group such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate.

[0100] In some cases, an asymmetric hairpin is a linear polynucleotide molecule comprising an antisense region, a loop region containing nucleotides or non-nucleotides, and a sense region, wherein the sense region contains fewer nucleotides than the antisense region, to the extent that it has enough complementary nucleotides to base-pair with the antisense region and to form a double helix with a loop. For example, an asymmetric hairpin polynucleotide molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) having a loop region that is long enough to mediate RNAi in a cell or in vitro and contains about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, an asymmetric hairpin polynucleotide molecule further contains a chemically modified 5' terminal phosphate group. In further cases, the loop region of an asymmetric hairpin polynucleotide molecule contains nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0101] In some embodiments, an asymmetric double helix is ​​a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region has nucleotides sufficiently complementary to the antisense region to form base pairs, and contains fewer nucleotides than the antisense region to the extent that it forms a double helix. For example, an asymmetric double polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) that is long enough to mediate RNAi in a cell or in vitro system, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0102] In some cases, universal bases refer to nucleotide base analogs that form base pairs with each of the nearly indistinguishable native DNA / RNA bases. Non-exclusive examples of universal bases include C-phenyl, C-naphthyl, and other aromatic derivatives known in the prior art, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437–2447).

[0103] Polynucleotide molecule synthesis In some embodiments, the polynucleic acid molecules described herein are constructed using procedures known in the art, by chemical synthesis and / or enzymatic ligation reactions. For example, polynucleic acid molecules are chemically synthesized using naturally occurring nucleotides, or using various modified nucleotides designed to increase the biostability of the molecule or to increase the physical stability of the double helix formed between the polynucleic acid molecule and the target nucleic acid. Exemplary methods include those described below: U.S. Patent Nos. 5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT International Publication No. WO2009099942; or European Patent Publication No. 1579015.Additional exemplary methods include those described below: Griffey et al., “2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J.Med.Chem.39(26):5100-5109(1997)); Obika, et al., “Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar puckering”. Tetrahedron Letters 38(50):8735 1997; Koizumi, M., “ENA oligonucleotides as therapeutics”. Current opinion in molecular therapeutics 8(2):144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits - antisense technologies: new "Chemical Possibilities," Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, polynucleotide molecules can be biologically generated using expression vectors in which the polynucleotide molecule is subcloned in antisense orientation (i.e., the transcribed RNA of the inserted polynucleotide molecule is antisense-oriented relative to the desired target polynucleotide molecule).

[0104] In some embodiments, polynucleic acid molecules are synthesized by a tandem synthesis method, where both strands are synthesized as a single adjacent oligonucleotide fragment or chain separated by a cleavage linker, which is then cleaved to yield separate fragments or chains that hybridize the double helix and allow for the purification of the double helix.

[0105] In some examples, polynucleic acid molecules are also assembled from two characteristic nucleic acid chains or fragments, where one fragment contains a sense region and the second fragment contains the molecule's antisense region.

[0106] For example, further modification methods for incorporating sugar, base, and phosphate modifications include: Eckstein et al., International Publication PCT No. WO 92 / 07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93 / 15187; Sproat, USPat. No. 5, 334, 711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97 / 26270; Beigelman et al.,USPat.No.5,716,824;Usman et al.,USPat.No.5,627,053;Woolf et al.,International PCT Publication No.WO 98 / 13526;Thompson et al.,USSer.No.60 / 082,404 which was filed on Apr.20,1998;Karpeisky et al. al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010. The above publication describes methods and tactics for determining the positions for incorporating sugar, base, and / or phosphate modifications into nucleic acid molecules without regulating catalytic activity.

[0107] In some cases, chemical modification of internucleotide bonds in polynucleic acid molecules with phosphorothioates, phosphorodithioates, and / or 5'-methylphosphonate bonds improves stability, while excessive modification often leads to toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide bonds may be minimized in some cases. In such cases, reducing the concentration of these bonds reduces the toxicity of these molecules and increases their efficacy and high specificity.

[0108] Polynucleic acid molecule conjugate In some embodiments, the polynucleic acid molecule (B) further binds to polypeptide A which is delivered to a desired site. In some cases, the polynucleic acid molecule binds to polypeptide A and optionally to a polymer moiety. In some examples, at least one polypeptide A binds to at least one B. In some examples, at least one polypeptide A binds to at least one B to form an AB conjugate. In some embodiments, at least one A binds 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 binds to at least two B. In some examples, at least one polypeptide A binds to at least two, three, four, five, six, seven, eight, or more B.

[0109] In some cases, a polynucleic acid molecule is conjugated to a polypeptide (A) and optionally a polymer moiety (C). In some embodiments, at least one polypeptide A is conjugated at one end of at least one B, while at least one C is conjugated at the opposite end of at least one B to form an ABC conjugate. In some examples, at least one polypeptide A is conjugated at one end of at least one B, while at least one C is conjugated at an internal site of at least one B. In some examples, at least one polypeptide A is directly conjugated to at least one C. In some examples, at least one B is indirectly conjugated to at least one polypeptide A via at least one C to form an ACB conjugate.

[0110] In some examples, at least one B and / or at least one C, and optionally at least one D, bind to at least one polypeptide A. In some examples, at least one B is bound to at least one polypeptide A terminally (e.g., 5' or 3' terminal) or via an internal site. In some cases, at least one C binds to at least one polypeptide A directly or indirectly by at least one B. Indirectly, by at least one B, at least one C binds at the same terminal as at least one polypeptide A on B, at a counter terminal from at least one polypeptide A, or independently via an internal site. In some examples, at least one additional polypeptide A further binds to at least one polypeptide A, B, or C. In further examples, at least one D optionally binds directly or indirectly to at least one polypeptide A, at least one B, or at least one C. If directly bound to at least one polypeptide A, at least one D may optionally bind to at least one B to form an ADB conjugate, or optionally bind to at least one B and at least one C to form an ADBC ​​conjugate. In some examples, to form a DABC conjugate, at least one D binds directly to at least one polypeptide A and indirectly to at least one B and at least one C. If indirectly bound to at least one polypeptide A, at least one D may optionally bind to at least one B to form an ABD conjugate, or optionally bind to at least one B and at least one C to form an ABDC conjugate. In some examples, at least one additional D further binds to at least one polypeptide A, B, or C.

[0111] joining part In some embodiments, the binding portion A is a polypeptide, peptide, or non-peptide ligand. In some examples, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some examples, the antibody or its antigen-binding fragment includes a humanized antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a monoclonal antibody or its antigen-binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triplicate antibody, a quadruplicate antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its antigen-binding fragment, a bispecific antibody or its binding fragment, or a chemically modified derivative thereof.

[0112] In some embodiments, binding site A is a bispecific antibody or its antigen-binding fragment. In some examples, the bispecific antibody is a trifunctional antibody or a bispecific mini-antibody. In some cases, the bispecific antibody is a trifunctional antibody. In some examples, the trifunctional antibody is a full-length monoclonal antibody containing binding sites for two different antigens.

[0113] In some cases, bispecific antibodies are bispecific miniantibodies. In some examples, bispecific miniantibodies include bivalent Fab2, F(ab)'3 fragments, bis-scFv, (scFv)2, diabodies, minibodies, triplicate antibodies, quadruplicate antibodies, or bispecific T cell engagers (BiTEs). In some embodiments, a bispecific T cell engager is a fusion protein containing two single-chain variable fragments (scFvs) in which two scFvs target epitopes of two different antigens.

[0114] In some embodiments, the binding portion A is a bispecific mini-antibody. In some examples, A is a bispecific Fab2. In some examples, A is a bispecific F(ab)'3 fragment. In some cases, A is a bispecific bis-scFv. In some embodiments, A is a bispecific diabody (scFv). In some embodiments, A is a bispecific mini-body. In some embodiments, A is a bispecific triplicate antibody. In other embodiments, A is a bispecific quadruplicate antibody. In other embodiments, A is a bispecific T-cell engager (BiTE).

[0115] In some embodiments, the binding portion A is a trispecific antibody. In some examples, the trispecific antibody includes an F(ab)'3 fragment or a trispecific antibody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, A is a trispecific antibody. In some embodiments, A is a trispecific antibody as described in Dimas, et al., “Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells,” Mol. Pharmaceuticals, 12(9):3490-3501 (2015).

[0116] In some embodiments, binding portion A is an antibody or its antigen-binding fragment that recognizes a cell surface protein. In some examples, binding portion A is an antibody or its antigen-binding fragment that recognizes a cell surface protein on muscle cells. In some cases, binding portion A is an antibody or its antigen-binding fragment that recognizes a cell surface protein on skeletal muscle cells.

[0117] In some embodiments, exemplary antibodies are, but are not limited to, anti-myosin antibodies, anti-transferrin receptor antibodies, and antibodies that recognize muscle-specific kinase (MuSK). In some examples, the antibody is an anti-transferrin receptor (anti-CD71) antibody.

[0118] In some embodiments, when the antibody is an anti-transferrin receptor (anti-CD71) antibody, the anti-transferrin receptor antibody specifically binds to the transferrin receptor (TfR), preferably specifically to transferrin receptor 1 (TfR1), or more preferably specifically to human transferrin receptor 1 (TfR1) (or human CD71).

[0119] In some cases, the anti-transferrin receptor antibody includes a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region including the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q, X2 is selected from Q or E, and the HCDR3 sequence includes SEQ ID NO: 19.

[0120] In some embodiments, the VH region of the anti-transferrin antibody includes sequences of HCDR1, HCDR2, and HCDR3 selected from Table 2.

[0121] [Table 2]

[0122] In some embodiments, the VH region includes an HCDR1 sequence containing sequence number 17, an HCDR2 sequence containing sequence number 18, 20, or 21, and an HCDR3 sequence containing sequence number 19. In some cases, the VH region includes an HCDR1 sequence containing sequence number 17, an HCDR2 sequence containing sequence number 18, and an HCDR3 sequence containing sequence number 19. The VH region includes an HCDR1 sequence containing sequence number 17, an HCDR2 sequence containing sequence number 20, and an HCDR3 sequence containing sequence number 19. In some cases, the VH region includes an HCDR1 sequence containing sequence number 17, an HCDR2 sequence containing sequence number 21, and an HCDR3 sequence containing sequence number 19.

[0123] In some embodiments, the VL region of the anti-transferrin receptor antibody comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, and if present, is F.

[0124] In some embodiments, the VL region of the anti-transferrin receptor antibody includes LCDR1, LCDR2, and LCDR3 sequences selected from Table 2.

[0125] [Table 3]

[0126] In some cases, the VL region includes the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence containing sequence numbers 23, 25, or 28, and the LCDR3 sequence containing sequence number 24 or 26, where X3 is selected from N or S.

[0127] In some cases, the VL region includes an LCDR1 sequence containing sequence number 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing sequence number 24 or 26, where X4 is selected from A or G and X5 is selected from D or E.

[0128] In some cases, the VL region includes the LCDR1 sequence containing sequence number 22 or 27, the LCDR2 sequence containing sequence number 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and if present, it is F.

[0129] In some cases, the VL region includes the LCDR1 sequence containing sequence number 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 is present or absent, and if present it is F.

[0130] In some cases, the VL region includes the LCDR1 sequence containing sequence number 22, the LCDR2 sequence containing sequence number 23, and the LCDR3 sequence containing sequence number 24.

[0131] In some cases, the VL region includes the LCDR1 sequence containing sequence number 22, the LCDR2 sequence containing sequence number 25, and the LCDR3 sequence containing sequence number 26.

[0132] In some cases, the VL region includes the LCDR1 sequence containing sequence number 27, the LCDR2 sequence containing sequence number 28, and the LCDR3 sequence containing sequence number 26.

[0133] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence EINPIX1GRSNYAX2KFQG where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence including SEQ ID NO: 19, the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence QHFWGTPLTX6 where X3 is selected from N or S, X4 is selected from A or G, X5 is selected from D or E, and X6 is present or absent, if present it is F.

[0134] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and the HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence containing SEQ ID NO: 24 or 26, where X3 is selected from N or S.

[0135] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, X1 selected from N or Q, X2 selected from Q or E, the HCDR2 sequence EINPIX1GRSNYAX2KFQG, and the HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22 or 27, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence containing SEQ ID NO: 24 or 26, X4 selected from A or G, and X5 selected from D or E.

[0136] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence EINPIX1GRSNYAX2KFQG, where X1 is selected from N or Q and X2 is selected from Q or E, and the HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22 or 27, the LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, is F.

[0137] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence EINPIX1GRSNYAX2KFQG where X1 is selected from N or Q and X2 is selected from Q or E, and the HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, X5 is selected from D or E, and X6 is present or absent, and if present it is F.

[0138] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence EINPIX1GRSNYAX2KFQG where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence including SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 22, an LCDR2 sequence including SEQ ID NO: 23, and an LCDR3 sequence including SEQ ID NO: 24.

[0139] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence EINPIX1GRSNYAX2KFQG where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence including SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 22, an LCDR2 sequence including SEQ ID NO: 25, and an LCDR3 sequence including SEQ ID NO: 26.

[0140] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence EINPIX1GRSNYAX2KFQG where X1 is selected from N or Q and X2 is selected from Q or E, and an HCDR3 sequence including SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 27, an LCDR2 sequence including SEQ ID NO: 28, and an LCDR3 sequence including SEQ ID NO: 26.

[0141] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 18, and an HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence containing SEQ ID NO: 24 or 26, where X3 is selected from N or S.

[0142] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 18, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 24 or 26, where X4 is selected from A or G and X5 is selected from D or E.

[0143] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 18, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22 or 27, an LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and if present, is F.

[0144] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence containing SEQ ID NO: 18, and the HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6; X5 is selected from D or E; and X6 is present or absent, and if present, is F.

[0145] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence containing SEQ ID NO: 18, and the HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22, the LCDR2 sequence containing SEQ ID NO: 23, and the LCDR3 sequence containing SEQ ID NO: 24.

[0146] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 18, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 21, and an LCDR3 sequence containing SEQ ID NO: 26.

[0147] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 18, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 27, an LCDR2 sequence containing SEQ ID NO: 28, and an LCDR3 sequence containing SEQ ID NO: 26.

[0148] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence containing SEQ ID NO: 24 or 26, where X3 is selected from N or S.

[0149] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 24 or 26, where X4 is selected from A or G and X5 is selected from D or E.

[0150] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22 or 27, an LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and if present, it is F.

[0151] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence containing SEQ ID NO: 20, and the HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6; X5 is selected from D or E; and X6 is present or absent, and if present, is F.

[0152] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 23, and an LCDR3 sequence containing SEQ ID NO: 24.

[0153] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 25, and an LCDR3 sequence containing SEQ ID NO: 26.

[0154] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 27, an LCDR2 sequence containing SEQ ID NO: 28, and an LCDR3 sequence containing SEQ ID NO: 26.

[0155] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 21, and an HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence containing SEQ ID NO: 24 or 26, where X3 is selected from N or S.

[0156] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 21, and an HCDR3 sequence containing SEQ ID NO: 19; the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence containing SEQ ID NO: 24 or 26, where X4 is selected from A or G and X5 is selected from D or E.

[0157] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 21, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22 or 27, an LCDR2 sequence containing SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence QHFWGTPLTX6, where X6 may or may not be present, and if present, it is F.

[0158] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising the HCDR1 sequence containing SEQ ID NO: 17, the HCDR2 sequence containing SEQ ID NO: 21, and the HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising the LCDR1 sequence containing SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, where X5 is selected from D or E, and X6 is present or absent, if present, F.

[0159] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 21, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 23, and an LCDR3 sequence containing SEQ ID NO: 24.

[0160] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 21, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 25, and an LCDR3 sequence containing SEQ ID NO: 26.

[0161] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 21, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 27, an LCDR2 sequence containing SEQ ID NO: 28, and an LCDR3 sequence containing SEQ ID NO: 26.

[0162] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the sequence of the VH region has approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 29-33, and the sequence of the VL region has approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs. 34-38.

[0163] In some embodiments, the VH region includes sequences selected from sequence numbers 29-33 (Table 4), and the VL region includes sequences selected from sequence numbers 34-38 (Table 5). The underlined regions in Tables 4 and 5 represent CDR1, CDR2, or CDR3 sequences, respectively.

[0164] [Table 4]

[0165] [Table 5]

[0166] In some embodiments, the anti-transferrin receptor antibody includes a VH region and a VL region, as illustrated in Table 6.

[0167] [Table 6]

[0168] In some embodiments, the anti-transferrin receptor antibodies described herein comprise an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some cases, the anti-transferrin receptor antibody comprises an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the anti-transferrin receptor antibody comprises an IgG1 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2a framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2b framework. In some cases, the anti-transferrin receptor antibody comprises an IgG3 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG4 framework.

[0169] In some cases, anti-transferrin receptor antibodies contain one or more mutations in the framework region, e.g., the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some cases, one or more mutations are intended to stabilize the antibody and / or extend its half-life. In some cases, one or more mutations are intended to modulate Fc receptor interactions and reduce or eliminate Fc effector functions such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional examples, one or more mutations are intended to modulate glycosylation.

[0170] In some embodiments, one or more mutations are located in the Fc region. In some cases, the Fc region contains mutations at residue positions L234, L235, or a combination thereof. In some cases, the mutations include L234 and L235. In some cases, the mutations include L234A and L235A. In some cases, the residue positions are relative to IgG1.

[0171] In some cases, the Fc region contains mutations at residue positions L234, L235, D265, N21, K46, L52, or P53, or combinations thereof. In some cases, the mutations include L234 and L235 in combination with mutations at residue positions K46, L52, or P53. In some cases, the Fc region contains mutations at L234, L235, and K46. In some cases, the Fc region contains mutations at L234, L235, and L52. In some cases, the Fc region contains mutations at L234, L235, and P53. In some cases, the Fc region contains mutations at D265 and N21. In some cases, the residue positions are relative to IgG1.

[0172] In some cases, the Fc region contains L234A, L235A, D265A, N21G, K46G, L52R, or P53G, or a combination thereof. In some cases, the Fc region contains L234A and L235A in combination with K46G, L52R, or P53G. In some cases, the Fc region contains L234A, L235A, and K46G. In some cases, the Fc region contains L234A, L235A, and L52R. In some cases, the Fc region contains L234A, L235A, and P53G. In some cases, the Fc region contains D265A and N21G. In some cases, the residue positions are relative to IgG1.

[0173] In some cases, the Fc region contains mutations or combinations of mutations at residue positions L235, L236, D265, N21, K46, L52, or P53. In some cases, the Fc region contains mutations at L235 and L236. In some cases, the Fc region contains mutations at L235 and L236 in combination with mutations at residue positions K46, L52, or P53. In some cases, the Fc region contains mutations at L235, L236, and K46. In some cases, the Fc region contains mutations at L235, L236, and L52. In some cases, the Fc region contains mutations at L235, L236, and P53. In some cases, the Fc region contains mutations at D265 and N21. In some cases, the residue positions are relative to IgG2b.

[0174] In some embodiments, the Fc region includes L235A, L236A, D265A, N21G, K46G, L52R, or P53G, or a combination thereof. In some cases, the Fc region includes L235A and L236A. In some cases, the Fc region includes L235A and L236A in combination with K46G, L52R, or P53G. In some cases, the Fc region includes L235A, L236A, and K46G. In some cases, the Fc region includes L235A, L236A, and L52R. In some cases, the Fc region includes L235A, L236A, and P53G. In some cases, the Fc region includes D265A and N21G. In some cases, the residue positions are relative to IgG2b.

[0175] In some embodiments, the Fc region contains mutations at residue positions L233, L234, D264, N20, K45, L51, or P52, where the residues correspond to positions 233, 234, 264, 296, 321, 327, and 328 of the SEQ ID NO: 233, L234, and P52. In some cases, the Fc region contains mutations at L233 and L234. In some cases, the Fc region contains mutations at L233 and L234 in combination with mutations at residue positions K45, L51, or P52. In some cases, the Fc region contains mutations at L233, L234, and K45. In some cases, the Fc region contains mutations at L233, L234, and L51. In some cases, the Fc region contains mutations at L233, L234, and K45. In some cases, the Fc region contains mutations at L233, L234, and P52. In some cases, the Fc region may contain mutations at D264 and N20. In some cases, the intended positions may correspond to residues L233, L234, D264, N20, K45, L51, or P52 in the IgG1, IgG2, IgG3, or IgG4 framework. In some cases, mutations to residues corresponding to residues L233, L234, D264, N20, K45, L51, or P52 in SEQ ID NO: 303 in the IgG1, IgG2, or IgG4 framework may also be intended.

[0176] In some embodiments, the Fc region comprises L233A, L234A, D264A, N20G, K45G, L51R, or P52G, where the residues correspond to positions 233, 234, 264, 20, 45, 51, and 52 in SEQ ID NO: 39. In some cases, the Fc region comprises L233A and L234A. In some cases, the Fc region comprises L233A and L234A in combination with K45G, L51R, or P52G. In some cases, the Fc region comprises L233A, L234A, and K45G. In some cases, the Fc region comprises L233A, L234A, and L51R. In some cases, the Fc region comprises L233A, L234A, and K45G. In some cases, the Fc region comprises L233A, L234A, and P52G. In some cases, the Fc region includes D264A and N20G.

[0177] In some embodiments, the human IgG constant region modifies antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC), for example, Natsume et al. Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4):2571-5; Moore et al., 2010 mAbs, 2(2):181-189; Lazar et al., 2006 PNAS, 103(11):4005-4010; Shields et al., 2001 JBC, 276(9):6591-6604; Stavenhagen et al., 2007 Cancer Res, 67(18):8882-8890; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48:152-164; Alegre et al. It can be modified using the amino acid modifications described in the review in al., 1992 J Immunol, 148:3461-3468; Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11.

[0178] In some embodiments, the anti-transferrin receptor antibodies described herein are full-length antibodies comprising a heavy chain (HC) and a light chain (LC). In some cases, the heavy chain (HC) contains a sequence selected from Table 7. In some cases, the light chain (LC) contains a sequence selected from Table 8. Underlined regions indicate the respective CDRs.

[0179] [Table 7] JPEG2023527638000019.jpg250170 JPEG2023527638000020.jpg254170 JPEG2023527638000021.jpg242170 JPEG2023527638000022.jpg181170

[0180] [Table 8]

[0181] In some embodiments, the anti-transferrin receptor antibodies described herein have an improved serum half-life compared to a reference anti-transferrin receptor antibody. In some cases, the improved serum half-life is longer than that of the reference anti-transferrin receptor antibody by at least 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, 30 days, or longer.

[0182] In some embodiments, binding site A binds to the polynucleic acid molecule (B) nonspecifically. In some examples, binding site A binds to the polynucleic acid molecule (B) in a non-site-specific manner via a lysine residue or a cysteine ​​residue. In some examples, binding site A binds to the polynucleic acid molecule (B) in a non-site-specific manner via a lysine residue (e.g., a lysine residue present at binding site A). In some cases, binding site A binds to the polynucleic acid molecule (B) in a non-site-specific manner via a cysteine ​​residue (e.g., a cysteine ​​residue present at binding site A).

[0183] In some embodiments, binding site A binds to the polynucleotide molecule (B) in a non-site-specific manner. In some examples, binding site A binds to the polynucleotide molecule (B) in a site-specific manner via lysine residues, cysteine ​​residues, at the 5'-terminus, at the 3'-terminus, with non-natural amino acids, or with enzymatically modified or enzymatically catalyzed residues. In some examples, binding site A binds to the polynucleotide molecule (B) in a site-specific manner via lysine residues (e.g., lysine residues present in binding site A). In some examples, binding site A binds to the polynucleotide molecule (B) in a site-specific manner via cysteine ​​residues (e.g., cysteine ​​residues present in binding site A). In some examples, binding site A binds to the polynucleotide molecule (B) in a site-specific manner at the 5'-terminus. In some examples, binding site A binds to the polynucleotide molecule (B) in a site-specific manner at the 3'-terminus. In some examples, binding site A binds to the polynucleotide molecule (B) in a site-specific manner via non-natural amino acids. In some cases, binding site A binds to the polynucleic acid molecule (B) via site-specific enzymatic modification or an enzymatically catalyzed residue.

[0184] In some embodiments, one or more polynucleotide molecules (B) bind to binding site A. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more polynucleotide molecules bind to one binding site A. In some examples, about 1 polynucleotide molecule binds to one binding site A. In some examples, about 2 polynucleotide molecules bind to one binding site A. In some examples, about 3 polynucleotide molecules bind to one binding site A. In some examples, about 4 polynucleotide molecules bind to one binding site A. In some examples, about 5 polynucleotide molecules bind to one binding site A. In some examples, about 6 polynucleotide molecules bind to one binding site A. In some examples, about 7 polynucleotide molecules bind to one binding site A. In some examples, about 8 polynucleotide molecules bind to one binding site A. In some examples, about 9 polynucleotide molecules bind to one binding site A. In some cases, approximately 10 polynucleotide molecules bind to one binding site A. In some cases, approximately 11 polynucleotide molecules bind to one binding site A. In some cases, approximately 12 polynucleotide molecules bind to one binding site A. In some cases, approximately 13 polynucleotide molecules bind to one binding site A. In some cases, approximately 14 polynucleotide molecules bind to one binding site A. In some cases, approximately 15 polynucleotide molecules bind to one binding site A. In some cases, approximately 16 polynucleotide molecules bind to one binding site A. In some cases, one or more polynucleotide molecules are the same. In other cases, one or more polynucleotide molecules are different.

[0185] In some embodiments, the number of polynucleotide molecules (B) bound to binding site A forms a certain ratio. In some examples, the ratio is called the DAR (drug-to-antibody) ratio, and the drug as referred herein is the polynucleotide molecule (B). In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 1 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 2 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 3 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 4 or more. In some examples, the DAR ratio of polynucleotide molecules (B) to binding site A is about 5 or more. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 6 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 7 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 8 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 9 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 10 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 11 or higher. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 12 or higher.

[0186] In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 1. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 2. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 3. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 4. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 5. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 6. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 7. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 8. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 9. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 10. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 11. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 12. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 13. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 14. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 15. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is approximately 16.

[0187] In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 1. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 2. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 4. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 6. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 8. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is 12.

[0188] In some cases, a conjugate containing polynucleotide molecule (B) and binding site A exhibited improved activity compared to a conjugate containing polynucleotide molecule (B) without binding site A. In some cases, the improved activity resulted in enhanced biologically relevant functions, such as improved stability, affinity, binding, functional activity, and efficacy in treating or preventing disease conditions. In some cases, the disease condition resulted from one or more mutated exons in a gene. In some cases, a conjugate containing polynucleotide molecule (B) and binding site A resulted in increased exon skipping of one or more mutated exons compared to a conjugate containing polynucleotide molecule (B) without binding site A. In some examples, exon skipping is increased by at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% in the conjugate containing the polynucleic acid molecule (B) and the binding site A, compared to the conjugate containing the polynucleic acid molecule (B) without binding site A.

[0189] In some embodiments, an antibody or its antigen-binding fragment is further modified, either alone or in combination, using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, or addition, and / or by recombination, and / or other modifications known in the art (e.g., post-translational and chemical modifications such as glycosylation and phosphorylation). In some examples, the modifications further include modifications to modulate the interaction with the Fc receptor. In some examples, one or more modifications include, for example, those described in International Publication WO97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence underlying the amino acid sequence of an antibody or its antigen-binding fragment are well known to those skilled in the art.

[0190] In some examples, the antigen-binding fragment further includes its derivatives and contains a polypeptide sequence comprising at least one CDR.

[0191] In some examples, the term “single-chain” as used herein means that the first and second domains of a bispecific single-chain construct are covalently linked in the form of a co-linear amino acid sequence, which can preferably be encoded by a single nucleic acid molecule.

[0192] In some examples, a bispecific single-chain antibody construct relates to a construct containing binding domains derived from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some examples, the scFv contains VH and VL domains linked by a linker peptide. In some examples, the linker is of sufficient length and sequence to allow each of the first and second domains to maintain its differential binding specificity independently of each other.

[0193] In some embodiments, as used herein, binding to or interaction by an antigen-interacting site defines the binding / interaction of at least two antigen-interacting sites to each other. In some examples, an antigen-interacting site defines a polypeptide motif that exhibits the ability to interact with a specific antigen or a specific group of antigens. In some cases, binding / interaction is also understood to define a specific recognition. In such cases, specific recognition refers to the fact that an antibody or its antigen-binding fragment can specifically interact with and / or bind to at least two amino acids of each of the target molecule. For example, specific recognition relates to the specificity of an antibody molecule or its ability to distinguish a specific range of target molecules. In additional examples, the specificity of an antigen-interacting site with an antigen results in the initiation of a signal, such as by induction of conformational changes or oligomerization of the antigen. In further embodiments, binding is illustrated by the specificity of the "key-lock principle". Therefore, in some cases, specific motifs in the antigen interaction site and the amino acid sequence of the antigen bind to each other as a result of their primary, secondary, or tertiary structure, as well as as a result of a second modification of the structure described above. In such cases, the specific interaction of the antigen interaction site with the antigen leads to the site's easy binding to the antigen.

[0194] In some cases, specific interactions further refer to reduced cross-reactivity or reduced off-target effects of an antibody or its antigen-binding fragment. For example, an antibody or its antigen-binding fragment that binds to a desired polypeptide / protein but does not bind to any other polypeptide, or does not bind to any other polypeptide, is considered specific to the desired polypeptide / protein. Specificity of the antigen interaction site: Examples of specific interactions with an antigen include the specificity of a ligand to its receptor, e.g., the interaction of antigenic determinants (epitopes) with the antigen-binding site of an antibody.

[0195] Therefore, in some cases, the polynucleic acid molecule conjugate includes an antisense strand that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1, an antisense strand having a sequence that is at least 80% identical to SEQ ID NO: 2, and an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to the polynucleic acid molecule, thereby mediating RNA interference against DMPK.

[0196] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense strand having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs. 3, 5, 7, 9, 11, 13, or 15, an antisense strand having the sequence of SEQ ID NOs. 4, 6, 8, 10, 12, 14, or 16, and the anti-transferrin receptor antibody or its antigen-binding The fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region comprising an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 23, and an LCDR3 sequence containing SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and polynucleic acid molecule are conjugated via a linker comprising 4-(N-maleimidomethyl)cyclohexane-1-amidate (SMCC).

[0197] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense strand having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NOs. 3, 5, 7, 9, 11, 13, or 15, and an antisense strand having the sequence of SEQ ID NOs. 4, 6, 8, 10, 12, 14, or 16, and anti The transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region contains at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0198] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense strand having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1, and an antisense strand having the sequence of SEQ ID NO: 2, wherein the sense strand has at least 3, 4, 5, or 6 consecutive 2'-O-methyl-modified nucleotides at its 5' end, and at least The anti-transferrin receptor antibody or its antigen-binding fragment comprises two or at least three 2'-F modified nucleotides, and includes a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0199] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense strand having the sequence of SEQ ID NO: 2, which is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1, and an antisense strand having at least two, at least three, at least four, or at least five consecutive 2'-O-methyl-modified nucleotides at its 3' end, and at least one, at least The anti-transferrin receptor antibody or its antigen-binding fragment comprises two, at least three, and at least four 2'-F modified nucleotides, and includes a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region includes an HCDR1 sequence containing SEQ ID NO: 17, an HCDR2 sequence containing SEQ ID NO: 20, and an HCDR3 sequence containing SEQ ID NO: 19, and the VL region includes an LCDR1 sequence containing SEQ ID NO: 22, an LCDR2 sequence containing SEQ ID NO: 23, and an LCDR3 sequence containing SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a maleimide linker.

[0200] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense strand having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1, and an antisense strand having the sequence of SEQ ID NO: 2, wherein the antisense strand contains 2'-O-methyl-modified nucleotides at its 5' and 3' ends. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region comprising an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence including SEQ ID NO: 18, and an HCDR3 sequence including SEQ ID NO: 19, and the VL region comprising an LCDR1 sequence including SEQ ID NO: 22, an LCDR2 sequence including SEQ ID NO: 3, and an LCDR3 sequence including SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and polynucleic acid molecules are conjugated via a maleimide linker.

[0201] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense strand having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1, and an antisense strand having the sequence of SEQ ID NO: 2, wherein the antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides at the 3' end and four 2'-F modified nucleotides, where four Any two of the 2'-F modified nucleotides are not consecutive, the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, the VH region having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3, and the VL region having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleic acid molecule are conjugated via a 6-amino-1-hexanol linker.

[0202] Additional connection In some embodiments, the binding site is a plasma protein. In some examples, the plasma protein includes albumin. In some examples, binding site A is albumin. In some examples, albumin is bound to the polynucleic acid molecule by one or more binding chemistry described herein. In some examples, albumin is bound to the polynucleic acid molecule by natural ligation chemistry. In some examples, albumin is bound to the polynucleic acid molecule by lysine linkage.

[0203] In some examples, binding site A is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, and hydrocarbons (including saturated, unsaturated, substituted, or combinations thereof). In some examples, the steroid is cholesterol. In some examples, the binding site is cholesterol. In some examples, cholesterol is bound to the polynucleic acid molecule by one or more binding chemistry described herein. In some examples, cholesterol is bound to the polynucleic acid molecule by natural ligation chemistry. In some examples, cholesterol is bound to the polynucleic acid molecule by lysine linkage.

[0204] In some examples, the binding site is a polymer containing a polynucleic acid molecule aptamer that binds to a specific surface marker on a cell, though not limited to this example. In this example, the binding site is a polynucleic acid that does not hybridize to a target gene or mRNA, but instead can selectively bind to the cell surface marker, similar to an antibody that binds to that specific epitope of the cell surface marker.

[0205] In some cases, the binding site is a peptide. In some cases, the peptide contains approximately 1 to 3 kDa. In some cases, the peptide contains approximately 1.2 to 2.8 kDa, approximately 1.5 to 2.5 kDa, or approximately 1.5 to 2 kDa. In some examples, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some examples, the binding site is a bicyclic peptide (e.g., bicycles in Bicycle Therapeutics).

[0206] In further cases, the binding portion is a small molecule. In some examples, the small molecule is an antibody-recruiting small molecule. In some cases, the antibody-recruiting small molecule includes a target binding end and an antibody binding end, where the target binding end can recognize and interact with cell surface receptors. For example, in some examples, a target binding end containing a glutamate urea compound enables interaction with PSMA, thereby enhancing antibody interactions with cells expressing PSMA. In some cases, the binding site is a small molecule described in Zhang et al., “A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules,” J Am Chem Soc. 132(36):12711-12716 (2010); or McEnaney, et al., “Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease,” ACS Chem Biol. 7(7):1139-1151 (2012).

[0207] Production of antibodies or their antigen-binding fragments In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments, anti-transferrin receptor antibodies or their antigen-binding fragments) are produced, in particular, by chemical synthesis or by recombinant expression using any method known in the art to facilitate the synthesis of polypeptides (e.g., antibodies), and preferably by recombinant expression techniques.

[0208] In some cases, antibodies or their antigen-binding fragments are recombinantly expressed, and the nucleic acids encoding the antibody or its antigen-binding fragment are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which include the synthesis of duplicate oligonucleotides containing a portion of the sequence encoding the antibody, annealing and ligation of the oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0209] Alternatively, nucleic acid molecules encoding antibodies can be arbitrarily generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing immunoglobulins) by PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence.

[0210] In some cases, antibodies or their antigen-bindings can be optionally generated by immunizing animals such as rabbits to produce polyclonal antibodies, or more preferably by producing monoclonal antibodies, as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), or by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, clones encoding at least the Fab portion of an antibody can be optionally obtained by screening a Fab expression library (e.g., Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind to specific antigens, or by screening an antibody library (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc.Natl.Acad.Sci.USA 94:4937).

[0211] In some embodiments, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used by splicing genes from mouse antibody molecules with appropriate antigen specificity together with genes from human antibody molecules with appropriate biological activity. Chimeric antibodies are molecules derived from various animal species, such as animal species, in which the different parts include a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region (e.g., a humanized antibody).

[0212] In some embodiments, techniques described for the production of single-chain antibodies (USPat. No. 4, 694, 778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are suitable for producing single-chain antibodies. Single-chain antibodies are formed by linking heavy or light chain fragments of the Fv region via amino acid bridges, resulting in single-chain polypeptides. Techniques for assembling functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0213] In some embodiments, an expression vector containing the antibody nucleotide sequence or the antibody nucleotide sequence itself is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

[0214] In some embodiments, various host expression vector systems are used to express the antibodies or their antigen-binding fragments described herein. Such host expression systems represent not only vehicles from which the antibody coding sequence is generated and subsequently purified, but also cells that express the antibody or its antigen-binding fragment in situ when deformed with or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with cosmid DNA expression vectors containing recombinant bacteriophage DNA, plasmid DNA, or the antibody or its binding fragment coding sequence; yeast (e.g., Saccharomyces picia) transformed with recombinant yeast expression vectors containing the antibody or its antigen-binding fragment coding sequence; insect cell lines (e.g., baculovirus) infected with recombinant virus expression vectors containing the antibody or its antigen-binding fragment coding sequence; and recombinant virus expression vectors (e.g., cauliflower mosaic). Plant cell lines infected with or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing an antibody or its antigen-binding fragment coding sequence; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) protecting recombinant expression constructs containing the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0215] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines that stably express antibodies are manipulated at will. Rather than using expression vectors containing viral replication origins, host cells are transformed with DNA controlled by appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of exogenous DNA, the cells are manipulated and grown in nutrient-enhanced medium for 1-2 days, then switched to selective medium. The selectable markers in the recombinant plasmid provide resistance to selection, allowing the cell to stably integrate the plasmid into its chromosome, grow, clone, and expand into a cell line, forming a focus. This method can be advantageously used to manipulate cell lines expressing antibodies or their antigen-binding fragments.

[0216] In some cases, but not limited to, many selection systems are used, including the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, which are utilized in tk-, hgprt-, or aprt- cells, respectively. Similarly, resistance to antimetabolites is used as a selection criterion for the following genes: dhfr, which gives resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which gives resistance to mycophenolate (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and neo, which gives resistance to aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy). 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215), as well as hygro (Santerre et al., 1984, Gene 30:147), which gives resistance to hygromycin.Known methods of recombinant DNA technology available in this field are generally described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J.Mol.Biol.150:1).

[0217] In some cases, antibody expression levels increase with vector amplification (see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987) for consideration). When a marker in an antibody-expressing vector system is amplified, an increase in the level of the inhibitor present in the host cell culture increases the number of copies of the marker gene. Since the amplified region is related to the antibody's nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0218] In some cases, any method known in the art for the purification or analysis of antibodies or antibody conjugates is used by, for example, chromatography (e.g., ion exchange, affinity, particularly affinity of protein A to specific antigens, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and high-performance protein liquid chromatography.

[0219] Conjugation Chemistry In some embodiments, polynucleotide molecule B is conjugated to a binding site. In some embodiments, polynucleotide molecule B is conjugated to a binding site in the formula AXB (where X is a linker that conjugates A and B). In some examples, the binding site includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and all analogs or derivatives of these classes of substances. Additional examples of binding sites further include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and steroids such as polysaccharides. In some examples, the binding site is an antibody or its antigen-binding fragment. In some examples, the polynucleotide molecule is further conjugated to a polymer and optionally to an endosomal soluble site.

[0220] In some embodiments, polynucleotide molecules are bound to the binding site by a chemical ligation process. In some examples, polynucleotide molecules are bound to the binding site by spontaneous ligation. In some cases, the conjugate is described as follows: Dawson, et al. “Synthesis of proteins by native chemical ligation,” Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives,” J.Am.Chem.Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.” Proc.Natl.Acad.Sci.USA 1999, 96, 10068-10073; or Wu, et al. “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew.Chem.Int.Ed. 2006, 45, 4116-4125. In some examples, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, polynucleic acid molecules bind to the binding site site-specifically or non-specifically via natural ligation chemistry.

[0221] In some cases, polynucleotide molecules bind to the binding site via a site-specific method utilizing "traceless" coupling technology (PhiloChem). In some cases, "traceless" coupling technology utilizes the aldehyde group-containing polynucleotide molecule and the N-terminal 1,2-aminothiol group of the binding site to be subsequently bound. (See Casi et al., “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibody for pharmacodelivery,” JACS 134(13):5887-5892(2012))

[0222] In some cases, polynucleic acid molecules bind to a binding site via a site-specific method utilizing unnatural amino acids introduced into the binding site. In some cases, the unnatural amino acid includes p-acetylphenylalanine (pAcPhe). In some cases, the keto group of pAcPhe selectively binds to an alkoxyamine-derived binding site to form an oxime bond. (See Axup et al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40):16101-16106(2012)).

[0223] In some cases, polynucleic acid molecules are bound to the binding site by site-specific methods utilizing enzyme-catalyzed processes. In some cases, site-specific methods utilize SMARTag® technology (Catalent, Inc.). In some cases, SMARTag® technology involves the production of formylglycine (FGly) residue from cysteine ​​by formylglycine-producing enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent binding of FGly to alkylhydrazine-functionalized polynucleic acid molecules via hydrazino-Pictet-Spengler (HIPS) ligation. (See Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9):3000-3005(2009); Agarwal, et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1):46-51(2013))

[0224] In some cases, the enzyme-catalyzed process involves microbial transglutaminase (mTG). In some cases, polynucleic acid molecules bind to the binding site using the microbial transglutaminase-catalyzed process. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and the primary amine of the functionalized polynucleic acid molecule. In some cases, mTG is produced from Streptomyces mobarensis. (See Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167(2013))

[0225] In some cases, polynucleic acid molecules bind to the binding site by a method described in PCT International Publication WO2014 / 140317, which utilizes sequence-specific transpeptidases.

[0226] In some examples, polynucleic acid molecules bind to the binding site in a manner described in U.S. Patent Publications 2015 / 0105539 and 2015 / 0105540.

[0227] Polymer bonding portion In some embodiments, polymer portion C further binds to polynucleic acid molecules described herein, binding portions described herein, or a combination thereof. In some examples, polymer portion C is a conjugated polynucleic acid molecule in the formula A-X1-B-X2-C (where X1 and X2 are two linkers conjugating A and B, and B and C, respectively). It binds to the polynucleic acid molecule. In some cases, polymer portion C binds to a binding portion. In other cases, polymer portion C binds to the polynucleic acid molecule binding portion molecule. In further cases, polymer portion C binds as illustrated above.

[0228] In some examples, polymer moiety C is a natural or synthetic polymer consisting of branched or unbranched monomers and / or long chains of crosslinked networks of two-dimensional or three-dimensional monomers. In some examples, polymer moiety C includes polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). In some examples, at least one polymer moiety C is, but is not limited to, alpha-, omega-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactidic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate (also known as poly(ethylene terephthalate), PET, PETG, or PETE), polytetramethylene glycol (PTG), or polyurethane, and mixtures thereof. As used herein, mixture refers to the use of various polymers within the same compound, as in the case of block copolymers. In some cases, a block copolymer is a polymer in which at least one part of the polymer is constructed from a monomer of another polymer. In some examples, polymer part C contains a polyalkylene oxide. In some examples, polymer part C contains PEG. In some examples, polymer part C contains polyethylene-imide (PEI) or hydroxyethyl starch (HES).

[0229] In some examples, C is the PEG portion. In some examples, the PEG portion is bound at the 5' end of the polynucleotide molecule, while the binding portion is bound at the 3' end. In some examples, the PEG portion is bound at the 3' end of the polynucleotide molecule, while the binding portion is bound at the 5' end. In some examples, the PEG portion binds to an internal site of the polynucleotide molecule. In some examples, the PEG portion, the binding portion, or a combination thereof binds to an internal site of the polynucleotide molecule. In some examples, the conjugate is a direct conjugate. In some examples, the binding is via a natural ligation.

[0230] In some embodiments, polyalkylene oxides (e.g., PEG) are polydisperse or monodisperse compounds. In some examples, polydisperse materials include a dispersed distribution of materials of different molecular weights, characterized by an average weight (weight-average) size and degree of dispersion. In some examples, monodisperse PEG contains molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weight represents the average molecular weight of the polyalkylene oxide (e.g., PEG) molecules.

[0231] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2 The values ​​are 700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000Da.

[0232] In some embodiments, C is a polyalkylene oxide (e.g., PEG), and the values ​​are approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 270 It has a molecular weight of 0, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG, and the values ​​are approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 29 It has molecular weights of 0, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some examples, the molecular weight of C is approximately 200 Da. In some examples, the molecular weight of C is approximately 300 Da. In some examples, the molecular weight of C is approximately 400 Da. In some examples, the molecular weight of C is approximately 500 Da. In some examples, the molecular weight of C is approximately 600 Da. In some examples, the molecular weight of C is approximately 700 Da. In some cases, the molecular weight of C is approximately 800 Da. In some cases, the molecular weight of C is approximately 900 Da. In some cases, the molecular weight of C is approximately 1000 Da. In some cases, the molecular weight of C is approximately 1100 Da. In some cases, the molecular weight of C is approximately 1200 Da. In some cases, the molecular weight of C is approximately 1300 Da.In some cases, the molecular weight of C is approximately 1400 Da. In some cases, the molecular weight of C is approximately 1450 Da. In some cases, the molecular weight of C is approximately 1500 Da. In some cases, the molecular weight of C is approximately 1600 Da. In some cases, the molecular weight of C is approximately 1700 Da. In some cases, the molecular weight of C is approximately 1800 Da. In some cases, the molecular weight of C is approximately 1900 Da. In some cases, the molecular weight of C is approximately 2000 Da. In some cases, the molecular weight of C is approximately 2100 Da. In some cases, the molecular weight of C is approximately 2200 Da. In some cases, the molecular weight of C is approximately 2300 Da. In some cases, the molecular weight of C is approximately 2400 Da. In some cases, the molecular weight of C is approximately 2500 Da. In some cases, the molecular weight of C is approximately 2600 Da. In some cases, the molecular weight of C is approximately 2700 Da. In some cases, the molecular weight of C is approximately 2800 Da. In some cases, the molecular weight of C is approximately 2900 Da. In some cases, the molecular weight of C is approximately 3000 Da. In some cases, the molecular weight of C is approximately 3250 Da. In some cases, the molecular weight of C is approximately 3350 Da. In some cases, the molecular weight of C is approximately 3500 Da. In some cases, the molecular weight of C is approximately 3750 Da. In some cases, the molecular weight of C is approximately 4000 Da. In some cases, the molecular weight of C is approximately 4250 Da. In some cases, the molecular weight of C is approximately 4500 Da. In some cases, the molecular weight of C is approximately 4600 Da. In some cases, the molecular weight of C is approximately 4750 Da. In some cases, the molecular weight of C is approximately 5000 Da. In some cases, the molecular weight of C is approximately 5500 Da. In some cases, the molecular weight of C is approximately 6000 Da. In some cases, the molecular weight of C is approximately 6500 Da. In some cases, the molecular weight of C is approximately 7000 Da. In some cases, the molecular weight of C is approximately 7500 Da. In some cases, the molecular weight of C is approximately 8000 Da. In some cases, the molecular weight of C is approximately 10,000 Da.In some cases, the molecular weight of C is approximately 12,000 Da. In some cases, the molecular weight of C is approximately 20,000 Da. In some cases, the molecular weight of C is approximately 35,000 Da. In some cases, the molecular weight of C is approximately 40,000 Da. In some cases, the molecular weight of C is approximately 50,000 Da. In some cases, the molecular weight of C is approximately 60,000 Da. In some cases, the molecular weight of C is approximately 100,000 Da.

[0233] In some embodiments, the polyalkylene oxide (e.g., PEG) contains separate ethylene oxide units (e.g., 4 to about 48 ethylene oxide units). In some examples, the polyalkylene oxide containing separate ethylene oxide units is linear. In other examples, the polyalkylene oxide containing separate ethylene oxide units is branched.

[0234] In some cases, polymer portion C is a polyalkylene oxide (e.g., PEG) containing separate ethylene oxide units. In some cases, polymer portion C contains about 4 to about 48 ethylene oxide units. In some cases, polymer portion C contains about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units.

[0235] In some cases, polymer portion C contains, for example, another PEG containing about 4 to about 48 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, or about 48 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 4 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 5 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 6 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 7 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 8 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 9 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 10 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 11 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 12 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 13 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 14 ethylene oxide units. In some cases, polymer portion C is, for example, a separate PEG containing about 15 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 16 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 17 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 18 ethylene oxide units.In some cases, polymer portion C is a separate PEG containing, for example, about 19 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 20 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 21 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 22 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 23 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 24 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 25 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 26 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 27 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 28 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 29 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 30 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 31 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 32 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 33 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 34 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 35 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 36 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 37 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 38 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 39 ethylene oxide units.In some cases, polymer portion C is a separate PEG containing, for example, about 40 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 41 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 42 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 43 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 44 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 45 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 46 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 47 ethylene oxide units. In some cases, polymer portion C is a separate PEG containing, for example, about 48 ethylene oxide units.

[0236] In some cases, polymer portion C is dPEG® (Quanta Biodesign Ltd).

[0237] In some embodiments, polymer portion C comprises a cationic mucin-based polymer (cMAP). In some examples, the cMAP comprises one or more subunits of at least one repeating subunit, the subunit structure of which is represented by formula (∨): [ka]

[0238] Here, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 at each occurrence, preferably 4-6 or 5; and n is independently 1, 2, 3, 4, or 5 at each occurrence. In some embodiments, m and n are, for example, about 10.

[0239] In some cases, cMAP further binds to the PEG moiety, forming cMAP-PEG copolymers, mPEG-cMAP-PEGm triblock polymers, or cMAP-PEG-cMAP triblock polymers. In some cases, the PEG moiety ranges from approximately 500 Da to approximately 50,000 Da. In some cases, the PEG moiety ranges from approximately 500 Da to approximately 1000 Da, greater than 1000 Da to approximately 5000 Da, greater than 5000 Da to approximately 10,000 Da, greater than 10,000 to approximately 25,000 Da, greater than 25,000 Da to approximately 50,000 Da, or any combination of two or more of these ranges.

[0240] In some cases, polymer portion C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, polymer portion C is a cMAP-PEG copolymer. In other cases, polymer portion C is an mPEG-cMAP-PEGm triblock polymer. In further cases, polymer portion C is a cMAP-PEG-cMAP triblock polymer.

[0241] In some embodiments, polymer portion C binds to a polynucleic acid molecule, a binding portion, and optionally an endosomal soluble portion, as illustrated above.

[0242] Endosomal lytic or cell membrane permeable portion In some embodiments, the molecule of formula (I): A-X1-B-X2-C further comprises an additional binding moiety. In some examples, the additional binding moiety is an endosomal soluble moiety and / or a cell membrane permeable moiety. In some cases, the endosomal soluble moiety is a cellular compartment-releasing component, a compound that can be released from any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum having cells. In some cases, the endosomal soluble moiety comprises an endosomal soluble polypeptide, an endosomal soluble polymer, an endosomal soluble lipid, or an endosomal soluble small molecule. In some cases, the endosomal soluble moiety comprises an endosomal soluble polypeptide. In other cases, the endosomal soluble moiety comprises an endosomal soluble polymer. In some cases, the cell membrane permeable moiety comprises a cell-permeable peptide (CPP). In other cases, the cell membrane permeable moiety comprises a cell-permeable lipid. In other cases, the cell membrane permeable moiety comprises a cell-permeable small molecule.

[0243] Endosomal-lytic and cell membrane-penetrating polypeptides In some embodiments, the molecule of formula (I): A-X1-B-X2-C is further bound to an endosomal soluble polypeptide. In some cases, the endosomal soluble polypeptide is a pH-dependent membrane-active peptide. In some cases, the endosomal soluble polypeptide is an amphiphilic polypeptide. In additional cases, the endosomal soluble polypeptide is a peptide mimetic. In some examples, the endosomal soluble polypeptide comprises INF, melittin, mucin, or derivatives thereof. In some examples, the endosomal soluble polypeptide comprises INF or its derivatives. In other cases, the endosomal soluble polypeptide comprises melittin or its derivatives. In further cases, the endosomal soluble polypeptide comprises mucin or its derivatives.

[0244] In some cases, INF7 is a 24-residue polypeptide, and these sequences include CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 67) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 68). In some cases, INF7 or its derivatives include the following sequences: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO: 69), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 70), or GLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 71).

[0245] In some cases, melittin is a 26-residue polypeptide, and this sequence includes CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 72) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 73). In some examples, melittin includes the polypeptide sequence described in U.S. Patent No. 8,501,930.

[0246] In some cases, mucins are antimicrobial peptides (AMPs) derived from the venom glands of the scorpion (Mesobuthus eupeus). In some cases, mucins consist of mucin-13 (these sequences include IFGAIAGLLKNIF-NH2 (SEQ ID NO: 74)) and mucin-18 (these sequences include FFGHLFKLATKIIPSLFQ (SEQ ID NO: 75)).

[0247] In some examples, the endosomal soluble polypeptide comprises a polypeptide, melittin or its derivative, or mucin or its derivative whose sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity with INF7 or its derivative. In some examples, the endosomal soluble portion comprises INF7 or its derivative, melittin or its derivative, or mucin or its derivative.

[0248] In some cases, the endosomal soluble portion is INF7 or a derivative thereof. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 67-71. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 67. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. In some cases, the endosomal soluble portion contains SEQ ID NOs. In some cases, the endosomal soluble portion contains SEQ ID NOs. 68-71. In some cases, the endosomal soluble portion consists of SEQ ID NOs. 68-71.

[0249] In some cases, the endosomal soluble portion is melittin or a derivative thereof. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 72 or 73. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 72. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 73. In some cases, the endosomal soluble portion contains SEQ ID NO: 72. In some cases, the endosomal soluble portion contains SEQ ID NO: 73. In some cases, the endosomal soluble portion consists of SEQ ID NO: 72. In some cases, the endosomal soluble portion consists of SEQ ID NO: 73.

[0250] In some cases, the endosomal soluble portion is mucin or a derivative thereof. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 74 or 75. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 74. In some cases, the endosomal soluble portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to SEQ ID NO: 75. In some cases, the endosomal soluble portion contains SEQ ID NO: 74. In some cases, the endosomal soluble portion contains SEQ ID NO: 75. In some cases, the endosomal soluble portion consists of SEQ ID NO: 74. In some cases, the endosomal soluble portion consists of SEQ ID NO: 75.

[0251] In some cases, the endosomal lytic portion contains sequences as illustrated in Table 9.

[0252] [Table 9] JPEG2023527638000026.jpg95170

[0253] In some cases, the endosome-lytic moiety comprises a Bak BH3 polypeptide that induces apoptosis through antagonizing inhibitors such as Bcl-2 and / or Bcl-xL. In some examples, the endosome-lytic moiety comprises the Bak BH3 polypeptide described in Albarran, et al., “Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier,” Reactive & Functional Polymers 71:261-265(2011).

[0254] In some examples, the endosome-lytic moiety comprises a polypeptide (e.g., a cell-penetrating polypeptide) as described in PCT International Publication No. WO2013 / 166155 or WO2015 / 069587.

[0255] Endosome-lytic lipid In some embodiments, the endosome-lytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, the molecule of formula (I): A-X1-B-X2-C further binds to an endosome-lytic lipid (e.g., a fusogenic lipid). Exemplary fusogenic lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), and N-methyl(2,2-di(9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di(9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (XTC).

[0256] In some examples, the endosome-lytic moiety is a lipid (e.g., a fusogenic lipid) described in PCT International Publication No. WO09 / 126,933.

[0257] Endosome-lytic small molecule In some embodiments, the endosomal soluble moiety is a small molecule. In some embodiments, the molecule of formula (I): A-X1-B-X2-C further binds to the endosomal soluble small molecule. Suitable exemplary small molecules as the endosomal soluble moiety include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), amopyroquine, primaquine, mefloquine, nivaquines, halophanthrin, quinone imines, or combinations thereof. In some examples, the quinoline endosome-soluble portion may include, but is not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline; 4-(4-diethylamino-1-butylamino)quinoline; 7-hydroxy C-4-(4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline; 7-Hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; Hydroxychloroquine phosphate; 7-Chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine); 7-Fur Oro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino(quinoline); 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino) Mino)quinoline; 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(1- Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline; 1-acetyl-1,2,3,4-tetrahydroquinoline; 8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride; 1-butyryl-1,2,3,4-tetrahydroquinoline;3-Chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline)(4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;3-Fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline(4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;4-(4-hydroxy-α,α'-bis(2-methyl-1- Pyrrolidinyl)-2,5-Xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinoline carboxyaldehyde; 1,1'-Pentamethylenequinolinium iodide; 8-Quinolinol sulfate; and amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs thereof. In some examples, the endosomal soluble moiety is a small molecule described by Naisbitt et al (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Patent No. 5,736,557.

[0258] Cell-permeable polypeptide (CPP) In some embodiments, the cell-permeable polypeptide comprises a short, positively charged peptide having 5 to 30 amino acids. In some embodiments, the cell-permeable polypeptide comprises an amino acid sequence rich in arginine or lysine. In some embodiments, the cell-permeable polypeptide comprises any polypeptide or combination thereof listed in Table 9.

[0259] [Table 10]

[0260] Linker In some embodiments, the linkers described herein are either severable or inseverable linkers. In some examples, the linker is severable. In other examples, the linker is inseverable.

[0261] In some cases, the linker is a non-polymer linker. A non-polymer linker refers to a linker that does not contain repeating monomer units produced by the polymerization process. Examples of non-polymer linkers include, but are not limited to, C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional crosslinker linkers, heterobifunctional crosslinker linkers, peptide linkers, traceless linkers, self-destructing linkers, maleimide-based linkers, or combinations thereof. In some cases, non-polymer linkers include C1-C6 alkyl groups (e.g., C5, C4, C3, C2, or C1 alkyl groups), homobifunctional crosslinker linkers, heterobifunctional crosslinker linkers, peptide linkers, traceless linkers, self-destructing linkers, maleimide-based linkers, or combinations thereof. In further cases, a non-polymer linker does not contain more than two linkers of the same type, e.g., more than two homobifunctional crosslinker linkers or more than two peptide linkers. In further cases, the nonpolymer linker optionally contains one or more reactive functional groups.

[0262] In some cases, the non-polymer linker does not contain the polymers described above. In some cases, the non-polymer linker does not contain the polymer encompassed by polymer portion C. In some cases, the non-polymer linker does not contain polyalkylene oxides (e.g., PEG). In some cases, the non-polymer linker does not contain PEG.

[0263] In some examples, the linker includes homobifunctional linkers. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagents dithiobis(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS), disuccinimidyl tartrate (DST), and disulfosuccinimidyl tartrate (sulfoDS). T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidylcarbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimerimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamide)butane (DPDPB), bismaleimide hexane (BMH), aryl halide-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene and 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamide)ethyl]disulfide (BASED ), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0264] In some embodiments, the linker includes a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-bridged linkers, e.g., N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), and water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimid ester (MBs), m-male Imidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidylCarbonyl reaction compounds such as 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyliodoacetate (NPIA), 4-(4-N-maleimidophenyl)butyrate hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), and 3-(2-pyridyldithio)propionylhydrazide (PDPH) Sulfhydryl-reactive crosslinkers, amine-reactive and photoreactive crosslinkers, e.g., N-hydroxysuccinimidyl-4-azidosalicylic acid (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamide)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamide)ethyl-1, 3'-Dithiopropionate (sAsD), N-Hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-Hydroxysulfosuccinimidyl-4-azidobenzoate (Sulfo-HsAB), N-Succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), Sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (Sulfo-sANPAH), N -5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamide)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl7-Azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyldiazopilbert (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinking linkers, e.g., 1-(ρ-azidosalicylamide)-4-(iodoacetamide)butane (AsIB), N-[4-(ρ-azidosalicylamide)butyl]-3'-(2'-pyridyldithio) Examples include propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl-reactive and photoreactive crosslinkers, such as ρ-azidobenzoylhydrazide (ABH), carboxylate salt-reactive and photoreactive crosslinkers, such as 4-(ρ-azidosalicylamide)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as ρ-azidophenylglyoxal (APG).

[0265] In some examples, the linker contains a reactive functional group. In some cases, the reactive functional group contains a nucleophile that is reactive to an electrophile present in the bonding portion. Exemplary electrophiles include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophiles include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0266] In some embodiments, the linker contains a maleimide group. In some examples, the maleimide group is also called a maleimide spacer. In some examples, the maleimide group further contains caproic acid to form maleimidocaproyl (mc). In some cases, the linker contains maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other examples, the maleimide group contains a maleimidomethyl group such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC) as described above.

[0267] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some examples, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalytic action of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination via the retromichael reaction. In some examples, the self-stabilizing maleimide is the maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker contains a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0268] In some embodiments, the linker includes a peptide moiety. In some examples, the peptide moiety includes at least 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety includes at most 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety includes about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some examples, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some examples, the peptide moiety is an incleavable peptide moiety. In some examples, the peptide portion includes Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 106), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 107), or Gly-Phe-Leu-Gly (SEQ ID NO: 108). In some cases, the linker contains peptide moieties such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker contains Val-Cit. In some cases, the linker is Val-Cit.

[0269] In some embodiments, the linker includes a benzoic acid group or a derivative thereof. In some examples, the benzoic acid group or a derivative thereof includes para-aminobenzoic acid (PABA). In some examples, the benzoic acid group or a derivative thereof includes gamma-aminobutyric acid (GABA).

[0270] In some embodiments, the linker comprises one or more maleimide groups, peptide moieties, and / or benzoic acid groups in any combination. In some embodiments, the linker comprises a combination of maleimide groups, peptide moieties, and / or benzoic acid groups. In some examples, the maleimide group is maleimidocaproyl (mc). In some examples, the peptide group is val-cit. In some examples, the benzoic acid group is PABA. In some examples, the linker comprises an mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further cases, the linker comprises an mc-val-cit-PABA group.

[0271] In some embodiments, the linker is a self-destructing linker or a self-excluding linker. In some cases, the linker is a self-destructing linker. In other cases, the linker is a self-excluding linker (e.g., a cyclized self-excluding linker). In some examples, the linker includes a linker described in U.S. Publication No. 9,089,614 or PCT Publication No. WO2015038426.

[0272] In some embodiments, the linker is a dendritic linker. In some examples, the dendritic linker includes a branched, multifunctional linker moiety. In some examples, the dendritic linker is used to increase the molar ratio of polynucleotide B pairs to binding moiety A. In some examples, the dendritic linker includes a PAMAM dendrimer.

[0273] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker portion (e.g., an atom or linker group) on the binding portion A, polynucleotide B, polymer C, or endosomal soluble portion D after cleavage. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linkers. In some cases, the linker is a traceless aryl-triazene linker as described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem 11(15):2493-2497(2013). In some examples, the linker is a traceless linker as described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102:2607-2024(2002). In some cases, the linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[0274] In some embodiments, the linker is U.S. Patent No. 6,884,869; No. 7,498,298; No. 8,288,352; No. 8,609,105; or No. 8,697,688; U.S. Patent Publication No. 2014 / 0127239; No. 2013 / 028919; No. 2014 / 286970; No. 2013 / 0 Linkers listed in No. 309256; No. 2015 / 037360; or No. 2014 / 0294851; or PCT Publication No. WO2015057699; No. WO2014080251; No. WO2014197854; No. WO2014145090; or No. WO2014177042.

[0275] In some embodiments, X1 and X2 are each independently a single bond or a non-polymeric linker. In some examples, X1 and X2 are each independently a single bond. In some cases, X1 and X2 are each independently a non-polymeric linker.

[0276] In some examples, X1 comprises a single bond or a non-polymeric linker. In some examples, X1 is a single bond. In some examples, X1 is a non-polymeric linker. In some examples, the linker is a C1-C6 alkyl group. In some cases, X1 is, for example, a C1-C6 alkyl group such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. In the context of the linker, and particularly when used in the context of X1, alkyl means a saturated straight-chain or branched-chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X1 comprises a homobifunctional linker or a heterobifunctional linker as described above. In some cases, X1 comprises a heterobifunctional linker. In some cases, X1 comprises sMCC. In other examples, X1 comprises a heterobifunctional linker optionally attached to a C1-C6 alkyl group. In other examples, X1 comprises sMCC optionally attached to a C1-C6 alkyl group. In some additional examples, X1 does not comprise a homobifunctional linker or a heterobifunctional linker as described above.

[0277] In some examples, X2 is a single bond or a linker. In some examples, X2 is a single bond. In other cases, X2 is a linker. In further cases, X2 is a nonpolymer linker. In some embodiments, X2 is a C1-C6 alkyl group. In some examples, X2 is a homobifunctional linker or heterobifunctional linker as described above. In some examples, X2 is a homobifunctional linker as described above. In some examples, X2 is a heterobifunctional linker as described above. In some examples, X2 comprises a maleimide group such as maleimidocaproyl (mc) as described above, or a self-stabilizing maleimide group. In some examples, X2 comprises a peptide moiety such as Val-Cit. In some examples, X2 comprises a benzoic acid group such as PABA. In further examples, X2 comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In additional examples, X2 comprises an mc group. In the additional example, X2 contains an mc-val-cit group. In the additional example, X2 contains a val-cit-PABA group. In the additional example, X2 contains an mc-val-cit-PABA group.

[0278] How to use Muscular dystrophy refers to the loss of muscle mass and / or the progressive decline and degeneration of muscles. In some cases, the loss of muscle mass or the progressive decline and degeneration of muscles results from a high rate of protein breakdown, a low rate of protein synthesis, or a combination of both. In some cases, the high rate of muscle protein breakdown is due to muscle protein catabolism (i.e., the breakdown of muscle proteins to use amino acids as substrates for gluconeogenesis).

[0279] In one embodiment, muscular dystrophy refers to a significant loss of muscle strength. Significant loss of muscle strength means a decrease in the strength of the diseased, damaged, or unused muscle tissue of the subject compared to the same muscle tissue of a control subject. In one embodiment, significant loss of muscle strength is a decrease of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the same muscle tissue of a control subject. In another embodiment, significant loss of muscle strength means a decrease in the strength of unused muscle tissue compared to the muscle strength of the same muscle tissue of the same subject prior to the period of unavailability. In one embodiment, significant loss of muscle strength is a decrease of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the muscle strength of the same muscle tissue of the same subject prior to the period of unavailability.

[0280] In another embodiment, muscular dystrophy refers to a significant loss of muscle mass. Significant loss of muscle mass means a decrease in the muscle volume of the diseased, damaged, or unused muscle tissue of the subject compared to the same muscle tissue of a control subject. In one embodiment, significant loss of muscle volume is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the same muscle tissue of a control subject. In another embodiment, significant loss of muscle mass means a decrease in the muscle volume of unused muscle tissue compared to the muscle volume of the same muscle tissue of the same subject before the period of incapacity. In one embodiment, significant loss of muscle tissue is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, compared to the muscle volume of the same muscle tissue of the same subject before the period of incapacity. Muscle volume can be measured voluntarily by evaluating the cross-sectional area of ​​the muscle, for example, by magnetic resonance imaging (e.g., muscle volume / cross-sectional area (CSA) MRI).

[0281] Myotonic dystrophy is a multi-organ neuromuscular disease that includes two main types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by a dominant increase in "CTG" repeats of the DM protein kinase (DMPK) gene, which, when transcribed into mRNA, forms a hairpin that binds with high affinity to the Muscleblind-like (MBNL) protein family. MBNL proteins are involved in regulating post-transcriptional splicing and polyadenylation sites, and loss of MBNL protein function leads to the accumulation of downstream nuclear lesions and an increase in missplicing events, and subsequently myotonia and other clinical symptoms.

[0282] In some embodiments, methods for treating muscular dystrophy (e.g., DM1) in subjects are described herein, the methods comprising providing a polynucleic acid molecule or a polynucleic acid molecule conjugate as described herein, and treating muscular dystrophy by administering a therapeutically effective amount of the polynucleic acid molecule or polynucleic acid molecule conjugate to a subject in need, wherein the polynucleic acid conjugate reduces the amount of human DMPK mRNA transcript. In some embodiments, administration of the polynucleic acid molecule conjugate to a subject reduces the amount of human DMPK mRNA transcript by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, and at least 80% of the DMPK mRNA expression level in patients who have not received treatment with the polynucleic acid molecule conjugate.

[0283] Pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to a subject by multiple routes of administration, including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet another example, the pharmaceutical compositions described herein are formulated for nasal administration.

[0284] In some embodiments, the pharmaceutical composition includes, but is not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsed-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and mixed formulations of immediate-release and controlled-release.

[0285] In some cases, the pharmaceutical formulation includes multi-particle formulations. In some cases, the pharmaceutical formulation includes nanoparticle formulations. In some cases, the nanoparticles include cMAP, cyclodextrin, or lipids. In some cases, the nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, nanoparticles are nanoparticles of metals, such as scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations thereof, their alloys, or oxides.

[0286] In some examples, nanoparticles consist of a core, or a core and a shell, as in core-shell nanoparticles.

[0287] In some examples, the nanoparticles are further coated with molecules for the binding of functional elements (e.g., with one or more polynucleic acid molecules or binding sites described herein). In some examples, the coating includes chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, or dextrin or cyclodextrin. In some examples, the nanoparticles include graphene-coated nanoparticles.

[0288] In some cases, the nanoparticles have at least one dimension of approximately 500 nm, 400 nm, 300 nm, 200 nm, or less than 100 nm.

[0289] In some examples, nanoparticle formulations include paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, or quantum dots. In some examples, the polynucleic acid molecules or binding sites described herein are directly or indirectly bound to the nanoparticles. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding sites described herein are directly or indirectly bound to the nanoparticles.

[0290] In some embodiments, the pharmaceutical formulation includes a delivery vector, such as a recombinant vector for the delivery of polynucleic acid molecules to cells. In some examples, the recombinant vector is a DNA plasmid. In other examples, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. In some examples, the recombinant vector capable of expressing polynucleic acid molecules results in stable expression in target cells. In further examples, viral vectors that result in transient expression of polynucleic acid molecules are used.

[0291] In some embodiments, the pharmaceutical formulation comprises a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, humectants, and diluents. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, and pregelatinized starch. For example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HAand Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0292] In some examples, the pharmaceutical formulation further includes pH adjusters or buffers, such as acids including acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases including sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers including citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0293] In some cases, a pharmaceutical formulation contains one or more salts in amounts necessary to bring the osmotic pressure of the composition into an acceptable range. Such salts include sodium, potassium, or ammonium cations, as well as chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions. Suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0294] In some cases, pharmaceutical formulations further include diluents used to stabilize the compound, as they provide a more stable environment. Salts dissolved in buffers, including phosphate-buffered saline (which also provide pH control or maintenance), are used as diluents in the art, but are not limited to these. In certain cases, the diluent increases the size of the composition to facilitate compression or to create a sufficient bulk for homogeneous mixing for capsule filling. Such compounds include, for example, microcrystalline cellulose such as lactose, starch, mannitol, sorbitol, dextrose, and Avicel®; calcium hydrogen phosphate, calcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; mononucleotide calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[0295] In some cases, pharmaceutical formulations contain disintegration agents or disintegrants to promote the breakdown or disintegration of a substance. The term “disintegrate” includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluid. Examples of disintegrants include starch, e.g., natural starch, e.g., corn starch or potato starch; pregelatinized starch, e.g., National 1551 or Amijel®; or sodium starch glycolate, e.g., Promogel® or Explotab®; cellulose, e.g., wood products; methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Min This includes Tia (registered trademark) and Solka-Floc (registered trademark), methylcellulose, croscarmellose, or cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol (registered trademark)), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, such as cross-linked polymers such as sodium starch glycolate and crospovidone, cross-linked polyvinylpyrrolidone, alginates, such as alginic acid or salts of alginic acid such as sodium alginate, clays such as Veegum (registered trademark) HV (magnesium aluminum silicate), rubber, such as agar, guar, locust bean, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, natural sponges, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, and sodium lauryl sulfate combined with starch.

[0296] In some cases, pharmaceutical formulations contain fillers such as lactose, calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, and polyethylene glycol.

[0297] Lubricants and lubricants are also optionally included in the pharmaceutical formulations described herein for preventing, reducing, or inhibiting adhesion or friction between materials. Typical lubricants include, for example, hydrocarbons such as stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, and mineral oil; hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®); higher fatty acids; and alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol, such as Carbowax®, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium lauryl sulfate or sodium lauryl sulfate, colloidal silica such as Syloid®, Cab-O-Sil®, starches such as corn starch, silicone oils, and surfactants.

[0298] Plasticizers include compounds used to reduce the brittleness of microencapsulated materials or film coatings by softening them. Suitable plasticizers include, for example, polyethylene glycol such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, as well as stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers also function as dispersants or wetting agents.

[0299] The solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doxate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycoflor, transktol, propylene glycol, and dimethyl isosorbide.

[0300] Stabilizers include compounds such as any antioxidants, buffers, acids, and preservatives.

[0301] The suspending agent is polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having molecular weights from about 300 to about 6000, from about 3350 to about 4000, or from about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxymethylcellulose stearin The compounds include acid acetate, polysorbate 80, hydroxyethylcellulose, sodium alginate, rubber, such as tragacanth gum, acacia gum, guar gum, xanthan gum, sugars, cellulose compounds, such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

[0302] Surfactants include compounds such as sodium lauryl sulfate, sodium doxate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polaxomer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils (e.g., polyoxyethylene (60) hydrogenated castor oil); and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40, etc. Often, surfactants are included to enhance physical stability or for other purposes.

[0303] Viscosity enhancers include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0304] The humectants include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium doxate, sodium oleate, sodium lauryl sulfate, sodium doxate, triacetin, Tween 80, vitamin E TPGS, and ammonium salts.

[0305] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic purposes. In some embodiments, the pharmaceutical compositions are administered once daily, twice daily, three times daily, or more frequently. The pharmaceutical compositions are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, once every two months, once every three months, once every four months, once every five months, once every six months, or more frequently. The pharmaceutical compositions are administered for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years, or longer.

[0306] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, consecutively, or at intervals of time. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered consecutively. In further cases, one or more pharmaceutical compositions are administered at intervals of time (for example, the first administration of the first pharmaceutical composition is on day 1, followed by at least one, two, three, four, five days or more before the administration of at least a second pharmaceutical composition).

[0307] In some embodiments, two or more different pharmaceutical compositions are administered simultaneously. In some examples, two or more different pharmaceutical compositions are administered simultaneously. In some cases, two or more different pharmaceutical compositions are administered simultaneously in succession without any interval between administrations. In other cases, two or more different pharmaceutical compositions are administered simultaneously in succession with intervals of approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, and 2 days between administrations.

[0308] If the patient's condition is improving, the administration of the composition may be continued at the doctor's discretion; alternatively, the dose of the administered composition may be temporarily reduced or temporarily interrupted for a specific period (i.e., a “drug-free period”). In some cases, the length of the drug-free period may vary between 2 days and 1 year, including, but not limited to, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. The dose reduction during drug-free days ranges from 10% to 100%, including, but are not limited to, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0309] Once the patient's condition improves, a maintenance dose is administered as needed. Subsequently, the dose, frequency, or both can be reduced, depending on the symptoms, to a level at which the improved disease, impairment, or illness is maintained.

[0310] In some embodiments, the predetermined amount of drug corresponding to such a quantity depends on factors such as the specific compound, the severity of the disease, and the characteristics of the subject or host requiring treatment (e.g., body weight), but nevertheless, it is routinely determined in methods known in the art according to the specific environment surrounding the case, including, for example, the specific drug being administered, the route of administration, and the subject or host being treated. In some examples, the desired dose is conveniently presented as a single dose, or as divided doses administered simultaneously (or over a short period of time), or at appropriate intervals, for example, two, three, or four or more divided doses (sub-doses) per day.

[0311] Due to the large number of variables in each treatment regimen, the ranges mentioned above are merely suggestive, and significant deviations from these recommendations are not uncommon. Such dosages are not limited but depend on many variables, including the activity of the compound used, the disease or illness being treated, the mode of administration, the individual subject's requirements, the severity of the disease or illness being treated, and the physician's judgment.

[0312] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell culture or experimental animals, including, but not limited to, determining the LD50 (lethal dose for up to 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). The dose-to-toxicity ratio is the therapeutic index, which is expressed as the ratio between the LD50 and the ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate a range of doses for use in humans. The doses of such compounds are preferably located within a range of circulating concentrations that include an ED50 with minimal toxicity. The dose varies within this range depending on the dosage form used and the route of administration utilized.

[0313] Kit / Product In one embodiment, kits and products used in conjunction with one or more compositions and methods described herein are disclosed herein. Such kits include a partitioned carrier, packaging, or container for housing one or more containers such as vials, tubes, etc., each container comprising one of the distinct elements for using the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0314] Products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging materials suitable for the selected formulation and the intended mode of administration and treatment.

[0315] For example, a container contains a target nucleic acid molecule as described herein. Such a kit may optionally include an identification mark or label, or instructions for use in the manner described herein.

[0316] A kit typically includes a label listing the contents and / or instructions for use, and an accompanying document with instructions for use. A set of instructions is also typically included.

[0317] In one embodiment, the label is on or attached to the container. In one embodiment, the label is attached to the container if the letters, numbers, or other markings forming the label are affixed, molded, or engraved onto the container itself. The label is attached to the container when it is present, for example, in a receptacle or transport device that holds the container as an accompanying document. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic purpose. The label also indicates how to use the contents, for example, in the method described herein.

[0318] In one embodiment, a pharmaceutical composition is presented in a pack or dispenser device containing one or more unit dosage forms comprising the compounds provided herein. The pack includes, for example, metal or plastic foil such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accompanied by a notice attached to a container in a form specified by a government agency that controls the manufacture, use or sale of a pharmaceutical product, the notice reflecting the government agency's approval of the form of the drug for administration to humans or animals. Such a notice is, for example, a label approved by the U.S. Food and Drug Administration with respect to a prescription drug or approved package insert. In one embodiment, a composition comprising the compounds provided herein, formulated on a suitable pharmaceutical carrier, is also prepared, placed in a suitable container, and labeled for the treatment of the indicated disease.

[0319] Specific terms Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the subject matter. It will be understood that the above general statements and the following detailed statements are illustrative and descriptive only and are not limited to any subject matter. In this application, the use of the singular includes the plural unless otherwise specified. Where used in the specification and appended claims, the singular forms "a," "an," and "the" include the plural referent unless the context explicitly states otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" is not limited to other forms such as "include," "includes," and "included."

[0320] As used herein, ranges and quantities can be expressed as "approximately" specific values ​​or ranges. "Approximately" also includes exact quantities. Therefore, "approximately 5 μL" also means "approximately 5 μL" and "5 μL". Generally, the term "approximately" includes quantities that are expected to be within experimental error.

[0321] The paragraph headings used herein are for organizational purposes only and should not be construed as restricting the subject matter described herein.

[0322] As used herein, the terms “individual,” “subject,” and “patient” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. No term is limited to situations characterized by supervision (e.g., constant or intermittent) by a healthcare worker (e.g., a physician, registered nurse, clinical nurse, physician’s assistant, nursing assistant, or hospice staff).

[0323] The term "therapeutically effective dose" refers to the amount of polynucleotide conjugate sufficient to provide the desired therapeutic effect in a mammalian subject. In some cases, this dose is a single or multiple dose to a patient (such as a human) to treat, prevent, prevent the onset of, cure, delay, reduce the severity of, or improve at least one symptom of a disorder or recurrent disorder, or to extend the patient's survival beyond what would be expected without such treatment. Naturally, the dose level of a particular polynucleotide conjugate used to provide a therapeutically effective dose varies depending on the type of injury, the subject's age, weight, sex, the subject's medical condition, the severity of the disease, the route of administration, and the specific inhibitor used. In some examples, a therapeutically effective dose of polynucleotide conjugate is first evaluated from cell cultures and animal models, as described herein. For example, IC50 values ​​determined by cell culture methods can optionally serve as a starting point in animal models, while IC50 values ​​determined in animal models can optionally be used to find a therapeutically effective dose in humans.

[0324] Skeletal muscles, or voluntary muscles, are typically attached to bones by tendons and are usually used to bring about skeletal movement, such as when moving or maintaining posture. While multiple controls of skeletal muscles are generally maintained as unconscious reflexes (e.g., postural muscles or the diaphragm), skeletal muscles respond to conscious control. Smooth muscles, or involuntary muscles, are found within the walls of organs and structures such as the esophagus, stomach, intestines, uterus, urethra, and blood vessels.

[0325] Skeletal muscle is further classified into two broad types: Type I (or "slow contraction") and Type II (or "fast contraction"). Type I muscle fibers are densely packed in capillaries and are rich in mitochondria and myoglobin, which gives Type I muscle tissue its characteristic red color. In some cases, Type I muscle fibers carry more oxygen and use fat or carbohydrates as fuel to maintain aerobic activity. Type I muscle fibers contract for a long time, but with little force. Type II muscle fibers are further subdivided into three major subtypes (IIa, IIx, and IIb) that differ in contractile speed and the force produced. Type II muscle fibers contract quickly and forcefully but fatigue rapidly, thus resulting in very short bursts of intense anaerobic activity before muscle contraction becomes painful.

[0326] Unlike skeletal muscle, smooth muscle is not under conscious control.

[0327] Cardiac muscle is also involuntary muscle, but its structure is very similar to skeletal muscle and it is found only in the heart. Cardiac and skeletal muscle are striae in that they contain sarcomeres, which are bundles packed into highly regular arrangements. In contrast, the myofibrils of smooth muscle cells are not arranged in sarcomeres and are therefore not striae.

[0328] Myoblasts encompass all cells that contribute to muscle tissue. Exemplary myoblasts include myoblasts, satellite cells, myotubes, and myofibrils.

[0329] As used herein, muscle strength is proportional to cross-sectional area (CSA), and muscle velocity is proportional to muscle fiber length. Therefore, a comparison of cross-sectional area and muscle fiber length between different types of muscles can provide an indication of muscular atrophy. Various methods for measuring muscle strength and muscle weight are known in the art; see, for example, “Musculoskeletal assessment: Joint range of motion and manual muscle strength” by Hazel M. Clarkson, published by Lippincott Williams & Wilkins, 2000. The generation of tomographic images of selected muscle tissue by calculated transverse tomography and evaluation by ultrasound are further methods for measuring muscle strength. [Examples]

[0330] These embodiments are provided for illustrative purposes only and do not limit the scope of the claims.

[0331] Example 1. Antibody siRNA conjugate DMPK-AOC is an antibody-siRNA conjugate drug product formed by the conjugation of a humanized IgG1 antibody (anti-human transferrin receptor antibody) targeting human transferrin receptor 1 and a single double-stranded siRNA oligonucleotide (DMPK siRNA) targeting DMPK mRNA (Figure 1). The SMCC maleimide linker is located at the 5' end of the passenger strand and is conjugated to the antibody via a single cysteine ​​in the antibody amino acid sequence. The conjugate binds to the human transferrin receptor on the cell surface, is taken up into the cell, and delivers the siRNA oligonucleotide to the intracellular compartment. Once taken up into the cell, the siRNA is loaded into RISC and hydrolyzes the target pathogenic DMPK mRNA.

[0332] The anti-human transferrin receptor antibody and DMPK siRNA used to prepare DMPK-AOC are manufactured using well-established manufacturing processes by commercial GMP-compliant Contract Development and Manufacturing Organizations (CDMOs). The anti-human transferrin receptor antibody is generated using recombinant protein expression technology in CHO cells, and the DMPK siRNA is produced using standard phosphoramidite solid-phase synthesis chemistry. As used herein, DMPK siRNA is a double-stranded siRNA oligonucleotide targeting DMPK mRNA, which is also conjugated with an SMCC linker attached to the 5' end of the passenger strand. Each of these is fully characterized and formally released. DMPK-AOC is produced using a standard random cysteine ​​bioconjugation reaction with maleimide and DMPK siRNA of the anti-human transferrin receptor antibody, followed by anion exchange chromatography purification to separate the bulk conjugate, which is then converted to the finished DMPK-AOC. The finished DMPK-AOC is then formally released using standard methodologies for protein therapy. Once manufacturing, testing, and release are complete, the antibody and DMPKsiRNA are bioconjugated to form the active pharmaceutical ingredient.

[0333] Example 2. Production of antibody AV01mAb A stable research cell bank (RCB) of stable cell lines was constructed using CHOK1SV host working cells, and the absence of contamination by mycoplasma, bacteria, fungi, and yeast was confirmed. A master cell bank (MCB) of 200 vials was prepared using vials from the research cell bank.

[0334] Antibody production from master cell bank - Cells from ampoules in the master cell bank were gradually increased in volume using protein-free medium before inoculation into the production bioreactor. Downstream processing - Once cell culture was complete, cells and cell debris were removed by filtration of the culture.

[0335] Example 3. Structural characterization of anti-human transferrin receptor antibodies Structure - The amino acid sequences of both the heavy and light chains were determined from the translation of the nucleotide sequence of the anti-human transferrin receptor antibody.

[0336] Heavy chain sequence of anti-human transferrin receptor antibody - SEQ ID NO: 48 [ka]

[0337] Light chain sequence of anti-human transferrin receptor antibody - SEQ ID NO: 63 [ka]

[0338] Example 4: DMPK siRNA DMPK siRNA is a synthetic binucleotide comprising a 19-mer passenger strand and a complementary 21-mer guide strand with a 2-nucleotide overhang at the 3' end of the guide strand. A C6-SMCC linker {4-(N-maleimidomethyl)cyclohexane-1-carboxamide} binds to the 5' end of the passenger strand, enabling conjugate to an antibody intermediate. The nucleotide sequences and internucleotide bindings are shown in Table 11.

[0339] [Table 11]

[0340] Single-stranded RNA (guide and passenger strands) are generated separately via solid-phase synthesis using a well-established phosphoramidite solid-phase synthesis method. The purified and lyophilized single-stranded RNAs are then duplexed in equimolar ratios to produce double-stranded siRNA. The SMCC linker is conjugated to the primary amine conjugation handle at the 5' end of the sense strand of the siRNA using standard N-hydroxysuccinimide chemistry. Excess unreacted SMCC linker is removed using a UF / DF step, releasing the resulting SMCC-siRNA.

[0341] Example 5: Selection of Antibodies Antibody Selection Criteria: Several anti-human transferrin receptor 1 (TfR1) antibodies were tested by ELISA and found to bind to the receptor with high affinity. These antibodies were also tested for binding to cynomolgus monkey TfR1, and cross-reactivity between species was confirmed. Mouse IgG2a monoclonal antibodies (mAbs) that bind to both human and cynomolgus monkey TfR1 were evaluated for specificity by demonstrating the lack of binding to the closely related transferrin receptor 2 (TfR2) by ELISA (Figure 3). The commercially available anti-TfR2 antibody B-6 showed clear binding to TfR2 by ELISA, but the mouse anti-human TfR1 mAb did not bind to TfR2 even at concentrations up to 10 mM. Mouse anti-human TfR1 mAbs were also evaluated for binding in the presence of TfR1-binding ligands transferrin (Tf) and homeostatic iron regulator (HFE), and the TfR1 mAbs maintained potent binding to TfR1 even in the presence of TfR1 ligands (Figure 4). As shown in Figure 4, the antibodies bound either directly to TfR1 or to TfR1 pre-bound to the cofactor transferrin (Tf) or HFE. AF2474 is a commercially available antibody known to bind to the same TfR1 epitope as transferrin or HFE. Mouse anti-human TfR1 mAbs show some loss of binding during direct interactions with TfR1 compared to cofactor complexes, but the change in affinity is minimal compared to AF2474. Importantly, competition of TfR1 mAbs with native ligands for TfR1 is expected to be toxic, as it may block iron uptake into cells. Therefore, the identified TfR1 mAb needed to bind to an epitope on TfR1 that minimized competition with the native ligand. Considering that all of these selection criteria were met, the mouse IgG2a anti-human TfR1 mAb was transferred to a humanization program to develop an antibody suitable for clinical development.

[0342] Example 6: In vivo activity MPK-AOC was used in in vivo studies in mice. In vivo studies of AOC in mice utilized a surrogate anti-TfR1 antibody that binds to mouse TfR1, as the lead human antibody AV01Ab does not cross-react with mouse TfR1 (only in humans and monkeys). Mouse cross-reactive siDMPK.36 was conjugated to an anti-mouse TfR1 mAb, and this conjugate was administered by IV injection to wild-type female CD-1 mice (n=4 per group). Tissue was collected, and DMPK mRNA knockdown was evaluated 7 days post-administration. AOC was administered in dose-response forms (based on siRNA weight) of 3, 1, 0.3, and 0.1 mg / kg, with a dose of 3 mg / kg resulting in an 80% reduction in DMPK expression in skeletal muscle (Figure 5). DMPK-AOC showed potent activity with an ED50 < 1 mg / kg and an EC50 of approximately 3 nM. The negative control scrambled sequence siRNA did not show knockdown of DMPK mRNA, demonstrating specificity for DMPK-AOC activity.

[0343] Mouse cross-reactive DMPK-AOC was conjugated to anti-mouse TfR1 mAh, and this conjugate was administered to wild-type female CD-1 mice (n=4 per group) by IV injection at a dose of 3 mg / kg (based on siRNA weight). Tissue was collected, and DMPK mRNA knockdown was evaluated weekly for up to 5 weeks after administration (Figure 6). The maximum DMPK knockdown (approximately 75%) was achieved in skeletal muscle 7–35 days after administration. Slightly less DMPK knockdown was achieved in the heart (approximately 65%), but no knockdown was observed in the liver despite the presence of DMPK siRNA. Due to the long duration of activity after a single AOC administration, the frequency of administration to patients can be reduced.

[0344] In vivo pharmacological data in non-human primates: DMPK-AOC was administered to wild-type male cynomolgus monkeys (n=3 per group) via IV infusion over 30 minutes, and tissue was collected over 12 weeks post-administration. Skeletal muscle was surgically biopsied under ketamine / xylazine anesthesia. Following final blood and muscle biopsies at 12 weeks post-administration, sedated animals were euthanized by overdose of the solution. Terminal punch biopsies of multiple additional tissues were then collected. A 75% reduction in DMPK expression was sustained after a single IV administration of 2 mg / kg (based on siRNA weight) of AOC, and persisted up to 12 weeks post-administration (Figure 7).

[0345] While preferred embodiments of the Disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Many variations, alterations, and substitutions can be conceived by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in the practice of the Disclosure. The following claims define the scope of the Disclosure, and methods and structures within the scope of these claims and their equivalents are intended to be encompassed thereby.

Claims

1. A small interfering RNA (siRNA) molecular conjugate comprising an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugated to an siRNA molecule that hybridizes to a target sequence of DMPK mRNA, wherein the sense strand of the siRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15, and the antisense strand of the siRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, and 16, and wherein the siRNA molecular conjugate mediates RNA interference against DMPK.

2. The siRNA molecular conjugate of claim 1, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises a humanized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a multispecific antibody or antigen-binding fragment thereof.

3. The siRNA molecule conjugate of claim 2, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, a sc diabody, a sc diabody-CH3, a triple body, a mini-antibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, or an intrabody.

4. The siRNA molecule conjugate of claim 1, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof specifically binds to the human transferrin receptor (TfR).

5. The siRNA molecule conjugate of claim 1, comprising a linker connecting the anti-transferrin receptor antibody or antigen-binding fragment thereof to the siRNA molecule.

6. a. the siRNA molecule conjugate of claim 1, and b. Pharmaceutically acceptable excipients 13. A pharmaceutical composition comprising:

7. The pharmaceutical composition of claim 6, formulated for parenteral, oral, intranasal, buccal, rectal, intrathecal, or transdermal administration.

8. The pharmaceutical composition of claim 6, formulated for intravenous administration.

9. The siRNA molecule conjugate described in claim 1, wherein the siRNA comprises a sense strand comprising SEQ ID NO:3 and an antisense strand comprising SEQ ID NO:

4.

10. The siRNA molecule conjugate of claim 1, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof is conjugated to the siRNA molecule via a linker.

11. The siRNA molecule conjugate of claim 10, wherein the linker comprises a 4-(N-maleimidomethyl)cyclohexane-1-amidate linker.

12. The siRNA molecule conjugate of claim 10, wherein the linker is conjugated to the sense strand of the siRNA molecule.

13. The siRNA molecule conjugate of claim 12, wherein the linker is conjugated to the 5' end of the sense strand of the siRNA molecule.

14. The siRNA molecule conjugate of claim 10, wherein the linker comprises a 6-amino-1-hexanol linker.

15. A method of treating muscular dystrophy in a subject, comprising the step of administering to the subject a therapeutically effective amount of a small interfering RNA (siRNA) conjugate comprising an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugated to an siRNA that hybridizes to a target sequence of myotonic dystrophy protein kinase (DMPK) mRNA, wherein the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15, and the antisense strand comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, and 16, and wherein the siRNA conjugate mediates RNA interference against DMPK, thereby treating the muscular dystrophy in the subject.

16. The method of claim 15, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof.

17. The method of claim 16, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises an IgG-scFv, a nanobody, a diabody, a DART, a TandAb, a sc diabody, a sc diabody-CH3, a triple body, a mini-antibody, a minibody, a TriBi minibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, or an intrabody.

18. The method of claim 15, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof specifically binds to the human transferrin receptor (TfR).

19. The method of claim 15, wherein the siRNA conjugate comprises a linker connecting the anti-transferrin receptor antibody or antigen-binding fragment thereof to the siRNA.

20. The method of claim 15, wherein the siRNA conjugate is administered parenterally, orally, intranasally, bucally, rectally, intrathecally, intravenously, or transdermally.

21. The method of claim 20, wherein the siRNA conjugate is administered intravenously.

22. The method described in claim 15, wherein the sense strand comprises the sequence of SEQ ID NO: 3 and the antisense strand comprises the sequence of SEQ ID NO:

4.

23. The method of claim 15, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof is conjugated to the siRNA via a linker.

24. The method of claim 23, wherein the linker comprises a 4-(N-maleimidomethyl)cyclohexane-1-amidate linker.

25. The method of claim 23, wherein the linker is conjugated to the sense strand.

26. The method of claim 25, wherein the linker is conjugated to the 5' end of the sense strand.

27. The method of claim 23, wherein the linker comprises a 6-amino-1-hexanol linker.

28. The method described in claim 15, wherein the muscular dystrophy is myotonic dystrophy type 1 (DM1).

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