Compositions and methods for treating muscular dystrophy

Abstract

Polynucleic acid molecules, pharmaceutical compositions, and methods for treating muscular dystrophy are provided. [Solution] A conjugate is provided, the conjugate comprising: (i) an anti-transferrin receptor antibody or its antigen-binding fragment, (ii) an siRNA molecule comprising a guide strand and a passenger strand, and (iii) a linker, wherein the linker comprises a maleimide group that attaches the anti-transferrin receptor antibody or its antigen-binding fragment to the end of the guide strand or the passenger strand.

Description

[Technical Field]

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

[0002] Background of the Invention Gene suppression through RNA-induced gene silencing provides multiple levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA downregulation, and siRNA-induced transcriptional attenuation. In some instances, RNA interference (RNAi) exerts long-lasting effects that span multiple cell divisions. Therefore, RNAi represents a viable method for drug target validation, gene function analysis, pathway analysis, and disease treatment.

[0003] Incorporation by Reference I All publications, patents, and patent applications mentioned in this specification are herein incorporated 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, disclosed herein are polynucleic acid molecules and pharmaceutical compositions for regulating genes associated with muscle wasting disorders, particularly facioscapulohumeral muscular dystrophy (e.g., DM1). Also described herein, in some embodiments, are methods for treating muscle wasting disorders, particularly FSHD, with the polynucleic acid molecules or polynucleic acid molecule conjugates disclosed herein.

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

[0006] In some embodiments, 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 comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising one of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, and an HCDR3 sequence comprising SEQ ID NO: 19. In some embodiments, the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence QHFWGTPLTX6, wherein 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, or F if present. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:22, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and, if present, is F. In some embodiments, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:22 or SEQ ID NO:27, an LCDR2 sequence comprising SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:28, and an LCDR3 sequence comprising SEQ ID NO:24 or SEQ ID NO:26. In some embodiments, the VH region comprises 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, and the VL region comprises 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. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises 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.In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 21, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22, an LCDR2 sequence comprising SEQ ID NO: 25, and an LCDR3 sequence comprising SEQ ID NO: 26. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 27, an LCDR2 sequence comprising SEQ ID NO: 28, and an LCDR3 sequence comprising SEQ ID NO: 26. In some embodiments, the VH region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 29-33. In some embodiments, the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 34-38. In some embodiments, the VH region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34.

[0007] In some embodiments, the anti-transferrin receptor antibody 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. In some embodiments, the anti-transferrin receptor antibody comprises an IgG-scFv, a nanobody, a BiTE, a diabody, a DART, a TandAb, a scdiabody, a scdiabody-CH3, a triplebody, a mini-antibody, a minibody, a TriBi minibody, a scFv-CH3 KIH, a Fab-scFv-Fc KIH, a Fab-scFv, a scFv-CH-CL-scFv, a F(ab')2, a F(ab')2-scFv2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scdiabody-Fc, a diabody-Fc, a tandem scFv-Fc, or an intrabody. In some embodiments, the anti-transferrin receptor antibody comprises 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 comprises at least one mutation in the Fc region. In some embodiments, the at least one mutation modulates effector function or reduces or eliminates Fc-γ receptor binding. In some embodiments, the at least one mutation is at residue position D265, N297, K322, L328, or P329, where the residue positions are relative to IgG1. In some embodiments, the Fc region comprises two or more, three or more, or four or more mutations. In some embodiments, the Fc region comprises mutations at L233 and L234, residues corresponding to positions 233 and 234 of SEQ ID NO: 39. In some embodiments, the Fc region comprises mutations at D265 and N297. In some embodiments, the Fc region comprises mutations at D265 and N297. In some embodiments, the anti-transferrin receptor antibody comprises 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 comprise at least one 2'-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some embodiments, the sense strand comprises a 2'-O-methyl modified nucleotide at the 5'-terminus. Alternatively and / or additionally, the sense strand comprises at least two consecutive 2'-O-methyl modified nucleotides at the 5'-terminus. Alternatively and / or additionally, the sense strand comprises at least three, four, five, or six consecutive 2'-O-methyl modified nucleotides at the 5'-terminus. Alternatively and / or additionally, the sense strand comprises six consecutive 2'-O-methyl modified nucleotides at the 5'-terminus. Alternatively and / or additionally, the sense strand comprises at least one 2'-F modified nucleotide. Alternatively and / or additionally, the sense strand comprises at least two, or at least three, 2'-F modified nucleotides. Alternatively and / or additionally, the sense strand comprises at least two, or at least three, consecutive 2'-F modified nucleotides. Alternatively and / or additionally, the sense strand comprises a 2'-O-methyl modified nucleotide at the 3'-end. Alternatively and / or additionally, the sense strand comprises at least two consecutive 2'-O-methyl modified nucleotides at the 3'-end. Alternatively and / or additionally, the sense strand comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive 2'-O-methyl modified nucleotides at the 3'-end. Alternatively and / or additionally, the sense strand comprises 10 consecutive 2'-O-methyl modified nucleotides at the 3'-end. Alternatively and / or additionally, the sense strand comprises at least two phosphorothioate internucleotide linkages. Alternatively and / or additionally, the sense strand has the sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15.

[0010] In some embodiments, the antisense strand comprises 2'-O-methyl modified nucleotides at the 5'-end. Alternatively and / or additionally, the antisense strand comprises 2'-O-methyl modified nucleotides at the 3'-end. Alternatively and / or additionally, the antisense strand comprises at least two, at least three, at least four, or at least five consecutive 2'-O-methyl modified nucleotides at the 3'-end. Alternatively and / or additionally, the antisense strand comprises five consecutive 2'-O-methyl modified nucleotides at the 3'-end. Alternatively and / or additionally, the antisense strand comprises at least one, at least two, at least three, or at least four 2'-F modified nucleotides. Alternatively and / or additionally, the antisense strand comprises four 2'-F modified nucleotides, where any two of the four 2'-F modified nucleotides are not consecutive. Alternatively and / or additionally, the antisense strand comprises two overhanging nucleotides at the 3'-end. Alternatively and / or additionally, the antisense strand comprises at least two, at least three phosphorothioate internucleotide linkages.Alternatively and / or additionally, the antisense strand has the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0011] In some embodiments, the polynucleic acid molecule conjugate comprises a linker connecting the anti-transferrin receptor antibody or antigen-binding fragment thereof 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 derivatives thereof. In some embodiments, the linker comprises 4-(N-maleimidomethyl)cyclohexane-1-amidate (SMCC). In some embodiments, the linker is attached 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 antigen-binding fragment thereof. In some embodiments, the cysteine ​​residue is in the Fc domain of the anti-transferrin receptor antibody or antigen-binding fragment thereof. In some embodiments, the ratio between the polynucleic acid molecule and the anti-transferrin receptor antibody or antigen-binding fragment thereof is about 1:1, 2:1, 3:1, or 4:1.

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

[0013] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody, or antigen-binding fragment thereof, conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK. and a polynucleic acid molecule having a sense strand having the sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15 and an antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, wherein 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 comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises 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 antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a linker comprising 4-(N-maleimidomethyl)cyclohexane-1-amidate (SMCC).

[0014] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody, or antigen-binding fragment thereof, 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: 3, 5, 7, 9, 11, 13, or 15 and an antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; 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 has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34; and the anti-transferrin receptor antibody or antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a maleimide linker.

[0015] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody, or antigen-binding fragment thereof, 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 comprises at least 3, 4, 5, or 6 consecutive 2'-O-methyl modified nucleotides at its 5'-end and at least 2 or at least 3 2'-F modified nucleotides; 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 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34; and the anti-transferrin receptor antibody or antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a maleimide linker.

[0016] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody, or antigen-binding fragment thereof, 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 comprises 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 two, at least three, or at least four 2'-F modified nucleotides; 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 comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19; the VL region comprises 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 antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a maleimide linker.

[0017] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody or antigen-binding fragment thereof 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 comprises 2'-O-methyl modified nucleotides at the 5' and 3' ends, 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 comprises 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, and the VL region comprises 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 antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a maleimide linker.

[0018] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody, or antigen-binding fragment thereof, 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 comprises at least five consecutive 2'-O-methyl modified nucleotides at its 3' end and four 2'-F modified nucleotides, wherein any two of the four 2'-F modified nucleotides are not consecutive; 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 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34; and the anti-transferrin receptor antibody or antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a 6-amino-1-hexanol linker.

[0019] Disclosed herein, in certain embodiments, is a polynucleic acid molecule conjugate comprising an anti-transferrin receptor antibody, or antigen-binding fragment thereof, 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 comprises at least five consecutive 2'-O-methyl modified nucleotides and four 2'-F modified nucleotides at its 3' end, and wherein any two of the four 2'-F modified nucleotides are not consecutive; 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 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30, and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34; and the anti-transferrin receptor antibody or antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a 6-amino-1-hexanol linker.

[0020] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising the polynucleic acid molecule conjugates described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions are formulated as nanoparticle formulations. In some embodiments, the pharmaceutical compositions are formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

[0021] Disclosed herein, in certain embodiments, is a method for treating muscular dystrophy in a subject in need thereof by providing a polynucleic acid conjugate or pharmaceutical composition described herein and administering the polynucleic acid conjugate to the subject in need thereof to treat the muscular dystrophy, wherein the polynucleic acid conjugate reduces the amount of mRNA transcripts of human DMPK. In some embodiments, the polynucleic acid portion mediates RNA interference against human DMPK and regulates muscle atrophy in the subject. In some embodiments, the muscular dystrophy is myotonic dystrophy type 1 (DM1).

[0022] Disclosed herein, in certain embodiments, is the use of a polynucleic acid conjugate or pharmaceutical composition described herein for treating a subject diagnosed with or suspected of having myotonic dystrophy type 1 (DM1), or for the manufacture of a medicament for treating myotonic dystrophy type 1 (DM1). Disclosed herein, in certain embodiments, is a kit comprising a polynucleic acid conjugate or pharmaceutical composition described herein. [Brief explanation of the drawings]

[0023] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the disclosure are utilized, and the accompanying drawings. The patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] 1 illustrates a schematic diagram of an antibody-siRNA conjugate. [Figure 2] 1 illustrates the schematic structure of DMPK siRNA. [Figure 3] 1 illustrates a graph of TfR2 binding of anti-TfR antibodies by ELISA. [Figure 4] 1 illustrates a graph of binding of anti-TfR antibodies to TfR1 in the presence of cofactors. [Figure 5] 1 illustrates a graph of the in vivo dose response of AOC-mediated DMPK knockdown in mouse skeletal muscle. [Figure 6] Graphs illustrating the time course of AOC-mediated DMPK knockdown in mouse tissues (left) and the concentration of siDMPK.36 in mouse tissues over time (right). [Figure 7] 1 illustrates a graph showing AOC-mediated DMPK knockdown in cynomolgus monkey skeletal muscle over a time course up to 12 weeks post-administration. DETAILED DESCRIPTION OF THE INVENTION

[0024] DM1 is a rare, monogenic, autosomal dominant, repeat expansion disorder affecting approximately 1 in 8,000 people in the United States based on clinical confirmation. However, recent genetic studies have estimated the prevalence of DM1 in the United States to be 1 in 2,532. DM1 is caused by an expansion of a CTG triplet repeat in the 3' noncoding region of the myotonic dystrophy protein kinase (DMPK) gene. This expansion ranges from fewer than 35 cases in healthy individuals to thousands of cases in DM1 patients. When the mutant DMPK gene is translated into mRNA, the self-complementary CUG repeat induces the formation of a large hairpin loop, trapping DMPK mRNA in the nucleus and conferring a toxic gain-of-function function. This toxicity is not due to translation of the mRNA into a toxic protein, but rather to the presence of a high concentration of CUG repeats in the nucleus, which act as a trap for the key CUG-binding protein, muscleblind-like protein 1 (MBNL1). By binding to the nuclear-retained DMPK CUG repeats, MBNL1 is sequestered within the nucleus and is unable to perform its normal function of directing mRNA processing. This results in the misprocessing of multiple mRNAs encoding important proteins. The resulting atypical proteins translated from these misspliced ​​mRNAs are ultimately responsible for the phenotypic changes characteristic of this disease.

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

[0026] In some embodiments, the arrangement or order of the various components comprising the nucleic acid composition further affects cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, when the nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide), the order or arrangement of the binding moiety, polymer, and / or polynucleic acid molecule (or polynucleotide) (e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule) further affects cellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

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

[0028] Further embodiments described herein include methods of treating muscular dystrophy comprising administering to a subject a polynucleic acid molecule or polynucleic acid molecule conjugate described herein.

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

[0030] In some embodiments, the polynucleic acid molecule comprises 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 NO: 1. In some embodiments, the polynucleic acid molecule comprises 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 NO:2. In some embodiments, the polynucleic acid molecule comprises 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 embodiments, the polynucleic acid molecule comprises 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: 4, 6, 8, 10, 12, 14, or 16.

[0031] In some embodiments, the polynucleic acid 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 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 to a sequence selected from SEQ ID NO: 2. In some cases, the polynucleic acid 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 to a sequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, or 15. 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 to a sequence selected from SEQ ID NOs: 4, 6, 8, 10, 12, 14, or 16.

[0032] In some embodiments, a polynucleic acid molecule comprises 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) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 1. In some cases, the antisense strand (e.g., the guide strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NO: 2. In some embodiments, a polynucleic acid molecule comprises 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) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, or 15. In some cases, the antisense strand (e.g., guide strand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 4, 6, 8, 10, 12, 14, or 16. Table 1 provides 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 comprise RNA, DNA, or PMO. In some cases, the polynucleic acid molecule comprises RNA. In some instances, the RNA comprises 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 heterogeneous nuclear RNA (hnRNA). In some instances, the RNA comprises shRNA. In some instances, the RNA comprises miRNA. In some instances, the RNA comprises dsRNA. In some instances, the RNA comprises tRNA. In some instances, the RNA comprises rRNA. In some instances, the RNA comprises hnRNA. In some instances, the RNA comprises siRNA. In some instances, the polynucleic acid molecule comprises siRNA.

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

[0036] In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 19 nucleotides in length. In some instances, the polynucleic acid molecule is about 18 nucleotides in length. In some instances, the polynucleic acid molecule is about 17 nucleotides in length. In some instances, the polynucleic acid molecule is about 16 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 nucleotides in length. In some instances, the polynucleic acid molecule is about 14 nucleotides in length. In some instances, the polynucleic acid molecule is about 13 nucleotides in length. In some instances, the polynucleic acid molecule is about 12 nucleotides in length. In some instances, the polynucleic acid molecule is about 11 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 nucleotides in length. In some instances, the polynucleic acid molecule is about 8 nucleotides in length. In some instances, the polynucleic acid molecule is about 8 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 35 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 to about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 to about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 to about 30 nucleotides in length.In some instances, the polynucleic acid molecule is about 12 to about 30 nucleotides in length.

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

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

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

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

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

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

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

[0044] In some embodiments, the sequence of the polynucleic acid molecule has five or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has four or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has three or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has two or fewer mismatches to the target sequences described herein. In some embodiments, the sequence of the polynucleic acid molecule has one or fewer mismatches to the target sequences described herein.

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

[0046] In some embodiments, the polynucleic acid molecule has reduced off-target effects. In some instances, "off-target" or "off-target effect" refers to any instance in which a polynucleic acid polymer directed against a given target causes an unintended effect by directly or indirectly interacting with another mRNA sequence, DNA sequence, cellular protein, or other moiety. In some instances, an "off-target effect" occurs when there is simultaneous degradation of other transcripts due to partial homology or complementarity between the other transcripts and the sense and / or antisense strands of the polynucleic acid molecule.

[0047] In some embodiments, polynucleic acid molecules contain natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules contain a combination of DNA, RNA, and / or nucleotide analogs. In some instances, synthetic or artificial nucleotide analogs or bases contain modifications at one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0048] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some examples, the modification includes H, OR, R, halo, SH, SR, NH, NHR, NR, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some examples, the alkyl moiety further comprises a modification. In some examples, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some examples, the alkyl moiety further comprises a heterosubstitution. In some examples, a carbon of the heterocyclic group is replaced with nitrogen, oxygen, or sulfur. In some examples, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0049] In some cases, the modification of the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. Exemplary chemical structures of a 2'-O-methyl modification of an adenosine molecule and a 2'O-methoxyethyl modification of a uridine are illustrated below. [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 attaches the amine group to the 2' oxygen. In some instances, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving its cellular uptake properties due to its zwitterionic properties. A typical chemical structure of a 2'-O-aminopropyl nucleoside phosphoramidite is illustrated below. [ka]

[0051] In some instances, the modification of the 2' hydroxyl group is a locked or bridged ribose modification (e.g., Locked Nucleic Acid or LNA), in which the oxygen molecule attached at the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxy-methylene linked bicyclic ribonucleotide monomer. Representative examples of the chemical structure of LNA are illustrated below. The representative example shown on the left highlights the chemical connectivity of the LNA monomer. The representative example shown on the right shows a locked 3'-endo ( 3 E) emphasizes structure. [ka]

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

[0053] In some embodiments, additional modifications at the 2' hydroxyl group include 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, nucleotide analogs include, but are not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methoxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, Included are 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine, and 2-thiocytidine, dihydrouridine, pseudouridine, queusine, archaeosine, naphthyl and substituted naphthyl groups, O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine, 5-oxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides further include nucleotides modified at the sugar moiety, as well as nucleotides having non-ribosyl sugars or analogs thereof. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes those known in the art as universal bases.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0055] In some embodiments, the nucleotide analog further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or a combination thereof. Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics natural nucleic acid structures by deviating from normal sugar and phosphate structures. In some instances, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In some cases, backbone modifications remove all positive and negative charges, making morpholino neutral molecules capable of crossing cell membranes without the aid of cellular delivery agents, such as those used by charged oligonucleotides. [ka]

[0056] In some embodiments, peptide nucleic acids (PNAs) contain no sugar backbone rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating backbone charge. [ka]

[0057] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some examples, the modified internucleotide bond includes, but is not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'-alkylenephosphonates, 5'-methylphosphonates, 3'-alkylenephosphonates, borontrifluorides, 3'-5' or 2'-5' linked boranophosphates and selenophosphates, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate bonds, alkylphosphonates, alkylphosphonothioates, arylphosphorothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkylphosphoramidates, phosphoropipera Antisense oligonucleotides include didate, phosphoroanilothioate, phosphoroanilidate, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazo, methylenedimethyldimethylhydrazo, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, thioamidate, riboacetyl group bond, aminoethylglycine, silyl, or siloxane bond, e.g., saturated or unsaturated and / or substituted and / or heteroatom-containing alkyl or cycloalkyl bond of 1 to 10 carbon atoms with or without heteroatoms, morpholino bond, amide, polyamide in which bases are directly or indirectly bound to the aza nitrogen of the backbone, or combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides containing phosphorothioate linkages. Exemplary PS ASOs are described below. [ka]

[0058] In some instances, the modification is a methyl or thiol modification, such as a methyl phosphonate or thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left) and methyl phosphonate nucleotides (right) are illustrated below. [ka]

[0059] In some examples, modified nucleotides include, but are not limited to, 2'-fluoro N3-P5'-phosphoramidites, exemplified as follows: [ka]

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

[0061] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications of the 3'- or 5'-terminal nucleotide analog. For example, the 3'-terminal optionally includes a 3' cationic group, or the nucleoside is inverted at the 3'-terminal including a 3'-3' linkage. In another alternative, the 3'-terminal is optionally linked to an aminoalkyl group, such as a 3'C5-aminoalkyl dT. In a further alternative, the 3'-terminal is optionally linked to an abasic site, such as an apurinic or apyrimidinic acid site. In some instances, the 5'-terminal is linked to an aminoalkyl group, such as a 5'-O-aminoalkyl substituent. In some cases, the 5'-terminal is linked to an abasic site, such as an apurinic or apyrimidinic acid site.

[0062] In some embodiments, a polynucleic acid molecule comprises one or more artificial nucleotide analogs described herein. In some examples, a polynucleic acid molecule described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 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 nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or a combination 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 , modified 2'-ON-methylacetamide (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or a combination thereof, including 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 the group consisting of:In some embodiments, a polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methyl modified nucleotides. In some embodiments, a polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, a polynucleic acid molecule described herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more thiolphosphonate nucleotides.

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

[0064] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 90% modifications, about 20% to about 90% modifications, about 30% to about 90% modifications, about 40% to about 90% modifications, about 50% to about 90% modifications, about 60% to about 90% modifications, about 70% to about 90% modifications, and about 80% to about 100% modifications.

[0065] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 80% modifications, about 20% to about 80% modifications, about 30% to about 80% modifications, about 40% to about 80% modifications, about 50% to about 80% modifications, about 60% to about 80% modifications, and about 70% to about 80% modifications.

[0066] In some examples, the polynucleic acid molecule comprises at least one of the following: about 10% to about 70% modifications, about 20% to about 70% modifications, about 30% to about 70% modifications, about 40% to about 70% modifications, about 50% to about 70% modifications, and about 60% to about 70% modifications.

[0067] In some examples, the polynucleic acid molecule comprises at least one of the following: about 10% to about 60% modifications, about 20% to about 60% modifications, about 30% to about 60% modifications, about 40% to about 60% modifications, and about 50% to about 60% modifications.

[0068] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 50% modifications, about 20% to about 50% modifications, about 30% to about 50% modifications, and about 40% to about 50% modifications.

[0069] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 40% modifications, about 20% to about 40% modifications, and about 30% to about 40% modifications.

[0070] In some cases, the polynucleic acid molecule comprises at least one of the following: about 10% to about 30% modifications, and about 20% to about 30% modifications.

[0071] Optionally, the polynucleic acid molecule contains about 10% to about 20% modifications.

[0072] In some cases, the polynucleic acid molecule contains from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% modifications.

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

[0074] In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modifications.

[0075] In some examples, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modified nucleotides.

[0076] In some examples, about 5 to about 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 5% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 10% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 15% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 20% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 25% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 30% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 35% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 40% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 45% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 50% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 55% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 60% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 65% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 70% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 75% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 80% of the polynucleic acid molecules contain an artificial nucleotide analog described herein.In some examples, about 85% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 90% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 95% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 96% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 97% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 98% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 99% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some examples, about 100% of the polynucleic acid molecules contain an artificial nucleotide analog described herein. In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 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'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

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

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

[0079] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, are 2'-O-methyl purine nucleotides.

[0080] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein pyrimidine nucleotides, when present in the antisense strand, are 2'-deoxy-2'-fluoro pyrimidine nucleotides, and purine nucleotides, when present in the antisense strand, comprise 2'-deoxy-purine nucleotides.

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

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

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

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

[0085] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand comprises a terminal cap moiety at the 5' end, the 3' end, or both the 5' and 3' ends. In other embodiments, the terminal cap moiety is an inverted deoxyabasic moiety.

[0086] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the antisense strand comprises a phosphate backbone modification at the 3' end of the antisense strand. In some cases, the phosphate backbone modification is phosphorothioate. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, and the sense strand comprises at least two phosphorothioate internucleotide bonds. Alternatively and / or additionally, the antisense strand comprises at least two, at least three phosphorothioate internucleotide bonds.

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

[0088] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence of SEQ ID NO: 1 and the antisense strand comprises the sequence of SEQ ID NO: 2, and the sense strand comprises 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 the 5' end.

[0089] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence of SEQ ID NO: 1 and the antisense strand comprises the sequence of SEQ ID NO: 2, and the antisense strand comprises 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 two, at least three, or at least four 2'-F modified nucleotides.

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

[0091] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence of SEQ ID NO: 1 and the antisense strand comprises the sequence of SEQ ID NO: 2, and the antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides and four 2'-F modified nucleotides at its 3' end, wherein any two of the four 2'-F modified nucleotides are not consecutive.

[0092] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence of SEQ ID NO:1 and the antisense strand comprises the sequence of SEQ ID NO:2, and wherein the sense strand and / or the antisense strand comprise 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, respectively.

[0093] In some cases, one or more of the artificial nucleotide analogs described herein are more resistant to nucleases, such as ribonucleases, e.g., RNase H, deoxyribonucleases, e.g., DNase, or exonucleases, e.g., 5'-3' exonucleases and 3'-5' exonucleases, compared to naturally occurring polynucleic acid molecules. In some examples, the artificial nucleotide analogs are 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 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'-O-N-methylacetamide (2'-O-N-methylacetamide). Artificial nucleotide analogs, including 2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases, such as ribonucleases, e.g., RNase H, deoxyribonucleases, e.g., DNases, or exonucleases, e.g., 5'-3' exonucleases or 3'-5' exonucleases. In some examples, the 2'-O-methyl modified polynucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonucleases, or 3'-5' exonucleases resistant). In some instances, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some instances, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, 2'-deoxy modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-deoxy-2'-O-fluoro modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, LNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, ENA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some examples, HNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, morpholinos are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, PNA modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, methylphosphonate nucleotide modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, thiol phosphonate nucleotide modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some examples, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some examples, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0094] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity for their mRNA targets compared to a comparable naturally occurring polynucleic acid molecule. 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'-fluoro N3-P5'-phosphoramidite, have increased binding affinity for their mRNA targets compared to the equivalent naturally occurring polynucleic acid molecules. In some instances, 2'-O-methyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-aminopropyl modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-deoxy modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-deoxy-2'-fluoro modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable native polynucleic acid molecules.In some instances, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, LNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, ENA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, PNA modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, HNA-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, morpholino-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, methylphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, thiolphosphonate nucleotide-modified polynucleic acid molecules have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, polynucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity for their mRNA targets compared to comparable natural polynucleic acid molecules. In some instances, the increased affinity is exemplified by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

[0095] In some embodiments, the polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules or polynucleic acid molecules comprising a single enantiomer. In some instances, the polynucleic acid molecules comprise L-nucleotides. In some instances, the polynucleic acid molecules comprise D-nucleotides. In some instances, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its enantiomer. In some instances, the polynucleic acid molecule composition comprises 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some instances, the polynucleic acid molecules are polynucleic acid molecules described in U.S. Patent Application Publication Nos. 2014 / 194610 and 2015 / 211006; and PCT International Publication No. WO2015107425.

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

[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 instances, the polynucleic acid molecule is modified with one or more of the modifications described above to increase its stability. In some cases, the polynucleic acid molecule is modified at the two hydroxyl positions, such as with 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 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) modifications, or with locked or bridged ribose structures (e.g., LNA or ENA). In some cases, the polynucleic acid molecule is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, the polynucleic acid molecule further comprises morpholino, PNA, HNA, methyl phosphonate nucleotide, thiol phosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to increase its stability. In some cases, the polynucleic acid molecule is a chiral pure (or stereopure) polynucleic acid molecule. In some cases, the chiral pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications of RNA to increase the stability of delivery will be apparent to those skilled in the art.

[0098] In some cases, the polynucleic acid molecule is a double-stranded polynucleotide molecule, comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some cases, the polynucleic acid molecule is assembled from two separate polynucleotides, one strand is a sense strand, and the other strand is an antisense strand, wherein the antisense strand and the sense strand are self-complementary (e.g., each strand comprises a nucleotide sequence complementary to the nucleotide sequence of the other strand; such as when the antisense strand and the sense strand form a double-stranded (duplex) or double-stranded structure. For example, the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the polynucleic acid molecule can be assembled from a single oligonucleotide, with the self-complementary sense and antisense regions of the polynucleic acid molecule being joined by a nucleic acid-based or non-nucleic acid-based linker.

[0099] In some cases, the polynucleic acid molecule is a polynucleotide with a double, asymmetric double, hairpin, or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of another target nucleic acid molecule or a part thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof.In other cases, the polynucleic acid molecule is a circular single-stranded polynucleotide with two or more loop structures and a base comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a part thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a part thereof, and the circular polynucleotide is processed in vivo or in vitro to generate an active polynucleic acid molecule that can mediate RNAi. In further cases, the polynucleic acid molecule further comprises a single-stranded polynucleotide having a nucleotide sequence complementary to that of a target nucleic acid molecule or a portion thereof (e.g., such a polynucleic acid molecule need not be present within the polynucleic acid molecule of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-stranded polynucleotide further comprises a terminal phosphate group such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate.

[0100] In some instances, an asymmetric hairpin is a linear polynucleic acid molecule comprising an antisense region, a loop portion comprising nucleotides or non-nucleotides, and a sense region, where the sense region contains sufficient complementary nucleotides to base pair with the antisense region but fewer nucleotides than the antisense region to form a looped duplex. For example, an asymmetric hairpin polynucleic acid molecule may comprise an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in vitro system and having a loop region comprising about 4 to about 8 nucleotides, and a sense region of about 3 to about 18 nucleotides complementary to the antisense region. In some instances, the asymmetric hairpin polynucleic acid molecule further comprises a chemically modified 5'-terminal phosphate group. In further instances, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0101] In some embodiments, an asymmetric duplex is a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region has sufficient complementary nucleotides to base-pair with the antisense region but contains fewer nucleotides than the antisense region, sufficient to form a duplex. For example, an asymmetric duplex polynucleic acid molecule may comprise an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in an in vitro system, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0102] In some cases, universal base refers to the nucleotide base analogue that forms base pairs with each of the natural DNA / RNA bases that are almost indistinguishable.Non-limiting examples of universal base include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole, as known in the prior art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

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

[0104] In some embodiments, the polynucleic acid molecule is synthesized by a tandem synthesis method, where both strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker, which is then cleaved to provide separate fragments or strands that hybridize to the duplex and allow for purification of the duplex.

[0105] In some instances, the polynucleic acid molecule is also assembled from two distinct nucleic acid strands or fragments, where one fragment comprises the sense region and the second fragment comprises the antisense region of the molecule.

[0106] Further modification methods, for example, to incorporate sugar, base, and phosphate modifications, include the following: 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, US Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97 / 26270; Beigelman et al. 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 publication describes methods and strategies for determining the location for incorporating sugar, base, and / or phosphate modifications into nucleic acid molecules without modulating catalytic activity.

[0107] In some cases, chemical modification of internucleotide bonds of polynucleic acid molecules with phosphorothioate, phosphorodithioate, and / or 5'-methylphosphonate bonds improves stability, while excessive modification often causes toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide bonds is sometimes minimized. In such cases, reducing the concentration of these bonds reduces the toxicity of these molecules and increases their efficacy and specificity.

[0108] Polynucleic acid molecule conjugates In some embodiments, the polynucleic acid molecule (B) is further conjugated to a polypeptide A, which is delivered to a desired site. In some cases, the polynucleic acid molecule is conjugated to a polypeptide A, optionally with a polymer moiety. In some examples, at least one polypeptide A is conjugated to at least one B. In some examples, at least one polypeptide A is conjugated to at least one B to form an AB conjugate. In some embodiments, at least one A is conjugated to the 5' end of B, the 3' end of B, an internal site of B, or any combination thereof. In some examples, at least one polypeptide A is conjugated to at least two Bs. In some examples, at least one polypeptide A is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more Bs.

[0109] In some cases, the polynucleic acid molecule is conjugated to a polypeptide (A) and optionally a polymer moiety (C). In some embodiments, at least one polypeptide A is attached at one end of at least one B, while at least one C is attached at the opposite end of at least one B to form an ABC conjugate. In some examples, at least one polypeptide A is attached at one end of at least one B, while at least one C is attached at an internal site of at least one B. In some examples, at least one polypeptide A is directly attached to at least one C. In some examples, at least one B is indirectly attached 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, are linked to at least one polypeptide A. In some examples, at least one B is linked to at least one polypeptide A at a terminal end (e.g., the 5' end or the 3' end) or via an internal site. Optionally, at least one C is linked to at least one polypeptide A directly or indirectly via at least one B. Indirectly, via at least one B, at least one C is linked at the same end on B as at least one polypeptide A, at the opposite end from at least one polypeptide A, or independently at an internal site. In some examples, at least one additional polypeptide A is further linked to at least one polypeptide A, B, or C. In a further example, at least one D is optionally linked, directly or indirectly, to at least one polypeptide A, at least one B, or at least one C. When directly bound to at least one polypeptide A, at least one D is also optionally bound to at least one B to form an ADB conjugate, or optionally bound to at least one B and at least one C to form an ADBC ​​conjugate. In some examples, at least one D is directly bound to at least one polypeptide A and indirectly bound to at least one B and at least one C to form a DABC conjugate. When indirectly bound to at least one polypeptide A, at least one D is also optionally bound to at least one B to form an ABD conjugate, or optionally bound to at least one B and at least one C to form an ABDC conjugate. In some examples, at least one additional D is further bound to at least one polypeptide A, B, or C.

[0111] joining part In some embodiments, binding moiety 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 antigen-binding fragment thereof includes a humanized antibody or antigen-binding fragment thereof, a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a monoclonal antibody or antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, an (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or antigen-binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

[0112] In some embodiments, binding moiety A is a bispecific antibody or antigen-binding fragment thereof. In some instances, the bispecific antibody is a trispecific antibody or a bispecific miniantibody. In some instances, the bispecific antibody is a trispecific antibody. In some instances, the trispecific antibody is a full-length monoclonal antibody that contains binding sites for two different antigens.

[0113] In some instances, the bispecific antibody is a bispecific miniantibody. In some instances, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody, or bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single-chain variable fragments (scFvs), where the two scFvs target epitopes of two different antigens.

[0114] In some embodiments, binding moiety A is a bispecific miniantibody. In some instances, A is a bispecific Fab2. In some instances, A is a bispecific F(ab)'3 fragment. Optionally, A is a bispecific bis-scFv. Optionally, A is a bispecific (scFv). In some embodiments, A is a bispecific diabody. In some embodiments, A is a bispecific minibody. In some embodiments, A is a bispecific triabody. In other embodiments, A is a bispecific tetrabody. In other embodiments, A is a bispecific T cell engager (BiTE).

[0115] In some embodiments, binding moiety A is a trispecific antibody. In some examples, the trispecific antibody comprises a F(ab)'3 fragment or a trispecific antibody. In some examples, A is a trispecific F(ab)'3 fragment. In some cases, A is a trispecific antibody. In some embodiments, A is a trispecific antibody 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 moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein. In some instances, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein on a muscle cell. In some instances, binding moiety A is an antibody or antigen-binding fragment thereof that recognizes a cell surface protein on a skeletal muscle cell.

[0117] In some embodiments, exemplary antibodies include, but are not limited to, anti-myosin antibodies, anti-transferrin receptor antibodies, and antibodies that recognize muscle-specific kinase (MuSK). In some instances, 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 transferrin receptor (TfR), preferably specifically binds to transferrin receptor 1 (TfR1), or more preferably specifically binds to human transferrin receptor 1 (TfR1) (or human CD71).

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

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

[0121] [Table 2]

[0122] In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 18, 20, or 21, and an HCDR3 sequence comprising SEQ ID NO: 19. In some embodiments, the VH region comprises 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. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19. In some embodiments, the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 21, and an HCDR3 sequence comprising SEQ ID NO: 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, wherein 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 comprises an LCDR1, LCDR2, and LCDR3 sequence selected from Table 2.

[0125] [Table 3]

[0126] In some cases, the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence comprising SEQ ID NO: 24 or 26, wherein X3 is selected from N or S.

[0127] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 24 or 26, wherein X4 is selected from A or G and X5 is selected from D or E.

[0128] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent and, if present, is F.

[0129] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22, an LCDR2 sequence AATNLAX5, and an LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 is present or absent, and if present is F.

[0130] In some cases, the VL region comprises 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.

[0131] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:22, an LCDR2 sequence comprising SEQ ID NO:25, and an LCDR3 sequence comprising SEQ ID NO:26.

[0132] In some cases, the VL region comprises an LCDR1 sequence comprising SEQ ID NO:27, an LCDR2 sequence comprising SEQ ID NO:28, and an LCDR3 sequence comprising SEQ ID NO:26.

[0133] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19; and the VL region 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, or F if present.

[0134] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence RTSENIYX3NLA, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence comprising 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, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 24 or 26, where X4 is selected from A or G and X5 is selected from D or E.

[0136] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and an LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and if present is F.

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

[0138] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19, and the VL region comprises 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.

[0139] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22, an LCDR2 sequence comprising SEQ ID NO: 25, and an LCDR3 sequence comprising SEQ ID NO: 26.

[0140] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising 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 comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 27, an LCDR2 sequence comprising SEQ ID NO: 28, and an LCDR3 sequence comprising SEQ ID NO: 26.

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

[0142] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises 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; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 24 or 26, wherein 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, wherein the VH region comprises 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; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and is F if present.

[0144] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises 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 comprises an LCDR1 sequence comprising SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6; and 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, wherein the VH region comprises 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, and the VL region comprises 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.

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

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

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

[0149] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 24 or 26, wherein 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, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, where X6 is present or absent, and is F if present.

[0151] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19; the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6; and 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, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises 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.

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

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

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

[0156] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 21, and an HCDR3 sequence comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence AX4TNLAX5, and an LCDR3 sequence comprising SEQ ID NO: 24 or 26, wherein 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, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 21, and an HCDR3 sequence comprising SEQ ID NO: 19; and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22 or 27, an LCDR2 sequence comprising SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and, if present, is F.

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

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

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

[0161] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 21, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 27, an LCDR2 sequence comprising SEQ ID NO: 28, and an LCDR3 sequence comprising 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 about 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 comprises about 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 comprises a sequence selected from SEQ ID NOs:29-33 (Table 4), and the VL region comprises a sequence selected from SEQ ID NOs: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 comprises a VH region and a VL region as illustrated in Table 6.

[0167] [Table 6]

[0168] In some embodiments, the anti-transferrin receptor antibody described herein comprises 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, the anti-transferrin receptor antibody contains one or more mutations in a framework region, such as the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some cases, the one or more mutations are intended to stabilize the antibody and / or extend half-life. In some cases, the one or more mutations are intended to modulate Fc receptor interaction and reduce or eliminate Fc effector function, such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional examples, the 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 includes 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 comprises a mutation at residue positions L234, L235, D265, N21, K46, L52, or P53, or a combination thereof. In some cases, the mutation comprises L234 and L235 in combination with a mutation at residue position K46, L52, or P53. In some cases, the Fc region comprises mutations at L234, L235, and K46. In some cases, the Fc region comprises mutations at L234, L235, and L52. In some cases, the Fc region comprises mutations at L234, L235, and P53. In some cases, the Fc region comprises mutations at D265 and N21. In some cases, the residue positions are relative to IgG1.

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

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

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

[0175] In some embodiments, the Fc region comprises a mutation 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 a SEQ ID NO: 1. Optionally, the Fc region comprises a mutation at L233 and L234. Optionally, the Fc region comprises a mutation at L233 and L234 in combination with a mutation at residue position K45, L51, or P52. Optionally, the Fc region comprises a mutation at L233, L234, and K45. Optionally, the Fc region comprises a mutation at L233, L234, and L51. Optionally, the Fc region comprises a mutation at L233, L234, and K45. Optionally, the Fc region comprises a mutation at L233, L234, and P52. In some cases, the Fc region contains mutations at D264 and N20. In some cases, positions equivalent to residues L233, L234, D264, N20, K45, L51, or P52 in the IgG1, IgG2, IgG3, or IgG4 framework are contemplated. In some cases, mutations to residues corresponding to residues L233, L234, D264, N20, K45, L51, or P52 of SEQ ID NO: 303 in the IgG1, IgG2, or IgG4 framework are also contemplated.

[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 of SEQ ID NO: 39. In some embodiments, the Fc region comprises L233A and L234A. In some embodiments, the Fc region comprises L233A and L234A. In some embodiments, the Fc region comprises L233A and L234A in combination with K45G, L51R, or P52G. In some embodiments, the Fc region comprises L233A, L234A, and K45G. In some embodiments, the Fc region comprises L233A, L234A, and L51R. In some embodiments, the Fc region comprises L233A, L234A, and K45G. In some embodiments, the Fc region comprises L233A, L234A, and P52G. In some cases, the Fc region comprises D264A and N20G.

[0177] In some embodiments, human IgG constant regions are used to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), e.g., 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. 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). Optionally, the heavy chain (HC) comprises a sequence selected from Table 7. Optionally, the light chain (LC) comprises a sequence selected from Table 8. The underlined regions represent the respective CDRs.

[0179] [Table 7] JPEG2026009930000019.jpg250170 JPEG2026009930000020.jpg254170 JPEG2026009930000021.jpg242170 JPEG2026009930000022.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 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 more, longer than the reference anti-transferrin receptor antibody.

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

[0183] In some embodiments, binding moiety A binds to polynucleic acid molecule (B) in a non-site-specific manner. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via a lysine residue, a cysteine ​​residue, at the 5'-end, at the 3'-end, at an unnatural amino acid, or at an enzyme-modified or enzyme-catalyzed residue. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via a lysine residue (e.g., a lysine residue present in binding moiety A). In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via a cysteine ​​residue (e.g., a cysteine ​​residue present in binding moiety A). In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner at the 5'-end. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner at the 3'-end. In some examples, binding moiety A binds to polynucleic acid molecule (B) in a site-specific manner via an unnatural amino acid. In some instances, the binding moiety A is attached to the polynucleic acid molecule (B) via an enzyme-modified or enzyme-catalyzed residue in a site-specific manner.

[0184] In some embodiments, one or more polynucleic acid molecules (B) are bound to binding moiety A. In some instances, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acid molecules are bound to one binding moiety A. In some instances, about 1 polynucleic acid molecule is bound to one binding moiety A. In some instances, about 2 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 3 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 4 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 5 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 6 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 7 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 8 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 9 polynucleic acid molecules are bound to one binding moiety A. In some instances, about 10 polynucleic acid molecules bind to one binding moiety A. In some instances, about 11 polynucleic acid molecules bind to one binding moiety A. In some instances, about 12 polynucleic acid molecules bind to one binding moiety A. In some instances, about 13 polynucleic acid molecules bind to one binding moiety A. In some instances, about 14 polynucleic acid molecules bind to one binding moiety A. In some instances, about 15 polynucleic acid molecules bind to one binding moiety A. In some instances, about 16 polynucleic acid molecules bind to one binding moiety A. In some instances, one or more polynucleic acid molecules are the same. In other instances, one or more polynucleic acid molecules are different.

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

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

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

[0188] In some instances, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety A has improved activity compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some instances, the improved activity results in an enhancement of a biologically relevant function, for example, improved stability, affinity, binding, functional activity, and efficacy in treating or preventing a disease condition. In some instances, the disease condition is the result of one or more mutated exons of a gene. In some instances, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety A results in increased exon skipping of one or more mutated exons compared to a conjugate comprising a polynucleic acid molecule (B) without a binding moiety A. In some instances, exon skipping is increased by at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% in a conjugate comprising polynucleic acid molecule (B) and binding moiety A compared to a conjugate comprising polynucleic acid molecule (B) without binding moiety A.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof is further modified, alone or in combination, using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, or addition, and / or by recombination and / or other modifications known in the art (e.g., post-translational and chemical modifications such as glycosylation and phosphorylation). In some examples, the modifications further include modifications to modulate interaction with Fc receptors. In some examples, one or more modifications include, for example, those described in International Publication No. WO 97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into nucleic acid sequences underlying the amino acid sequence of an antibody or antigen-binding fragment thereof are well known to those of skill in the art.

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

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

[0192] In some instances, bispecific single-chain antibody constructs relate to constructs comprising binding domains from two antibodies. In such embodiments, the bispecific single-chain antibody construct is a tandem bi-scFv or diabody. In some instances, the scFv comprises a VH and a VL domain connected by a linker peptide. In some instances, the linker is of sufficient length and sequence to allow each of the first and second domains to retain their differential binding specificities independently of each other.

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

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

[0195] Thus, in some cases, the polynucleic acid molecule conjugate comprises 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, and an antisense strand having a sequence that is at least 80% identical to SEQ ID NO:2, and an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugated to the polynucleic acid molecule, such that the polynucleic acid molecule conjugate mediates RNA interference against DMPK.

[0196] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or antigen-binding fragment thereof 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: 3, 5, 7, 9, 11, 13, or 15, and an antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, and wherein the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or antigen-binding fragment thereof 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: 3, 5, 7, 9, 11, 13, or 15, and an antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16. The fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises a region comprising an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises 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 antigen-binding fragment thereof and the 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 antigen-binding fragment thereof 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: 3, 5, 7, 9, 11, 13, or 15, and an antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; The 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 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30 and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34, and the anti-transferrin receptor antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 and two, at least three 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 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 30 and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 34, and the anti-transferrin receptor antibody or antigen-binding fragment thereof 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 antigen-binding fragment thereof conjugated to a polynucleic acid molecule that hybridizes to a target sequence of DMPK, wherein the polynucleic acid molecule has a sense 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 the sequence of SEQ ID NO: 2, and the antisense strand has 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 antigen-binding fragment thereof comprises two, at least three, or 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 comprises an HCDR1 sequence comprising SEQ ID NO: 17, an HCDR2 sequence comprising SEQ ID NO: 20, and an HCDR3 sequence comprising SEQ ID NO: 19, and the VL region comprises 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 antigen-binding fragment thereof 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 antigen-binding fragment thereof 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 2'-O-methyl modified nucleotides at the 5' and 3' ends; 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 comprises 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, and the VL region comprises an LCDR1 sequence comprising SEQ ID NO: 22, an LCDR2 sequence comprising SEQ ID NO: 3, and an LCDR3 sequence comprising SEQ ID NO: 24, and the anti-transferrin receptor antibody or antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a maleimide linker.

[0201] In certain embodiments, the polynucleic acid molecule conjugate comprises an anti-transferrin receptor antibody or antigen-binding fragment thereof 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 contiguous 2'-O-methyl modified nucleotides at its 3' end and four 2'-F modified nucleotides, wherein the four any two of the 2'-F modified nucleotides in SEQ ID NO:3 are not contiguous; 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 comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:3 and the VL region comprises at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO:34; and the anti-transferrin receptor antibody or antigen-binding fragment thereof and the polynucleic acid molecule are conjugated via a 6-amino-1-hexanol linker.

[0202] Additional binding moieties In some embodiments, the binding moiety is a plasma protein. In some instances, the plasma protein includes albumin. In some instances, binding moiety A is albumin. In some instances, albumin is bound to the polynucleic acid molecule by one or more of the binding chemistries described herein. In some instances, albumin is bound to the polynucleic acid molecule by native ligation chemistry. In some instances, albumin is bound to the polynucleic acid molecule by a lysine bond.

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

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

[0205] In some instances, the binding moiety is a peptide. In some instances, the peptide comprises about 1 to about 3 kDa. In some instances, the peptide comprises about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some instances, the peptide is a bicyclic peptide. In some instances, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the binding moiety is a bicyclic peptide (e.g., Bicycles from Bicycle Therapeutics).

[0206] In further instances, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some instances, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, where the target-binding end is capable of recognizing and interacting with a cell surface receptor. For example, in some instances, the target-binding end comprises a glutamate urea compound, which allows interaction with PSMA, thereby enhancing antibody interaction with cells expressing PSMA. In some examples, the binding moiety is a small molecule described in Zhang et al., "A remote arene-binding site on prostate-specific membrane antigen revealed by antibody-recruiting small molecules," J Am Chem Soc. 132(36):12711-12716 (2010); or McEnaney, et al., "Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease," ACS Chem Biol. 7(7):1139-1151 (2012).

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

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

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

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

[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 a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region (e.g., humanized antibodies).

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

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

[0214] In some embodiments, various host-expression vector systems are utilized to express the antibodies or antigen-binding fragments thereof described herein. Such host-expression systems not only represent vehicles in which antibody coding sequences are generated and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or antigen-binding fragments thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or antigen-binding fragment coding sequences; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or antigen-binding fragment coding sequences; insect cell systems (e.g., baculovirus) infected with recombinant viral expression vectors containing the antibody or antigen-binding fragment coding sequences; and recombinant viral expression vectors (e.g., cauliflower mosaic virus). Plant cell lines infected with viruses (CaMV and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing antibody or antigen-binding fragment coding sequences; or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (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 instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, cells are engineered to grow in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes, grow, and form foci that are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or antigen-binding fragments thereof.

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

[0217] In some instances, antibody expression levels are increased by vector amplification (for a review, see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is related to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

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

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

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

[0221] In some instances, polynucleic acid molecules are coupled to binding moieties in a site-directed manner using "traceless" coupling technology (PhiloChem). In some instances, the "traceless" coupling technology utilizes an N-terminal 1,2-aminothiol group on a polynucleic acid molecule that contains an aldehyde group and is then coupled to a binding moiety. (See Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacovigilance," JACS 134(13):5887-5892 (2012)).

[0222] In some instances, polynucleic acid molecules are conjugated to binding moieties in a site-directed manner utilizing unnatural amino acids introduced into the binding moiety. In some instances, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some instances, the keto group of pAcPhe selectively binds to alkoxy-amine derived binding moieties to form oxime bonds. (See Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40):16101-16106 (2012)).

[0223] In some instances, the polynucleic acid molecule is attached to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Catalent, Inc.). In some instances, SMARTag™ technology involves the generation of a formylglycine (FGly) residue from cysteine ​​by formylglycine generating enzyme (FGE) via an oxidation process in the presence of an aldehyde tag, and the subsequent attachment of FGly to an alkylhydrazine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation. (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 examples, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some examples, the polynucleic acid molecule is conjugated to the binding moiety using a microbial transglutaminase-catalyzed process. In some examples, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine in the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some examples, mTG is produced by Streptomyces mobaraensis. (See Strop et al., "Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates," Chemistry and Biology 20(2) 161-167 (2013)).

[0225] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by methods such as those described in PCT International Publication No. WO2014 / 140317, which utilize sequence-specific transpeptidases.

[0226] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by methods such as those described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540.

[0227] Polymer-binding moiety In some embodiments, polymer moiety C is further linked to a polynucleic acid molecule described herein, a binding moiety described herein, or a combination thereof. In some examples, polymer moiety C is a conjugated polynucleic acid molecule of the formula A-X1-B-X2-C (X1, X2 are two linkers conjugating A and B, B and C, respectively). Binds to a polynucleic acid molecule. In some cases, polymer moiety C is linked to a binding moiety. In other cases, polymer moiety C is linked to a polynucleic acid molecule binding moiety. In further cases, polymer moiety C is linked as illustrated above.

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

[0229] In some instances, C is a PEG moiety. In some instances, the PEG moiety is attached at the 5' end of the polynucleic acid molecule, while the linking moiety is attached at the 3' end of the polynucleic acid molecule. In some instances, the PEG moiety is attached at the 3' end of the polynucleic acid molecule, while the linking moiety is attached at the 5' end of the polynucleic acid molecule. In some instances, the PEG moiety is attached to an internal site of the polynucleic acid molecule. In some instances, the PEG moiety, the linking moiety, or a combination thereof, is attached to an internal site of the polynucleic acid molecule. In some instances, the conjugate is a direct conjugate. In some instances, the attachment is via native ligation.

[0230] In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some instances, a polydisperse material comprises a dispersed distribution of materials of different molecular weights, characterized by average weight (weight average) size and dispersity. In some instances, a monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the molecular weight indicated represents the average molecular weight of the polyalkylene oxide (e.g., PEG) molecules.

[0231] In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 83 700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

[0232] In some embodiments, C is a polyalkylene oxide (e.g., PEG) and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 830 and having a molecular weight of 0, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 850 In some instances, the molecular weight of C is about 200 Da. In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da.In some instances, the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1450 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da. In some instances, the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da.In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da.

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

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

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

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

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

[0239] In some examples, cMAP is further conjugated to a PEG moiety to form a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some examples, the PEG moiety ranges from about 500 Da to about 50,000 Da. In some examples, the PEG moiety is about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 Da to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.

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

[0241] In some embodiments, the polymer moiety C is linked to a polynucleic acid molecule, a binding moiety, and optionally an endosomolytic moiety, as exemplified above.

[0242] Endosomolytic or cell membrane-penetrating moiety 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 endosomolytic moiety and / or a cell membrane-penetrating moiety. In some cases, the endosomolytic moiety is a compound capable of releasing a cellular compartment-releasing component, e.g., any of the cellular compartments known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other endoplasmic reticulum comprising a cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer. In some cases, the cell membrane-penetrating moiety comprises a cell-penetrating peptide (CPP). In other cases, the cell membrane-penetrating moiety comprises a cell-penetrating lipid. In other cases, the cell membrane-penetrating moiety comprises a cell-penetrating small molecule.

[0243] Endosomolytic and cell membrane-penetrating polypeptides In some embodiments, the molecule of Formula (I): A-X1-B-X2-C is further linked to an endosomolytic polypeptide. Optionally, the endosomolytic polypeptide is a pH-dependent membrane-active peptide. Optionally, the endosomolytic polypeptide is an amphipathic polypeptide. In additional instances, the endosomolytic polypeptide is a peptidomimetic. In some instances, the endosomolytic polypeptide comprises INF, melittin, mucin (meucin), or their respective derivatives. In some instances, the endosomolytic polypeptide comprises INF or a derivative thereof. In other instances, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further instances, the endosomolytic polypeptide comprises mucin or a derivative thereof.

[0244] In some instances, INF7 is a 24-residue polypeptide, and these sequences include CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 67), or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 68). In some instances, INF7 or a derivative thereof includes 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 instances, melittin is a 26-residue polypeptide, and the sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 72), or alternatively, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 73). In some instances, melittin comprises a polypeptide sequence described in U.S. Patent No. 8,501,930.

[0246] In some instances, the mucins are antimicrobial peptides (AMPs) derived from the venom glands of the scorpion Mesobuthus eupeus. In some instances, the mucins are comprised of mucin-13 (the sequence of which comprises IFGAIAGLLKNIF-NH2 (SEQ ID NO: 74)) and mucin-18 (the sequence of which comprises FFGHLFKLATKIIPSLFQ (SEQ ID NO: 75)).

[0247] In some examples, the endosomolytic polypeptide comprises a polypeptide whose sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to INF7 or a derivative thereof, a melittin or derivative thereof, or a mucin or derivative thereof. In some examples, the endosomolytic moiety comprises INF7 or a derivative thereof, a melittin or derivative thereof, or a mucin or derivative thereof.

[0248] In some examples, the endosomolytic moiety is INF7 or a derivative thereof. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 67-71. In some examples, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 67. In some examples, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 68-71. Optionally, the endosomolytic portion comprises SEQ ID NO: 67. Optionally, the endosomolytic portion comprises SEQ ID NO: 68-71. Optionally, the endosomolytic portion consists of SEQ ID NO: 67. Optionally, the endosomolytic portion consists of SEQ ID NO: 68-71.

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

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

[0251] In some instances, the endosomolytic moiety comprises a sequence as illustrated in Table 9.

[0252] [Table 9] JPEG2026009930000026.jpg95170

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

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

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

[0256] In some examples, the endosomolytic moiety is a lipid (eg, a fusogenic lipid) described in PCT International Publication No. WO 09 / 126,933.

[0257] Endosomolytic small molecules In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, a molecule of Formula (I): A-X1-B-X2-C is further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amodiaquine (carnoquines), ampicillin, primaquine, mefloquine, nivaquines, halofantrine, quinoneimines, or combinations thereof. In some examples, quinoline endosomolytic moieties include, but are not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-1-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-1-butylamino)quinoline; 4-(4-diethylamino-1-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline 7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino-)quinoline;7-Hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;Hydroxychloroquine phosphate;7-Chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline (desmethylhydroxychloroquine);7-Fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline 7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-butylamino)quinoline;7-Fluoro ... 4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-butylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-butylamino)quinoline;7-Chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-methylbutylamino)quinoline;7-Fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)amino-1-methylbutylamino)quinoline;4-(1- Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-Hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloridequinoline;1-Acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-Butyryl-1,2,3,4-tetrahydroquinoline;3-chloro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline)(4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline; 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline)(4-[(4-diethylamino)-1-methylbutylamino]-6-methoxyquinoline; 4-(4-hydroxy-α,α'-bis(2-methyl-1- pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinolinecarboxaldehyde; 1,1'-pentamethylenequinolinium iodide; 8-quinolinol sulfate and amino, aldehyde, carboxylic acid, hydroxyl, halogen, keto, sulfhydryl, and vinyl derivatives or analogs thereof. In some examples, the endosomolytic moiety is a small molecule described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Pat. No. 5,736,557.

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

[0259] [Table 10]

[0260] Linker In some embodiments, the linkers described herein are cleavable or non-cleavable linkers. In some examples, the linker is a cleavable linker. In other examples, the linker is a non-cleavable linker.

[0261] In some cases, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not contain repeating units of a monomer produced by a polymerization process. Exemplary non-polymeric linkers include, but are not limited to, a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In some cases, the non-polymeric linker includes a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-immolative linker, a maleimide-based linker, or a combination thereof. In further cases, the non-polymeric linker does not include more than two linkers of the same type, for example, more than two homobifunctional cross-linkers or more than two peptide linkers. In further instances, the non-polymeric linker optionally includes one or more reactive functional groups.

[0262] In some instances, the non-polymeric linker does not include a polymer as described above. In some instances, the non-polymeric linker does not include a polymer encompassed by polymer moiety C. In some instances, the non-polymeric linker does not include a polyalkylene oxide (e.g., PEG). In some instances, the non-polymeric linker does not include PEG.

[0263] In some instances, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDS), and the like. T), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds such as 1,5-difluoro-2,4-dinitrobenzene, 1,3-difluoro-4,6-dinitrobenzene (DFDNB), 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED ), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic 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 comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl-crosslinking linkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamide]hexanoate (sulfo-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs ... Imidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidylCarbonyl-reactive and -reactive amines such as 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH) and sulfhydryl-reactive cross-linkers, amine-reactive and photoreactive cross-linkers, such as N-hydroxysuccinimidyl-4-azidosalicylate (NH-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NH-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NH-LC-AsA), sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1, 3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N -5-Azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl7-Azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP), ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive cross-linkers, such as 1-(ρ-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio) propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as ρ-azidophenylglyoxal (APG).

[0265] In some instances, the linker comprises a reactive functional group. In some instances, the reactive functional group comprises a nucleophilic group reactive to an electrophilic group present in the linking moiety. Exemplary electrophilic groups include carbonyl groups such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

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

[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 catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing retro-Michael elimination. In some examples, the self-stabilizing maleimide is a maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol. 32(10):1059-1062 (2014). In some examples, the linker comprises a self-stabilizing maleimide. In some examples, the linker is a self-stabilizing maleimide.

[0268] In some embodiments, the linker comprises a peptide moiety. In some examples, the peptide moiety comprises at least 2, 3, 4, 5, or more than 6 amino acid residues. In some examples, the peptide moiety comprises at most 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some examples, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some examples, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some examples, the peptide moiety is a non-cleavable peptide moiety. In some examples, the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (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 examples, the linker comprises a peptide moiety such as Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

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

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

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

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

[0273] In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or linker group) on the binding moiety A, the polynucleotide B, the polymer C, or the endosomolytic moiety D after cleavage. Exemplary traceless linkers include, but are not limited to, a germanium linker, a silicon linker, a sulfur linker, a selenium linker, a nitrogen linker, a phosphorus linker, a boron linker, a chromium linker, or a phenylhydrazide linker. In some cases, the linker is a traceless aryl-triazene linker 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 described in Blaney, et al., "Traceless solid-phase organic synthesis," Chem. Rev. 102:2607-2024 (2002). In some examples, the linker is a traceless linker as described in US Pat. No. 6,821,783.

[0274] In some embodiments, the linker is a polymerizable compound as described in U.S. Patent Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 2013 / 0 309256; 2015 / 037360; or 2014 / 0294851; or PCT Publication Nos. WO2015057699; WO2014080251; WO2014197854; WO2014145090; or 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 instances, 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 a C1-C6 alkyl group, such as a C5, C4, C3, C2, or C1 alkyl group. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. When used in the context of a linker, particularly in the context of X1, alkyl refers to a saturated, straight- or branched-chain hydrocarbon radical containing up to 6 carbon atoms. In some examples, X1 comprises a homobifunctional linker or a heterobifunctional linker 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 bonded to a C1-C6 alkyl group. In other examples, X1 comprises sMCC optionally bonded to a C1-C6 alkyl group. In some additional examples, X1 does not comprise a homobifunctional linker or a heterobifunctional linker 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 non-polymeric linker. In some embodiments, X2 is a C1-C6 alkyl group. In some examples, X2 is a homobifunctional linker or a heterobifunctional linker described above. In some examples, X2 is a homobifunctional linker described above. In some examples, X2 is a heterobifunctional linker described above. In some examples, X2 comprises a maleimide group, such as maleimidocaproyl (mc), 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 an additional example, X2 comprises a mc-val-cit group. In an additional example, X2 comprises a val-cit-PABA group. In an additional example, X2 comprises a mc-val-cit-PABA group.

[0278] How to use Muscular dystrophies refer to the loss of muscle mass and / or the progressive weakening and degeneration of muscle. In some cases, the loss of muscle mass or the progressive weakening and degeneration of muscle is caused by 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 protein to use amino acids as substrates for gluconeogenesis).

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

[0280] In another embodiment, muscular dystrophy refers to a significant loss of muscle mass. By significant loss of muscle mass, we mean a decrease in muscle volume in a subject's diseased, damaged, or unused muscle tissue compared to the same muscle tissue in a control subject. In some embodiments, the 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 in a control subject. In another embodiment, the significant loss of muscle mass refers to a decrease in muscle volume in unused muscle tissue compared to the muscle volume of the same muscle tissue in the same subject prior to the period of disuse. In some embodiments, the 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 in the same subject prior to the period of disuse. Muscle volume is optionally measured by assessing muscle cross-sectional area, such as by magnetic resonance imaging (eg, by muscle volume / cross-sectional area (CSA) MRI method).

[0281] Myotonic dystrophy is a multisystemic neuromuscular disease that includes two major types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by a dominantly inherited "CTG" repeat expansion in the 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 post-transcriptional splicing and polyadenylation site regulation, and loss of MBNL protein function leads to the accumulation of downstream nuclear foci and increased mis-splicing events, resulting in myotonia and other clinical symptoms.

[0282] In some embodiments, methods of treating muscular dystrophy (e.g., DM1) in a subject are described herein, the methods comprising providing a polynucleic acid molecule described herein or a polynucleic acid molecule conjugate described herein, and administering a therapeutically effective amount of the polynucleic acid molecule or polynucleic acid molecule conjugate to a subject in need thereof to treat the muscular dystrophy, wherein the polynucleic acid conjugate reduces the amount of human DMPK mRNA transcript. In some embodiments, administering 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%, or at least 80% of the DMPK mRNA expression level in a patient not treated 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 routes of administration. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet other examples, the pharmaceutical compositions described herein are formulated for nasal administration.

[0284] In some embodiments, pharmaceutical compositions include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolve formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and combined immediate and controlled release formulations.

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

[0286] In some instances, the nanoparticles comprise a core, or alternatively, a core and a shell, such as in core-shell nanoparticles.

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

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

[0289] In some examples, the nanoparticle formulation comprises a paramagnetic nanoparticle, a superparamagnetic nanoparticle, a metal nanoparticle, a fullerene-like material, an inorganic nanotube, a dendrimer (such as one with a covalently bound metal chelate), a nanofiber, a nanohorn, a nano-onion, a nanorod, a nanorope, or a quantum dot. In some examples, a polynucleic acid molecule or binding moiety described herein is directly or indirectly attached to the nanoparticle. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleic acid molecules or binding moieties described herein are directly or indirectly attached to the nanoparticle.

[0290] In some embodiments, the pharmaceutical formulation comprises a delivery vector, e.g., a recombinant vector for delivery of a polynucleic acid molecule to a cell. In some instances, the recombinant vector is a DNA plasmid. In other instances, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. In some instances, the recombinant vector capable of expressing a polynucleic acid molecule provides stable expression in target cells. In a further example, a viral vector is used that provides transient expression of a polynucleic acid molecule.

[0291] In some embodiments, pharmaceutical formulations include a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier substances include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, humectants, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholate, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. 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 comprises a pH adjusting or buffering agent, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in the amount necessary to maintain the pH of the composition within an acceptable range.

[0293] In some instances, the pharmaceutical formulation contains one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

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

[0295] In some cases, pharmaceutical formulations contain disintegration agents or disintegrants to promote the break-up or disintegration of materials. The term "disintegrate" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starch, such as 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, methyl crystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Min Tia®, and Solka-Floc®), methylcellulose, croscarmellose, or crosslinked cellulose, e.g., crosslinked starch such as crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®), crosslinked carboxymethylcellulose, or crosslinked croscarmellose, crosslinked polymers such as sodium starch glycolate, crospovidone, crosslinked polyvinylpyrrolidone, alginates, e.g., alginic acid or salts of alginic acid such as sodium alginate, clays such as Veegum® HV (magnesium aluminum silicate), gums such as agar, guar, locust bean, karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, natural sponge, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, etc.

[0296] In some instances, the pharmaceutical formulation comprises a filler such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0297] Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein to prevent, reduce, or inhibit adhesion or friction of materials. Typical lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as 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, e.g., 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 oil, surfactants, and the like.

[0298] Plasticizers include compounds used to soften microencapsulation materials or film coatings, thereby reducing their brittleness. Suitable plasticizers include, for example, polyethylene glycols such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers also function as dispersing or wetting agents.

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

[0300] Stabilizers include any antioxidant, buffer, acid, preservative, and like compounds.

[0301] Suspending agents include polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having a molecular weight of from about 300 to about 6000, from about 3350 to about 4000, or from about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose stearin. Acid acetate, polysorbate 80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth, gum arabic, guar gum, xanthan including xanthan gum, sugars, cellulosics such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, and the like.

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

[0303] Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0304] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, docusate sodium, sodium oleate, sodium lauryl sulfate, docusate sodium, triacetin, Tween 80, vitamin E TPGS, ammonium salts, and the like.

[0305] Treatment regimen In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic use. In some embodiments, the pharmaceutical compositions are administered once a day, twice a day, three times a day, or more. The pharmaceutical compositions are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, once every two months, once every three months, once every four months, once every five months, once every six months, or more. The pharmaceutical compositions are administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[0306] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at an interval (e.g., a first administration of a first pharmaceutical composition is on day 1, followed by at least 1, 2, 3, 4, 5, or more days before administration of at least a second pharmaceutical composition).

[0307] In some embodiments, two or more different pharmaceutical compositions are administered simultaneously.In some instances, two or more different pharmaceutical compositions are administered simultaneously.In some cases, two or more different pharmaceutical compositions are administered simultaneously without any interval between administrations.In other cases, two or more different pharmaceutical compositions are administered simultaneously consecutively with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, or 2 days between administrations.

[0308] Alternatively, the dose of the administered composition may be temporarily reduced or temporarily suspended for a specified period of time (i.e., a "drug holiday"). In some instances, the length of the drug holiday may vary between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can be 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0309] Once the patient's condition has improved, a maintenance dose is administered if necessary, after which the dosage and / or frequency of administration can be reduced, depending on the symptoms, to a level at which the improved disease, disorder, or condition is maintained.

[0310] In some embodiments, the amount of a given agent corresponding to such an amount will depend on factors such as the particular compound, the severity of the disease, the nature (e.g., body weight) of the subject or host requiring treatment, and the like, but will nevertheless be routinely determined by methods known in the art according to the particular circumstances surrounding the case, including, for example, the specific agent being administered, the route of administration, and the subject or host being treated. In some instances, the desired dosage is conveniently presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., two, three, four or more sub-doses per day.

[0311] Because of the large number of variables associated with any particular treatment regimen, the foregoing ranges are only suggestive, and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on many variables, including, but not limited to, the activity of the compound being used, the disease or condition being treated, the mode of administration, the requirements of the particular subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

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

[0313] Kits / Products In some embodiments, the present disclosure provides kits and products that can be used with one or more compositions and methods described herein. Such kits include a carrier, packaging, or container that is partitioned to contain one or more containers, such as vials, tubes, etc., each container containing one of the individual components for using the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is made of various materials, such as glass or plastic.

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

[0315] For example, a container contains a target nucleic acid molecule described herein. Such kits optionally include an identifying description or label or instructions for use in the methods described herein.

[0316] Kits typically include a label listing the contents and / or instructions for use, and a package insert with instructions for use. A set of instructions is also typically included.

[0317] In one embodiment, a label is on or associated with the container. In one embodiment, a label is attached to a container when letters, numbers, or other indicia forming the label are affixed to, molded into, or engraved into the container itself. A label is associated with a container when it is present in a receptacle or carrier that holds the container, for example, as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates how to use the contents, for example, in the manner described herein.

[0318] In some embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms comprising a compound provided herein. The pack may, for example, comprise metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accompanied by a notice attached to the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the drug form for human or animal administration. Such notice may, for example, be labeling approved by the U.S. Food and Drug Administration for prescription drugs or approved package inserts. In one embodiment, compositions comprising a compound provided herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated disease.

[0319] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter belongs. It is understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used within the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is non-limiting.

[0320] As used herein, ranges and amounts can be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

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

[0322] As used herein, the terms "individual," "subject," and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. No term is limited to situations characterized by the supervision (e.g., full-time or intermittent) of a health care professional (e.g., a physician, registered nurse, bedside nurse, physician assistant, nursing assistant, or hospice worker).

[0323] The term "therapeutically effective amount" refers to an amount of a polynucleic acid molecule conjugate sufficient to provide a desired therapeutic effect in a mammalian subject. In some cases, this amount is a single or multiple dose administered to a patient (e.g., a human) to treat, prevent, prevent the onset of, cure, delay, reduce the severity of, or ameliorate at least one symptom of a disorder or recurrent disorder, or to prolong the patient's survival beyond that expected in the absence of such treatment. Naturally, the dosage level of a particular polynucleic acid molecule conjugate used to provide a therapeutically effective amount will vary depending on the type of injury, the subject's age, weight, and sex, the subject's medical condition, the severity of the disease, the route of administration, and the particular inhibitor used. In some examples, a therapeutically effective amount of a polynucleic acid molecule conjugate is initially assessed using cell culture and animal models, as described herein. For example, IC50 values ​​determined using cell culture methods optionally serve as a starting point in animal models, while IC50 values ​​determined in animal models are optionally used to find therapeutically effective amounts in humans.

[0324] Skeletal, or voluntary, muscles are usually anchored to bones by tendons and are commonly used to effect skeletal movement, such as during locomotion or posture. Although some control of skeletal muscles is generally maintained as an involuntary reflex (e.g., postural muscles or the diaphragm), skeletal muscles can respond to conscious control. Smooth, 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 twitch") and type II (or "fast twitch"). Type I muscle fibers are densely packed with capillaries and rich in mitochondria and myoglobin, which give type I muscle tissue its characteristic red color. In some cases, type I muscle fibers carry more oxygen and use fat or carbohydrates for fuel to sustain aerobic activity. Type I muscle fibers contract for a longer period but with less force. Type II muscle fibers are further subdivided into three main subtypes (IIa, IIx, and IIb) that differ in the speed of contraction and the force generated. Type II muscle fibers contract fast and powerfully but fatigue rapidly, thus resulting in a very short, intense period of anaerobic activity before the muscle contraction becomes painful.

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

[0327] Cardiac muscle is also an involuntary muscle, but it is structurally very similar to skeletal muscle and is found only in the heart. Cardiac and skeletal muscles are striated in that they contain sarcomeres packed into highly ordered bundles. In contrast, the myofibrils of smooth muscle cells are not arranged in sarcomeres and therefore are not striated.

[0328] Muscle cells include any cell that contributes to muscle tissue. Exemplary muscle cells include myoblasts, satellite cells, myotubes, and myofibrillar tissue.

[0329] As used herein, muscle strength is proportional to cross-sectional area (CSA), and muscle velocity is proportional to muscle fiber length. Therefore, comparison of cross-sectional area and muscle fiber size between various muscle types can provide an indication of muscle atrophy. Various methods for measuring muscle strength and muscle mass 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. Computed transverse tomography (CT) for generating cross-sectional images of selected muscle tissue and sonographic evaluation are additional methods for measuring muscle strength. [Example]

[0330] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims.

[0331] Example 1. Antibody siRNA conjugates DMPK-AOC is an antibody-siRNA conjugate drug product formed by conjugation of a humanized IgG1 antibody (anti-human transferrin receptor antibody) targeting human transferrin receptor 1 and one double-stranded siRNA oligonucleotide (DMPK siRNA) targeting DMPK mRNA (Figure 1). An SMCC maleimide linker is located at the 5' end of the passenger strand, which is conjugated to the antibody via one cysteine ​​in the antibody amino acid sequence. The conjugate binds to the human transferrin receptor on the cell surface, is internalized, and delivers the siRNA oligonucleotide to the intracellular compartment. Once internalized, 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 create DMPK-AOC are produced using well-established manufacturing processes by a commercial, GMP-compliant contract development and manufacturing organization (CDMO). The anti-human transferrin receptor antibody is produced 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 that is also conjugated with an SMCC linker attached to the 5' end of the passenger strand. Each of these is fully characterized and officially released. DMPK-AOC is produced using a standard random cysteine ​​bioconjugation reaction of the anti-human transferrin receptor antibody with DMPK siRNA using maleimide, followed by anion exchange chromatography purification to isolate the bulk conjugate, which is then converted to the finished DMPK-AOC. The finished DMPK-AOC is then officially released using standard methodologies for protein therapeutics. Once manufacturing, testing, and release are complete, the antibody and DMPK siRNA are each bioconjugated to form a drug substance.

[0333] Example 2. Production of antibody AV01mAb Cell Bank - A stable research cell bank (RCB) of stable cell lines was constructed using CHOK1SV host working cells and confirmed to be free of mycoplasma, bacteria, mold, and yeast contamination. A 200-vial master cell bank (MCB) was prepared using vials from the research cell bank.

[0334] Antibody production from the master cell bank - Cells from an ampoule of the master cell bank were gradually increased in volume using protein-free medium before inoculating the production bioreactor. Downstream processing - Upon completion of cell culture, 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 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 duplex oligonucleotide containing a 19-mer passenger strand and a complementary 21-mer guide strand with a two-nucleotide overhang at the 3' end of the guide strand. A C6-SMCC linker {4-(N-maleimidomethyl)cyclohexane-1-carboxamide} is attached to the 5' end of the passenger strand, allowing for conjugation to an antibody intermediate. The nucleotide sequence and internucleotide linkages are shown in Table 11.

[0339] [Table 11]

[0340] The single-stranded RNAs (guide and passenger strands) are each generated separately via solid-phase synthesis using the well-established phosphoramidite solid-phase synthesis method. The purified and lyophilized single strands are then duplexed in an equimolar ratio to generate double-stranded siRNA. An 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: Antibody Selection 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, confirming cross-species reactivity. A mouse IgG2a monoclonal antibody (mAb) that binds to both human and cynomolgus monkey TfR1 was evaluated for specificity by demonstrating a lack of binding to the closely related transferrin receptor 2 (TfR2) by ELISA (Figure 3). While the commercially available anti-TfR2 antibody B-6 shows clear binding to TfR2 by ELISA, mouse anti-human TfR1 mAb does not bind to TfR2 even at concentrations up to 10 mM. The mouse anti-human TfR1 mAb was also evaluated for binding in the presence of the TfR1-binding ligands transferrin (Tf) and homeostatic iron regulator (HFE). The TfR1 mAb maintained strong binding to TfR1 even in the presence of TfR1 ligands (Figure 4). As shown in Figure 4, the antibody either bound directly to TfR1 or to TfR1 pre-bound to the cofactors transferrin (Tf) or HFE. AF2474 is a commercially available antibody known to bind to the same TfR1 epitope as transferrin or HFE. While the mouse anti-human TfR1 mAb shows some loss of binding during direct interaction with TfR1 compared to the cofactor complex, the change in affinity is minimal compared to AF2474. Importantly, competition of the TfR1 mAb with the natural ligand for TfR1 is expected to be toxic, potentially blocking iron uptake into cells. Therefore, the identified TfR1 mAb must bind to an epitope on TfR1 that minimizes competition with the natural ligand. Considering that it fulfilled all these selection criteria, we proceeded to a humanization program for the mouse IgG2a anti-human TfR1 mAb to develop an antibody suitable for clinical development.

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

[0343] A mouse cross-reactive DMPK-AOC was conjugated to an anti-mouse TfR1 mAh, and the conjugate was administered to wild-type female CD-1 mice (n = 4 per group) at 3 mg / kg by IV injection (based on siRNA weight). Tissues were harvested, and DMPK mRNA knockdown was assessed weekly for up to 5 weeks post-administration (Figure 6). Maximum DMPK knockdown (approximately 75%) was achieved in skeletal muscle between days 7 and 35 post-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 there. The long duration of activity of AOC after a single administration allows for less frequent dosing in patients.

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

[0345] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the disclosure described herein may be utilized in practicing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of the claims and their equivalents be covered thereby.

Claims

1. A conjugate, wherein the conjugate is (i) an anti-transferrin receptor antibody or its antigen-binding fragment, (ii) a siRNA molecule comprising a guide strand and a passenger strand, and (iii) a linker, Here, the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region including HCDR1 containing SEQ ID NO: 17, HCDR2 containing SEQ ID NO: 20, and HCDR3 containing SEQ ID NO:

19. Here, the anti-transferrin receptor antibody or its antigen-binding fragment includes a variable light chain (VL) region comprising LCDR1 containing SEQ ID NO: 22, LCDR2 containing SEQ ID NO: 23, and LCDR3 containing SEQ ID NO:

24. Here, the passenger strand of the siRNA includes SEQ ID NO: 1, and the guide strand of the siRNA includes SEQ ID NO: 2, or here, the passenger strand of the siRNA includes SEQ ID NO: 3, and the guide strand of the siRNA includes SEQ ID NO: 4, and Here, the anti-transferrin receptor antibody or its antigen-binding fragment is bound to the end of the guide chain or the passenger chain via the linker. Conjugate.

2. A conjugate, wherein the conjugate is (i) an anti-transferrin receptor antibody or its antigen-binding fragment, (ii) siRNA comprising a guide strand and a passenger strand, and (iii) a linker, Here, the anti-transferrin receptor antibody or its antigen-binding fragment comprises the variable heavy chain (VH) sequence of SEQ ID NO: 30 and the variable light chain (VL) sequence of SEQ ID NO:

34. Here, the passenger strand of the siRNA includes SEQ ID NO: 1, and the guide strand of the siRNA includes SEQ ID NO: 2, or here, the passenger strand of the siRNA includes SEQ ID NO: 3, and the guide strand of the siRNA includes SEQ ID NO: 4, and Here, the anti-transferrin receptor antibody or its antigen-binding fragment is bound to the end of the guide chain or the passenger chain via the linker. Conjugate.

3. The conjugate according to claim 1 or claim 2, wherein the anti-transferrin receptor antibody or its antigen-binding fragment is a full-length anti-transferrin receptor antibody.

4. The conjugate according to claim 3, wherein the full-length anti-transferrin receptor antibody is a humanized anti-transferrin receptor antibody or a human anti-transferrin receptor antibody.

5. The conjugate according to claim 3 or claim 4, wherein the full-length anti-transferrin receptor antibody comprises a mutation selected from the group consisting of L233A, L234A, and L327R in the heavy chain constant region.

6. The conjugate according to claim 5, wherein the full-length anti-transferrin receptor antibody comprises L233A, L234A, and L327R mutations in the heavy chain constant region.

7. The conjugate according to claim 1 or claim 2, wherein the anti-transferrin receptor antibody or its antigen-binding fragment is selected from the group consisting of monovalent Fab', bivalent Fab 2, and single-chain variable fragments (scFv).

8. A conjugate, wherein the conjugate is (i) an anti-transferrin receptor antibody, (ii) a siRNA molecule including a guide strand and a passenger strand, and (iii) a linker, Here, the anti-transferrin receptor antibody comprises two heavy chains each containing SEQ ID NO: 48 and two light chains each containing SEQ ID NO:

63. Here, the passenger strand of the siRNA includes SEQ ID NO: 1, and the guide strand of the siRNA includes SEQ ID NO: 2, or here, the passenger strand of the siRNA includes SEQ ID NO: 3, and the guide strand of the siRNA includes SEQ ID NO: 4, and Here, the anti-transferrin receptor antibody is bound to the end of the guide strand or passenger strand of the siRNA via the linker. Conjugate.

9. The conjugate according to any one of claims 1 to 8, wherein the linker includes a non-polymer linker.

10. The conjugate according to any one of claims 1 to 9, wherein the linker includes an uncuttable linker.

11. The conjugate according to any one of claims 1 to 10, wherein the linker comprises a heterobifunctional crosslinking linker.

12. The conjugate according to any one of claims 1 to 11, wherein the linker includes a C6 linker.

13. The conjugate according to claim 12, wherein the C6 linker is a 6-amino-1-hexanol linker.

14. The linker is The conjugate according to any one of claims 1 to 13, comprising, where * indicates a binding site to a cysteine ​​residue of the anti-transferrin receptor antibody, and ** indicates a direct or indirect binding site to the 5' end of the passenger strand of the siRNA molecule.

15. The linker is The conjugate according to any one of claims 1 to 14, comprising, where * indicates a binding site to a cysteine ​​residue of the anti-transferrin receptor antibody, and ** indicates a direct or indirect binding site to the 5' end of the passenger strand of the siRNA molecule.

16. The conjugate according to any one of claims 1 to 15, wherein the drug-to-antibody ratio (DAR) of the siRNA molecule to the anti-transferrin receptor antibody or its antigen-binding fragment is about 1.

17. A pharmaceutical composition for treating muscular dystrophy, comprising a plurality of conjugates according to any one of claims 1 to 16 and a pharmaceutically acceptable excipient.

18. Use of the pharmaceutical composition according to claim 17 in the manufacture of a pharmaceutical for treating muscular dystrophy.

19. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is formulated for intravenous administration.

20. The use according to claim 18, wherein the pharmaceutical composition is formulated for intravenous administration.

21. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is formulated for subcutaneous administration.

22. The use according to claim 18, wherein the pharmaceutical composition is formulated for subcutaneous administration.

23. The use according to any one of claims 18, 20, or 22, wherein the muscular dystrophy is myotonic dystrophy type 1 (DM1).

24. The pharmaceutical composition according to any one of claims 17, 19, or 21, wherein the pharmaceutical composition is for treating muscular dystrophy.

25. The pharmaceutical composition according to claim 24, wherein the muscular dystrophy is myotonic dystrophy type 1 (DM1).

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