Compositions and methods for treating muscular dystrophy

Abstract

The invention relates to a composition and a method for treating muscular dystrophy. In particular, disclosed herein are polynucleic acid molecules, pharmaceutical compositions, and methods for treating muscular dystrophy (DM1).

Description

[0001] This application is a divisional application of Chinese patent application No. 202180038837.3, entitled "Composition and Method for Treating Muscular Dystrophy" (the corresponding PCT application was filed on March 26, 2021, with application number 202180038837.3). Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 001,211, filed March 27, 2020, which is incorporated herein by reference in its entirety. Background Technology

[0003] Gene repression via RNA-induced gene silencing offers several levels of control: transcriptional inactivation, small interfering RNA (siRNA)-induced mRNA degradation, and siRNA-induced transcriptional attenuation. In some cases, RNA interference (RNAi) provides durable effects on multiple cell divisions. Therefore, RNAi represents a viable approach for drug target validation, gene function analysis, pathway analysis, and disease treatment. Incorporation

[0004] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is expressly and individually indicated to be incorporated by reference. Summary of the Invention

[0005] In some embodiments, this document discloses polynucleotide molecules and pharmaceutical compositions for regulating genes associated with muscular dystrophy (e.g., DM1). In some embodiments, this document also describes methods for treating muscular atrophy using the polynucleotide molecules or polynucleotide conjugates disclosed herein.

[0006] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPK Hybridization to the target sequence. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand having a sequence with at least 80% identity to SEQ ID NO: 1, and the antisense strand having a sequence with at least 80% identity to SEQ ID NO: 2. The polynucleotide molecule conjugate mediates hybridization to the target sequence. DMPK RNA interference.

[0007] In some embodiments, the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence of SEQ ID NO: 19. In some embodiments, the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of one of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19. In some embodiments, the VL region 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 may or may not be present, or if present, it is F. In some embodiments, the VL region comprises the LCDR1 sequence including SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E, and X6 may or may not be present, or if present, it is F. In some embodiments, the VL region comprises the LCDR1 sequence including SEQ ID NO: 22 or SEQ ID NO: 27, the LCDR2 sequence including SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 28, and the LCDR3 sequence including SEQ ID NO: 24 or SEQ ID NO: 26. In some embodiments, the VH region includes the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region includes the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24. In some embodiments, the VH region includes the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region includes the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24.In some embodiments, the VH region includes the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region includes the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 25, and the LCDR3 sequence of SEQ ID NO: 26. In some embodiments, the VH region includes the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region includes the LCDR1 sequence of SEQ ID NO: 27, the LCDR2 sequence of SEQ ID NO: 28, and the LCDR3 sequence of SEQ ID NO: 26. In some embodiments, the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequences selected from SEQ ID NO: 29-33. In some embodiments, the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with sequences selected from SEQ ID NO: 34-38. In some embodiments, the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34.

[0008] In some embodiments, the anti-transferrin receptor antibody comprises a humanized antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment. In some embodiments, the anti-transferrin receptor antibody comprises IgG-scFv, nanobodies, BiTE, biantibodies, DART, TandAb, scDiabody, scDiabody-CH3, triantibodies, microantibodies, microantibodies, TriBi microantibodies, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intracellular antibodies. 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 the IgG2b framework. Alternatively, in some embodiments, the anti-transferrin receptor antibody comprises the IgG4 framework.

[0009] 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 regulates effector function, or attenuates or eliminates Fc-γ receptor binding. In some embodiments, the at least one mutation is located at residue positions D265, N297, K322, L328, or P329, wherein the residue positions refer 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, wherein the residues correspond 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 NO: 39-62 and a light chain (LC) sequence selected from SEQ ID NO: 63-66. In some embodiments, the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

[0010] In some embodiments, the sense strand and the antisense strand each independently comprise at least one 2'-modified nucleotide, at least one modified nucleotide linker, or at least one reverse abase-free moiety. In some embodiments, the sense strand comprises a 2'-O-methyl-modified nucleotide at its 5' end. Alternatively and / or additionally, the sense strand comprises at least two consecutive 2'-O-methyl-modified nucleotides at its 5' end. Alternatively and / or additionally, the sense strand comprises at least three, four, five, or six consecutive 2'-O-methyl-modified nucleotides at its 5' end. Alternatively and / or additionally, the sense strand comprises six consecutive 2'-O-methyl-modified nucleotides at its 5' end. 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 its 3' end. Alternatively and / or additionally, the sense strand comprises at least two consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or additionally, the sense strand comprises at least three, four, five, six, seven, eight, nine, or ten consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or additionally, the sense strand comprises ten consecutive 2'-O-methyl modified nucleotides at its 3' end. Alternatively and / or additionally, the sense strand comprises at least two phosphate thioester nucleotides linked together. Alternatively and / or additionally, the sense strand has the sequence SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15.

[0011] In some embodiments, the antisense strand comprises a 2'-O-methyl modified nucleotide at its 5' end. Alternatively and / or additionally, the antisense strand comprises a 2'-O-methyl modified nucleotide at its 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 its 3' end. Alternatively and / or additionally, the antisense strand comprises five consecutive 2'-O-methyl modified nucleotides at its 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, wherein any two of the four 2'-F modified nucleotides are not consecutive. Alternatively and / or additionally, the antisense strand comprises two overhanging nucleotides at its 3' end. Alternatively and / or additionally, the antisense strand comprises at least two or at least three phosphate thioester nucleotides linked together. Alternatively and / or additionally, the antisense strand has the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0012] In some embodiments, the polynucleotide conjugate includes a linker connecting an anti-transferrin receptor antibody or its antigen-binding fragment to the polynucleotide 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 homo- or hetero-bifunctional linker, a maleimide group, a dipeptide moiety, a benzoic acid group, or a derivative thereof. In some embodiments, the linker comprises 4-(N-maleimidomethyl)cyclohexane-1-amidate (SMCC). In some embodiments, the linker is coupled to the 5' end of the sense chain. In some embodiments, the polynucleotide molecule is conjugated to a cysteine ​​residue of the anti-transferrin receptor antibody or its antigen-binding fragment. In some embodiments, the cysteine ​​residue is located within the Fc domain of the anti-transferrin receptor antibody or its antigen-binding fragment. In some embodiments, the ratio of the polynucleic acid molecule to the anti-transferrin receptor antibody or its antigen-binding fragment is about 1:1, 2:1, 3:1 or 4:1.

[0013] In some implementations, the polynucleotide moiety mediates targeting of humans DMPKRNA interference with a gene regulates muscle atrophy in the target. In some embodiments, RNA interference includes reducing the expression of the DMPK gene mRNA transcript by at least 50%, at least 60%, or at least 70% compared to the amount of the DMPK gene mRNA transcript in cells affected by muscular dystrophy. In some embodiments, the muscular dystrophy is myotonic dystrophy type 1 (DM1).

[0014] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPK Hybridization to the target sequence. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15, the antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16, the antiferrin receptor antibody or its antigen-binding fragment comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence including SEQ ID NO: 17, the HCDR2 sequence including SEQ ID NO: 20, and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence including SEQ ID NO: 22, the LCDR2 sequence including SEQ ID NO: 23, and the SEQ ID NO: 24. The LCDR3 sequence of 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleotide molecule are conjugated by a linker comprising 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).

[0015] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPKHybridization to the target sequence. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15, the antisense strand having the sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16, and the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0016] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPK The target sequence is hybridized. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand has the sequence of SEQ ID NO: 1, the antisense strand has the sequence of SEQ ID NO: 2, the sense strand comprises at least three, four, five or six consecutive 2'-O-methyl modified nucleotides at the 5' end and at least two or three 2'-F modified nucleotides, the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleotide molecule are conjugated via a maleimide linker.

[0017] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPKHybridization of the target sequence. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1, and the antisense strand having the sequence of SEQ ID NO: 2. 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 and at least one, at least two, at least three, or at least four 2'-F-modified nucleotides. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19. The VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24. The anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleotide molecule are conjugated via a maleimide linker.

[0018] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPK Hybridization to the target sequence. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1, the antisense strand having the sequence of SEQ ID NO: 2, the antisense strand containing 2'-O-methyl modified nucleotides at the 5' and 3' ends, the anti-transferrin receptor antibody or its antigen-binding fragment comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleotide molecule are conjugated via a maleimide linker.

[0019] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPKThe target sequence is hybridized. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand has the sequence of SEQ ID NO: 1, the antisense strand has the sequence of SEQ ID NO: 2, the antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides at the 3' end and four 2'-F modified nucleotides, wherein any two of the four 2'-F modified nucleotides are not consecutive, and the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleotide molecule are conjugated via a 6-amino-1-hexanol linker.

[0020] In some embodiments, this document discloses a polynucleotide conjugate comprising an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, wherein the polynucleotide molecule is conjugated to... DMPK The target sequence is hybridized. The polynucleotide molecule has a sense strand and an antisense strand, the sense strand has the sequence of SEQ ID NO: 1, the antisense strand has the sequence of SEQ ID NO: 2, the antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides at the 3' end and four 2'-F modified nucleotides, wherein any two of the four 2'-F modified nucleotides are not consecutive, and the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34, and the anti-transferrin receptor antibody or its antigen-binding fragment and the polynucleotide molecule are conjugated via a 6-amino-1-hexanol linker.

[0021] In some embodiments, this document discloses a pharmaceutical composition comprising a polynucleic acid molecular conjugate as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, oral, rectal, or transdermal administration.

[0022] In some embodiments, this document discloses a method for treating muscular dystrophy in a subject of need, the method comprising providing the polynucleotide conjugate or pharmaceutical composition described herein and administering the polynucleotide conjugate to the subject of need to treat muscular dystrophy, wherein the polynucleotide conjugate reduces the body's... DMPK The amount of mRNA transcripts. In some implementations, the polynucleotide moiety mediates targeting human... DMPK RNA interference regulates muscle atrophy in the target. In some implementations, the muscular dystrophy is type 1 myotonic dystrophy (DM1).

[0023] In some embodiments, this document discloses the use of the polynucleotide conjugates or pharmaceutical compositions described herein for treating subjects diagnosed with or suspected of having myotonic dystrophy type 1 (DM1), or for manufacturing a medicament for treating subjects diagnosed with or suspected of having myotonic dystrophy type 1 (DM1). In some embodiments, this document discloses a kit comprising the polynucleotide conjugates or pharmaceutical compositions described herein. Attached Figure Description

[0024] The various aspects of this disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of this disclosure, and the accompanying drawings. This patent application document contains at least one color-drawn drawing. Upon request and payment of the necessary fees, the Patent Office will provide a copy of the published text of this patent application with color drawings.

[0025] Figure 1 A schematic diagram of the antibody-siRNA conjugate is shown.

[0026] Figure 2 A schematic structure of DMPK siRNA is shown.

[0027] Figure 3 A graph showing the binding of anti-TfR antibodies to TfR2 via ELISA is shown.

[0028] Figure 4 The diagram shows the binding of anti-TfR antibody to TfR1 in the presence of cofactor.

[0029] Figure 5 A graph showing the in vivo dose-response to AOC-mediated DMPK knockdown in mouse skeletal muscle is presented.

[0030] Figure 6 The graph shows the time progression of AOC-mediated DMPK knockdown in mouse tissue (left) and the change in siDMPK.36 concentration in mouse tissue over time (right).

[0031] Figure 7 A graph showing AOC-mediated DMPK knockdown in cynomolgus monkey skeletal muscle over a time course up to 12 weeks after administration is presented. Detailed Implementation

[0032] DM1 is a rare, single-gene, autosomal dominant, repetitive expansion disorder, clinically determined to affect approximately 1 in 8,000 people in the United States. However, a recent genetic study estimated the prevalence of DM1 in the US to be 1 in 2,532. DM1 is caused by the amplification of the CTG triplet repeat found in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The amplification ranges from <35 in healthy individuals to thousands in DM1 patients. When the mutated DMPK gene is translated into mRNA, the self-complementary CTG repeat sequence induces the formation of a large hairpin loop, trapping the DMPK mRNA in the cell nucleus and conferring toxicity acquisition function. The toxicity is not due to the conversion of the mRNA into a toxic protein, but rather to the presence of high concentrations of the CTG repeat sequence in the cell nucleus, which acts as a trap for the key CTG-coupled protein, muscle blindlike protein 1 (MBNL1). By binding to the DMPK CUG repeat sequence remaining in the nucleus, MBNL1 is isolated in the nucleus and unable to perform its normal function of guiding mRNA processing. As a result, multiple mRNAs encoding key proteins are misprocessed. The resulting atypical proteins translated from these misspliced ​​mRNAs are the ultimate cause of the disease's phenotypic characteristics.

[0033] Nucleic acid (e.g., RNAi) therapy is a highly selective and specific targeted therapy. However, in some cases, nucleic acid therapy is also hampered by poor intracellular uptake, limited blood stability, and nonspecific immune stimulation. To address these issues, various modifications to nucleic acid compositions have been explored, such as novel linkers for better stability and / or reduced toxicity, optimization of binding moieties for improved target specificity and / or target delivery, and nucleic acid polymer modifications for improved stability and / or reduced off-target effects.

[0034] In some embodiments, the arrangement or sequence of the different components constituting the nucleic acid composition further affects intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation. For example, if the nucleic acid component includes a binding moiety, a polymer, and a polynucleotide molecule (or polynucleotide), the order or arrangement of the binding moiety, polymer, and / or polynucleotide molecule (or polynucleotide) (e.g., binding moiety-polynucleotide molecule-polymer, binding moiety-polymer-polynucleotide molecule, or polymer-binding moiety-polynucleotide molecule) further affects intracellular uptake, stability, toxicity, efficacy, and / or nonspecific immune stimulation.

[0035] In some embodiments, the description herein includes polynucleotide molecules and polynucleotide conjugates for the treatment of muscular dystrophy. In some cases, the polynucleotide conjugates described herein enhance intracellular uptake, stability, and / or therapeutic efficacy. In some cases, the polynucleotide conjugates comprise an anti-transferrin receptor antibody or its antigen-binding fragment conjugated to a polynucleotide molecule, which... DMPK Hybridization with the target sequence. In some cases, polynucleotide conjugates include molecules of formula (I): A-X1-B.

[0036] Other implementation methods described herein include methods for treating muscular dystrophy, including administering the polynucleotide molecules or polynucleotide conjugates described herein to a subject.

[0037] Polynucleic acid molecules In some embodiments, the polynucleotide molecule hybridizes with the target sequence of a muscular dystrophy-related gene. Preferably, among muscular dystrophy-related genes, the polynucleotide molecule described herein hybridizes with the myotonic dystrophy protein kinase gene (MDK). DMPK Hybridization of the target sequence of the gene (also known as DM, DM1, DM1PK, DMK, MDPK, MT-PK, Dm15, dystrophic protein kinase, DM1 protein kinase gene).

[0038] In some embodiments, the polynucleotide molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 1. In some embodiments, the polynucleotide molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 2. In some embodiments, the polynucleotide molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15. In some embodiments, the polynucleotide molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0039] In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 1. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 2. In some cases, the polynucleotide molecule comprises both a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 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 with a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16.

[0040] In some embodiments, the polynucleotide molecule comprises a sense strand (e.g., a guest strand) and an antisense strand (e.g., a guide strand). In some cases, the sense strand (e.g., the guest 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 with 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 with a sequence selected from SEQ ID NO: 2. In some embodiments, the polynucleotide molecule comprises a sense strand (e.g., a guest strand) and an antisense strand (e.g., a guide strand). In some cases, the sense strand (e.g., the transient strand) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15. In some cases, the antisense strand (e.g., the guide strand) contains a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16. Table 1 presents the nucleic acid sequences and modified sequences of SEQ ID NO: 1-16.

[0041] Table 1

[0042] mX = 2'-O-methylribonucleoside fX = 2'-Fluoronucleotide -(s)- = Phosphothioester nucleotide linker - = Phosphodiester nucleotide linker In some embodiments, the polynucleotide molecules described herein comprise RNA, DNA, or PMO. In some cases, the polynucleotide molecules comprise RNA. In some cases, the RNA includes short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterologous nuclear RNA (hnRNA). In some cases, the RNA includes shRNA. In some cases, the RNA includes miRNA. In some cases, the RNA includes dsRNA. In some cases, the RNA includes tRNA. In some cases, the RNA includes rRNA. In some cases, the RNA includes hnRNA. In some cases, the RNA includes siRNA. In some cases, the polynucleotide molecules comprise siRNA.

[0043] In some embodiments, the length of the polynucleotide molecule is about 8 to about 50 nucleotides. In some embodiments, the length of the polynucleotide molecule is about 10 to about 50 nucleotides. In some cases, the length of the polynucleotide 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.

[0044] In some implementations, the polynucleotide molecule is approximately 50 nucleotides long. In some cases, the polynucleotide molecule is approximately 45 nucleotides long. In some cases, the polynucleotide molecule is approximately 40 nucleotides long. In some cases, the polynucleotide molecule is approximately 35 nucleotides long. In some cases, the polynucleotide molecule is approximately 30 nucleotides long. In some cases, the polynucleotide molecule is approximately 25 nucleotides long. In some cases, the polynucleotide molecule is approximately 20 nucleotides long. In some cases, the polynucleotide molecule is approximately 19 nucleotides long. In some cases, the polynucleotide molecule is approximately 18 nucleotides long. In some cases, the polynucleotide molecule is approximately 17 nucleotides long. In some cases, the polynucleotide molecule is approximately 16 nucleotides long. In some cases, the polynucleotide molecule is approximately 15 nucleotides long. In some cases, the polynucleotide molecule is approximately 14 nucleotides long. In some cases, the polynucleotide molecule is approximately 13 nucleotides long. In some cases, the polynucleotide molecule is approximately 12 nucleotides long. In some cases, the polynucleotide molecule is approximately 11 nucleotides long. In some cases, the length of a polynucleotide molecule is about 10 nucleotides. In some cases, the length is about 8 nucleotides. In some cases, the length is between about 8 and about 50 nucleotides. In some cases, the length is between about 10 and about 50 nucleotides. In some cases, the length is between about 10 and about 45 nucleotides. In some cases, the length is between about 10 and about 40 nucleotides. In some cases, the length is between about 10 and about 35 nucleotides. In some cases, the length is between about 10 and about 30 nucleotides. In some cases, the length is between about 10 and about 25 nucleotides. In some cases, the length is between about 10 and about 20 nucleotides. In some cases, the length is between about 15 and about 25 nucleotides. In some cases, the length is between about 15 and about 30 nucleotides. In some cases, the length of polynucleotide molecules is between about 12 and about 30 nucleotides.

[0045] In some implementations, the polynucleotide molecule comprises a first polynucleotide. In some cases, the polynucleotide molecule comprises a second polynucleotide. In some cases, the polynucleotide molecule comprises both a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide is a sense strand or a transit strand. In some cases, the second polynucleotide is an antisense strand or a guide strand.

[0046] In some embodiments, the polynucleotide molecule is the first polynucleotide. In some embodiments, the length of the first polynucleotide is about 8 to about 50 nucleotides. In some embodiments, the length of the first polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of 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.

[0047] In some cases, the length of the first polynucleotide is approximately 50 nucleotides. In some cases, the length of the first polynucleotide is approximately 45 nucleotides. In some cases, the length of the first polynucleotide is approximately 40 nucleotides. In some cases, the length of the first polynucleotide is approximately 35 nucleotides. In some cases, the length of the first polynucleotide is approximately 30 nucleotides. In some cases, the length of the first polynucleotide is approximately 25 nucleotides. In some cases, the length of the first polynucleotide is approximately 20 nucleotides. In some cases, the length of the first polynucleotide is approximately 19 nucleotides. In some cases, the length of the first polynucleotide is approximately 18 nucleotides. In some cases, the length of the first polynucleotide is approximately 17 nucleotides. In some cases, the length of the first polynucleotide is approximately 16 nucleotides. In some cases, the length of the first polynucleotide is approximately 15 nucleotides. In some cases, the length of the first polynucleotide is approximately 14 nucleotides. In some cases, the length of the first polynucleotide is approximately 13 nucleotides. In some cases, the length of the first polynucleotide is approximately 12 nucleotides. In some cases, the length of the first polynucleotide is approximately 11 nucleotides. In some cases, the length of the first polynucleotide is about 10 nucleotides. In some cases, the length of the first polynucleotide is about 8 nucleotides. In some cases, the length of the first polynucleotide is between about 8 and about 50 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 50 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 45 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 40 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 35 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 30 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 25 nucleotides. In some cases, the length of the first polynucleotide is between about 10 and about 20 nucleotides. In some cases, the length of the first polynucleotide is between about 15 and about 25 nucleotides. In some cases, the length of the first polynucleotide is between about 15 and about 30 nucleotides. In some cases, the length of the first polynucleotide is between about 12 and about 30 nucleotides.

[0048] In some embodiments, the polynucleotide molecule is a second polynucleotide. In some embodiments, the length of the second polynucleotide is about 8 to about 50 nucleotides. In some embodiments, the length of the second polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of 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.

[0049] In some cases, the length of the second polynucleotide is approximately 50 nucleotides. In some cases, the length of the second polynucleotide is approximately 45 nucleotides. In some cases, the length of the second polynucleotide is approximately 40 nucleotides. In some cases, the length of the second polynucleotide is approximately 35 nucleotides. In some cases, the length of the second polynucleotide is approximately 30 nucleotides. In some cases, the length of the second polynucleotide is approximately 25 nucleotides. In some cases, the length of the second polynucleotide is approximately 20 nucleotides. In some cases, the length of the second polynucleotide is approximately 19 nucleotides. In some cases, the length of the second polynucleotide is approximately 18 nucleotides. In some cases, the length of the second polynucleotide is approximately 17 nucleotides. In some cases, the length of the second polynucleotide is approximately 16 nucleotides. In some cases, the length of the second polynucleotide is approximately 15 nucleotides. In some cases, the length of the second polynucleotide is approximately 14 nucleotides. In some cases, the length of the second polynucleotide is approximately 13 nucleotides. In some cases, the length of the second polynucleotide is approximately 12 nucleotides. In some cases, the length of the second polynucleotide is approximately 11 nucleotides. In some cases, the length of the second polynucleotide is about 10 nucleotides. In some cases, the length of the second polynucleotide is about 8 nucleotides. In some cases, the length of the second polynucleotide is between about 8 and about 50 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 50 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 45 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 40 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 35 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 30 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 25 nucleotides. In some cases, the length of the second polynucleotide is between about 10 and about 20 nucleotides. In some cases, the length of the second polynucleotide is between about 15 and about 25 nucleotides. In some cases, the length of the second polynucleotide is between about 15 and about 30 nucleotides. In some cases, the length of the second polynucleotide is between about 12 and about 30 nucleotides.

[0050] In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the polynucleotide molecule further comprises blunt ends, overhangs, or combinations thereof. In some cases, the blunt end is a 5' blunt end, a 3' blunt end, or both. In some cases, the overhang is a 5' overhang, a 3' overhang, or both. In some cases, the overhang contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the overhang contains 1 non-base-paired nucleotide. In some cases, the overhang contains 2 non-base-paired nucleotides. In some cases, the overhang contains 3 non-base-paired nucleotides. In some cases, the overhang contains 4 non-base-paired nucleotides. In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises two non-base-paired nucleotides as 3' overhangs, while the sense strand has no overhangs. Optionally, in such embodiments, the non-base-paired nucleotides have a sequence of TT, dTdT, or UU. In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand has one or more nucleotides complementary to the antisense sequence at the 5' end.

[0051] In some embodiments, the sequence of the polynucleotide molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 50% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 60% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 70% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 80% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 90% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 95% complementary to the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule is at least 99% complementary to the target sequence described herein. In some cases, the sequence of the polynucleotide molecule is 100% complementary to the target sequence described herein.

[0052] In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with the target sequence described herein. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has three or fewer mismatches with the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has two or fewer mismatches with the target sequence described herein. In some cases, the sequence of the polynucleotide molecule has one or fewer mismatches with the target sequence described herein.

[0053] In some implementations, the specificity of the polynucleotide molecule hybridizing with the target sequence described herein is that the polynucleotide molecule has 95%, 98%, 99%, 99.5%, or 100% sequence complementarity with the target sequence. In some cases, the hybridization is performed under highly stringent conditions.

[0054] In some implementations, polynucleotide molecules exhibit reduced off-target effects. In some cases, "off-target" or "off-target effect" refers to any situation where a polynucleotide polymer targeting a given target causes an unintended effect through direct or indirect interaction with another mRNA sequence, DNA sequence, or cellular protein or other part. In some cases, an "off-target effect" occurs when other transcripts are simultaneously degraded due to partial homology or complementarity between the sense and / or antisense strands of the polynucleotide molecule.

[0055] In some embodiments, the polynucleotide molecule comprises natural or synthetic or artificial nucleotide analogs or bases. In some cases, the polynucleotide molecule comprises a combination of DNA, RNA, and / or nucleotide analogs. In some cases, the synthetic or artificial nucleotide analogs or bases contain modifications at one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0056] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a modified nucleic acid at the 2' hydroxyl group of the ribose moiety. In some cases, the modification includes H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary amines), amides, ethers, esters, alcohols, and oxygen. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification includes an azo group, a ketone group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic group (e.g., imidazole, hydrazine, or hydroxyamino), an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide). In some cases, the alkyl moiety further comprises a heterosubstitution. In some cases, the carbon of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some cases, the heterocyclic substitution includes, but is not limited to, morpholino, imidazole, and pyrrolidine groups.

[0057] In some cases, the modification at the 2' hydroxyl group is either 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 2'-O-methyl modifications in adenosine molecules and 2'-O-methoxyethyl modifications in uridine molecules are shown below.

[0058] 2'-O-methyl-adenosine 2'-O-methoxyethyluridine In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, in which an extended amine group of the propyl linker binds the amine group to the 2' oxygen. In some cases, this modification neutralizes the total negative charge derived from the phosphate ester of the oligonucleotide molecule by introducing a positive charge from the amine group with each sugar, and improves cellular uptake properties due to its zwitterionic nature. An exemplary chemical structure of 2'-O-aminopropyl nucleotide phosphoramidide is shown below.

[0059]

[0060] 2'-O-aminopropyl nucleoside phosphoramidone In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), in which an oxygen molecule bound to the 2' carbon is linked to the 4' carbon via a methylene group, thereby forming a 2'- C ,4'- C-O-methylene linked bicyclic ribonucleotide monomers. An exemplary representation of the chemical structure of LNA is shown below. The representation on the left highlights the chemical linkage of the LNA monomer. The representation on the right highlights the locked 3'-endo-( ) furanose ring of the LNA monomer. 3 E) Conformation.

[0061]

[0062] LNA (Locked Nucleic Acid) In some cases, modifications at the 2' hydroxyl group include ethylidene nucleic acids (ENAs), such as 2'-4'-ethylidene-bridged nucleic acids, which lock the sugar conformation to a C3'-puckering conformation. ENAs are part of a class of modified nucleic acids that are bridged nucleic acids and also contain LNAs. Exemplary chemical structures of ENAs and bridged nucleic acids are shown below.

[0063]

[0064] In some embodiments, additional modifications at the 2' hydroxyl group include 2'-deoxy, 2'-deoxy-2'-fluorine, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA).

[0065] In some embodiments, the nucleotide analog comprises modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenine, 2-methyladenine, 3-methylcytidine, 6-methyluridine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 5-methylaminoethyluridine, 5-methyloxyuridine, and denitronucleotides such as 7-denitro-adenine. Glycosides, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio-based bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, braided glycoside, archapurin, naphthyl and substituted naphthyl, any O-alkylated and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-hydroxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as G-clamp nucleotides, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those with modified sugar moieties, as well as nucleotides having a non-ribosyl sugar or analogue. For example, in some cases, the sugar moieties are or are based on mannose, arabinose, pyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbon rings. The term nucleotide also includes universal bases known in the art. For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or muscarin.

[0066] In some embodiments, the nucleotide analogue further comprises morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoroN3-P5'-phosphamide, 1',5'-anhydrohexadiol nucleic acid (HNA), or combinations thereof. Morpholino or phosphodiamidomorpholino oligonucleotides (PMOs) comprise synthetic molecules whose structure mimics the structure of native nucleic acids but deviates from the normal sugar and phosphate ester structures. In some cases, the five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is linked by a phosphodiamid group instead of a phosphate ester group. In such cases, the skeletal alteration removes all positive and negative charges, allowing the neutral morpholino molecule to cross the cell membrane without the aid of a cell delivery agent, such as the one used by charged oligonucleotides.

[0067]

[0068] In some implementations, the peptide nucleic acid (PNA) does not contain sugar rings or phosphate ester linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating skeletal charge.

[0069]

[0070] In some embodiments, one or more modifications optionally occur at the internucleotide linker. In some cases, the modified internucleotide linker includes, but is not limited to, thiophosphates, dithiophosphates, methylphosphonates, 5'-alkylphosphonates, 5'-methylphosphonates, 3'-alkylphosphonates, boron trifluoridates, 3'-5' linked or 2'-5' linked boron phosphates and selenophosphates, triphosphates, thioalkyl phosphates, hydrophosphonates, alkylphosphonates, alkylthiophosphonates, arylthiophosphonates, selenophosphates, diselenophosphates, hypophosphite, phosphoramides, 3'-alkylphosphamides, aminoalkylphosphamides, thiophosphamides, etc. Piperazine phosphate, thioaniline phosphate, aniline phosphate, ketones, sulfones, sulfonamides, carbonates, carbamates, methylene hydrazine, methylene dimethyl hydrazine, methyl acetal, thiomethyl acetal, oxime, methylene imino, methylene methyl imino, thioamide, linkage with a ribose acetyl group, aminoethylglycine, silyl or siloxane linkage, linkage with an alkyl or cycloalkyl group of 1 to 10 carbons with or without heteroatoms (which is saturated or unsaturated and / or substituted and / or contains heteroatoms), linkage with a morpholino structure, amides, polyamides (wherein the base is directly or indirectly linked to a nitrogen atom in the main chain), and combinations thereof. Thiophosphate antisense oligonucleotides (PSASOs) are antisense oligonucleotides containing a thiophosphate linkage. Exemplary PS ASOs are shown below.

[0071]

[0072] In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate or a thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left) and methylphosphonate nucleotides (right) are shown below.

[0073]

[0074] In some cases, the modified nucleotides include, but are not limited to, 2'-fluoroN3-P5'-phosphoramide as shown below:

[0075] In some cases, the modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-dehydrated hexitol nucleic acids (HNA)) as shown below:

[0076] In some embodiments, one or more modifications may further optionally include modifications to the ribose moiety, phosphate backbone, and nucleoside, or modifications to a nucleotide analog at the 3' or 5' end. For example, the 3' end may optionally contain a 3' cationic group, or a reverse nucleoside linked at the 3' end in 3'-3'. In another alternative, the 3' end may optionally be conjugated to an aminoalkyl group, such as 3'C5-aminoalkyl dT. In yet another alternative, the 3' end may optionally be conjugated to a baseless site, such as a depurinyl or depyrimidine site. In some cases, the 5' end may be conjugated to an aminoalkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5' end may be conjugated to a baseless site, such as a depurinyl or depyrimidine site.

[0077] In some embodiments, the polynucleotide molecule comprises one or more of the artificial nucleotide analogs described herein. In some cases, the polynucleotide molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analogues include nucleotides modified 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-methylacetamido (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphamide, or combinations thereof. In some cases, polynucleotide molecules contain 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 following: 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- Nucleotides modified with dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoroN3-P5'-phosphinate, or combinations thereof. In some cases, polynucleotide molecules contain 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 cases, polynucleotide molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more nucleotides modified with 2'-O-methoxyethyl (2'-O-MOE).

[0078] In some cases, polynucleotide molecules contain 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.

[0079] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification.

[0080] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification.

[0081] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and about 60% to about 70% modification.

[0082] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification.

[0083] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification.

[0084] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification.

[0085] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 30% of modifications and about 20% to about 30% of modifications.

[0086] In some cases, polynucleotide molecules contain approximately 10% to 20% modifications.

[0087] In some cases, polynucleotide molecules contain about 15% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% of modifications.

[0088] In other cases, polynucleotide molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of modifications.

[0089] In some implementations, the polynucleotide molecule contains at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications.

[0090] In some cases, polynucleotide molecules contain at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides.

[0091] In some cases, approximately 5% to approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 5% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 10% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 15% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 20% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 25% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 30% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 35% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 40% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 45% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 50% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 55% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 60% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 65% ​​of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 70% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 75% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 80% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 85% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 90% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 95% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 96% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 97% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 98% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 99% of polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 100% of polynucleotide molecules contain the artificial nucleotide analogs described herein.In some embodiments, the artificial nucleotide analogues include nucleotides modified 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-methylacetamido (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphamide, or combinations thereof.

[0092] In some embodiments, the polynucleotide molecule comprises about 1 to about 25 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 1 modification, wherein the modification comprises the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 2 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 3 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 4 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 5 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 6 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 7 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 8 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 9 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 10 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 11 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 12 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 13 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 14 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 15 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 16 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 17 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 18 modifications, wherein the modifications comprise the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 19 modifications, wherein these modifications include the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 20 modifications, wherein these modifications include the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 21 modifications, wherein these modifications include the artificial nucleotide analogs described herein.In some embodiments, the polynucleotide molecule comprises about 19 modifications, wherein these modifications include the artificial nucleotide analogs described herein. In some embodiments, the polynucleotide molecule comprises about 22 modifications, wherein these modifications include the artificial nucleotide analogs described herein.

[0093] In some embodiments, the polynucleotide molecule is assembled from two separate polynucleotides, one polynucleotide containing a sense strand and the second polynucleotide containing an antisense strand of the polynucleotide molecule. In some embodiments, the polynucleotide molecule contains a sense strand and an antisense strand, and the pyrimidine nucleotide in the sense strand contains a 2'-O-methylpyrimidine nucleotide, and the purine nucleotide in the sense strand contains a 2'-deoxypurine nucleotide. In some embodiments, the polynucleotide molecule contains a sense strand and an antisense strand, and the pyrimidine nucleotide present in the sense strand contains a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide present in the sense strand contains a 2'-deoxypurine nucleotide.

[0094] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, and the pyrimidine nucleotide, when present in the antisense strand, is 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, when present in the antisense strand, is 2'-O-methylpurine nucleotide.

[0095] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, and the pyrimidine nucleotide, when present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, when present in the antisense strand, comprises a 2'-deoxy-purine nucleotide.

[0096] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand contains a 2'-O-methyl-modified nucleotide at its 5' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand contains at least two consecutive 2'-O-methyl-modified nucleotides at its 5' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand contains at least three, four, five, or six consecutive 2'-O-methyl-modified nucleotides at its 5' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand contains six consecutive 2'-O-methyl-modified nucleotides at its 5' end.

[0097] Alternatively and / or additionally, the polynucleotide 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 polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises at least two or at least three 2'-F modified nucleotides. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises at least two or at least three consecutive 2'-F modified nucleotides.

[0098] In some embodiments, the polynucleotide molecule includes a sense strand and an antisense strand, wherein the sense strand contains a 2'-O-methyl-modified nucleotide at its 3' end. Alternatively and / or additionally, the polynucleotide molecule includes a sense strand and an antisense strand, wherein the sense strand contains at least two consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or additionally, the polynucleotide molecule includes a sense strand and an antisense strand, wherein the sense strand contains at least three, four, five, six, seven, eight, nine, or ten consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or additionally, the polynucleotide molecule includes a sense strand and an antisense strand, wherein the sense strand contains ten consecutive 2'-O-methyl-modified nucleotides at its 3' end.

[0099] Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains a 2'-O-methyl-modified nucleotide at its 5' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains a 2'-O-methyl-modified nucleotide at its 3' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains at least two, at least three, at least four, or at least five consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains five consecutive 2'-O-methyl-modified nucleotides at its 3' end. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains at least one, at least two, at least three, or at least four 2'-F-modified nucleotides. Alternatively and / or additionally, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises four 2'-F modified nucleotides, any two of which are not consecutive. In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises two overhanging nucleotides at the 3' end.

[0100] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand. In some embodiments, the sense strand comprises a terminal cap portion at the 5' end, 3' end, or both the 5' and 3' ends. In other embodiments, the terminal cap portion is an inverted deoxygenated, baseless portion.

[0101] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains a phosphate backbone modification at its 3' end. In some cases, the phosphate backbone modification is a thiophosphate. In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, and the sense strand comprises at least two thiophosphate nucleotides linked together. Alternatively and / or additionally, the antisense strand comprises at least two, or at least three, thiophosphate nucleotides linked together.

[0102] In some implementations, the polynucleotide molecule contains a sense strand and an antisense strand, wherein the antisense strand contains a glycerol group modification at its 3' end.

[0103] In some embodiments, the polynucleotide 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 three, four, five or six consecutive 2'-O-methyl modified nucleotides at the 5' end and at least two or at least three 2'-F modified nucleotides.

[0104] In some embodiments, the polynucleotide 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 the 3' end and at least one, at least two, at least three, or at least four 2'-F modified nucleotides.

[0105] In some embodiments, the polynucleotide 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 nucleotides modified with 2'-O-methyl at the 5' and 3' ends.

[0106] In some embodiments, the polynucleotide 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 at the 3' end and four 2'-F modified nucleotides, wherein any two of the four 2'-F modified nucleotides are not consecutive.

[0107] In some embodiments, the polynucleotide 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 and / or antisense strand comprises 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% of the modified nucleotides of the corresponding sequences of SEQ ID NO: 3 and / or SEQ ID NO: 4, respectively.

[0108] In some cases, one or more artificial nucleotide analogs described herein are resistant to the following nucleases compared to natural polynucleotide molecules: for example, ribonucleases (e.g., RNase H), deoxyribonucleases (e.g., DNase), or exonucleases (e.g., 5'-3' exonucleases and 3'-5' exonucleases). In some cases, artificial nucleotide analogs containing 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-methylacetamido (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphamide, or combinations thereof, are resistant to the following nucleases: for example, ribonucleases (e.g., RNase). RNase H), deoxyribonuclease (e.g., DNase), or exonuclease (e.g., 5'-3' and 3'-5' exonucleases). In some cases, 2'-O-methyl modified polynucleotides exhibit nuclease resistance (e.g., RNase H, DNase, 5'-3' or 3'-5' exonuclease resistance). In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides exhibit nuclease resistance (e.g., RNase H, DNase, 5'-3' or 3'-5' exonuclease resistance). In some cases, 2'-O-aminopropyl modified polynucleotides exhibit nuclease resistance (e.g., RNase H, DNase, 5'-3' or 3'-5' exonuclease resistance). In some cases, 2'-deoxy-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-deoxy-2'-fluorinated polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl (2'-O-AP) modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE) exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleotide molecules modified with 2'-ON-methylacetamide (2'-O-NMA) exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, LNA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, morpholinonucleotides exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate nucleotide-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiolphosphonate nucleotide-modified polynucleotide molecules exhibit nuclease resistance (e.g., resistance to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramide exhibit nuclease resistance (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistance). In some cases, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some cases, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0109] In some implementations, one or more artificial nucleotide analogs described herein have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. Nucleotides containing 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-methylacetamido (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, or 2'-fluoroN3-P5'-phosphinoamide have increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. In some cases, 2'-O-methyl-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, 2'-O-aminopropyl-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, 2'-deoxy-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, 2'-deoxy-2'-fluoro-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, polynucleotide molecules modified with 2'-ON-methylacetamido (2'-O-NMA) exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides.In some cases, LNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, ENA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, PNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, HNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, morpholino-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, methylphosphonate nucleotide-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, thiol phosphonate nucleotide-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to their equivalent natural polynucleotides. In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramide exhibit increased binding affinity to their mRNA targets relative to equivalent native polynucleotide molecules. In some cases, this increased affinity is described by lower Kd, higher melting temperature (Tm), or a combination thereof.

[0110] In some embodiments, the polynucleotide molecules described herein are chiral (or stereopure) polynucleotide molecules, or polynucleotide molecules comprising a single enantiomer. In some cases, the polynucleotide molecule comprises an L-nucleotide. In some cases, the polynucleotide molecule comprises a D-nucleotide. In some cases, the polynucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomers. In some cases, the polynucleotide molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some cases, the polynucleotide molecule is the polynucleotide molecule described in U.S. Patent Publications 2014 / 194610 and 2015 / 211006 and PCT Publication WO2015107425.

[0111] In some embodiments, the polynucleotide molecules described herein are further modified to include an aptamer conjugate portion. In some cases, the aptamer conjugate portion is a DNA aptamer conjugate portion. In some cases, the aptamer conjugate portion is an alphamer (Centauri Therapeutics) that includes an aptamer portion that recognizes a specific cell surface target and a portion that presents a specific epitope for linking to a circulating antibody. In some cases, the polynucleotide molecules described herein are further modified to include an aptamer conjugate portion as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0112] In other embodiments, the polynucleotide molecules described herein are modified to increase their stability. In some embodiments, the polynucleotide molecule is RNA (e.g., siRNA). In some cases, the polynucleotide molecule is modified by one or more of the above modifications to increase its stability. In some cases, the polynucleotide molecule is modified at the 2' hydroxyl position, for example by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, 2'-deoxy-2'-fluorine, 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-methylacetamido (2'-O-NMA) modification or by a locked or bridged ribose conformation (e.g., LNA or ENA). In some cases, polynucleotide molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, polynucleotide molecules also include morpholino, PNA, HNA, methylphosphonate nucleotides, thiophosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphamide to increase their stability. In some cases, the polynucleotide molecules are chiral (or stereopure) polynucleotide molecules. In some cases, chiral (or stereopure) polynucleotide molecules are modified to increase their stability. Appropriate modifications to RNA to increase delivery stability are readily apparent to those skilled in the art.

[0113] In some cases, a polynucleotide molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region contains a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, a polynucleotide molecule is assembled from two separate polynucleotides, one strand being a sense strand and the other an antisense strand, wherein the antisense and sense strands are self-complementary (e.g., each strand contains a nucleotide sequence complementary to a nucleotide sequence in the other strand; e.g., where the antisense and sense strands form a double helix or double-stranded structure, e.g., where the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand contains a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule, and the sense strand contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, a polynucleotide molecule is assembled from a single oligonucleotide, wherein the self-complementary sense and antisense regions of the polynucleotide molecule are linked by a nucleic acid-based or non-nucleic acid-based linker.

[0114] In some cases, polynucleotide molecules are polynucleotides with double strands, asymmetric double strands, hairpins, or asymmetric hairpin secondary structures, possessing self-complementary sense and antisense regions. The antisense region contains a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in a separate target nucleic acid molecule, and the sense region has a nucleotide sequence corresponding to or partially corresponding to the target nucleic acid sequence. In other cases, polynucleotide molecules are cyclic single-stranded polynucleotides having two or more loop structures and a stem containing self-complementary sense and antisense regions. The antisense region contains a nucleotide sequence complementary to or partially complementary to a nucleotide sequence in a target nucleic acid molecule, and the sense region has a nucleotide sequence corresponding to or partially corresponding to the target nucleic acid sequence. The cyclic polynucleotide is processed in vivo or in vitro to generate an active polynucleotide molecule capable of mediating RNAi. In other cases, polynucleotide molecules also contain single-stranded polynucleotides having a nucleotide sequence complementary to or partially complementary to the nucleotide sequence in the target nucleic acid molecule (e.g., when the polynucleotide molecule does not require the presence of a nucleotide sequence corresponding to or partially corresponding to the target nucleic acid sequence within the polynucleotide molecule), wherein the single-stranded polynucleotide further comprises a terminal phosphate group, such as 5'-phosphate (see, for example, Martinez et al., 2002). Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell 10, 537-568) or 5',3'-bisphosphonic acid.

[0115] In some cases, asymmetric hairpins are linear polynucleotide molecules containing an antisense region, a loop portion containing nucleotides or nonnucleotides, and a sense region containing fewer nucleotides than the antisense region, such that the sense region has enough complementary nucleotides to base-pair with the antisense region and form a double helix with the loop. For example, an asymmetric hairpin polynucleotide molecule contains an antisense region (e.g., about 19 to about 22 nucleotides) long enough to mediate RNAi in a cellular or in vitro system, a loop region containing about 4 to about 8 nucleotides, and a sense region containing about 3 to about 18 nucleotides complementary to the antisense region. In some cases, asymmetric hairpin polynucleotide molecules also contain a chemically modified 5' phosphate group. In other cases, the loop portion of the asymmetric hairpin polynucleotide molecule contains nucleotides, nonnucleotides, linker molecules, or conjugate molecules.

[0116] In some implementations, an asymmetric duplex is a polynucleotide molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region, to such an extent that the sense region has sufficient complementary nucleotides to base-pair with the antisense region and form a duplex. For example, an asymmetric duplex polynucleotide molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cellular or in vitro system and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0117] In some cases, a universal base refers to a nucleotide base analog that forms a base pair with every natural DNA / RNA base, and there is little difference between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitrazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, for example, Loakes, 2001). Nucleic Acids Research, 29, 2437-2447).

[0118] Polynucleic acid molecule synthesis In some embodiments, the polynucleotide molecules described herein are constructed using procedures known in the art, employing chemical synthesis and / or enzymatic ligation reactions. For example, polynucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the double strand formed between the polynucleotide molecule and a target nucleic acid. Exemplary methods include those described in U.S. Patent Nos. 5,142,047; 5,185,444; 5,889,136; 6,008,400 and 6,111,086; PCT Publication No. WO2009099942; or European Publication No. 1579015. Additional exemplary methods include those described in: Griffey et al., “2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J Med. Chem . 39(26):5100-5109 (1997)); Obika et al. "Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclicnucleosides having a fixed C3, -endo sugar puckering". Tetrahedron Letters 38(50): 8735 (1997); Koizumi, M. "ENA oligonucleotides as therapeutics". Current opinion in molecular therapeutics8 (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, expression vectors are used to biologically generate polynucleotide molecules, in which the polynucleotide molecule has been subcloned into the expression vector in an antisense orientation (i.e., the RNA transcribed from the inserted polynucleotide molecule will be antisense to the target polynucleotide molecule of interest).

[0119] In some implementations, polynucleotide molecules are synthesized via a tandem synthesis approach, in which two strands are synthesized as single, continuous oligonucleotide fragments or chains separated by cleavable linkers, which are then cleaved to provide individual fragments or chains that hybridize and allow for the purification of the duplex.

[0120] In some cases, polynucleotide molecules are also assembled from two different nucleic acid chains or fragments, one of which includes a sense region and the second of which includes the antisense region of the molecule.

[0121] Other modification methods used for incorporation, such as sugar, base, and phosphate modifications, include: Eckstein et al., International Publication PCT No. WO 92 / 07065; Perrault et al., Nature 1990, 344, 565-568; Pieken et al. Science 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al., International Publication PCT No. WO 93 / 15187; Sproat, U.S. Patent 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., U.S. Patent No. 5,716,824; Usman et al., U.S. Patent No. 5,627,053; Woolf et al., International PCT Publication No. WO 98 / 13526; Thompson et al., U.S. Serial No. 60 / 082,404, filed April 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett.,39, 1131; Earnshaw and Gait, 1998, Biopolymers ( Nucleic Acid Sciences ), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999–2010. These publications describe general methods and strategies for determining the positions of sugar, base, and / or phosphate modifications incorporated into nucleic acid molecules without modulating catalysis.

[0122] In some cases, while chemical modifications to the internucleotide linkages of polynucleotide molecules using thiophosphates, dithiophosphates, and / or 5'-methylphosphonates improve stability, over-modification can sometimes lead to toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages is sometimes minimized. In such cases, reducing the concentration of these linkages results in reduced toxicity, increased efficacy, and improved specificity of the molecules.

[0123] Polynucleic acid conjugates In some embodiments, the polynucleotide molecule (B) is further conjugated to a polypeptide (A) for delivery to the site of interest. In some cases, at least one polypeptide A is conjugated to at least one B. In some cases, 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 on B, or any combination thereof. In some cases, at least one polypeptide A is conjugated to at least two Bs. In some cases, at least one polypeptide A is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more Bs.

[0124] In some embodiments, at least one polypeptide A is conjugated to one end of at least one B, while at least one C is conjugated to the opposite end of at least one B to form an ABC conjugate. In some cases, at least one polypeptide A is conjugated to one end of at least one B, while at least one of the Cs is conjugated at an internal or terminal site of at least one B. In some cases, at least one polypeptide A is directly conjugated to at least one C. In some cases, at least one B is indirectly conjugated to at least one polypeptide A via at least one C to form an ACB conjugate.

[0125] In some cases, at least one B and / or at least one C, and optionally at least one D, are conjugated to at least one polypeptide A. In some cases, at least one B is conjugated to at least one polypeptide A at its terminal (e.g., 5' or 3' end) or via an internal site. In some cases, at least one C is directly conjugated to at least one polypeptide A, or indirectly conjugated to at least one polypeptide A via at least one B. If conjugated indirectly via at least one B, then at least one C is conjugated to B at the same terminal as at least one polypeptide A, to at least one polypeptide A at the opposite terminal, or independently at an internal site. In some cases, at least one additional polypeptide A is further conjugated to at least one polypeptide A, B, or C. In other cases, at least one D is optionally conjugated directly or indirectly to at least one polypeptide A, at least one B, or at least one C. If directly conjugated to at least one polypeptide A, then at least one D is also optionally conjugated to at least one B to form an ADB conjugate, or optionally conjugated to at least one B and at least one C to form an ADBC ​​conjugate. In some cases, at least one D is directly conjugated to at least one polypeptide A and indirectly conjugated to at least one B and at least one C to form a DABC conjugate. If indirectly conjugated to at least one polypeptide A, at least one D may also optionally be conjugated to at least one B to form an ABD conjugate, or optionally conjugated to at least one B and at least one C to form an ABDC conjugate. In some cases, at least one additional D is further conjugated to at least one polypeptide A, B, or C.

[0126] Combined part In some embodiments, binding portion A is a polypeptide, peptide, or non-peptide ligand. In some cases, the polypeptide is an antibody or a fragment thereof. In some cases, the fragment is a binding fragment. In some cases, the antibody or its antigen-binding fragment comprises a humanized antibody or its antigen-binding fragment, a mouse antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a monoclonal antibody or its antigen-binding fragment, a monovalent Fab', a bivalent Fab2, an F(ab)'3 fragment, a single-chain variable fragment (scFv), a bis-scFv, (scFv)2, a biantibody, a microantibody, a nanobody, a triantibody, a tetraantibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camelid antibody or its antigen-binding fragment, a bispecific antibody or its binding fragment, or a chemically modified derivative thereof.

[0127] In some embodiments, binding portion A is a bispecific antibody or its antigen-binding fragment. In some cases, the bispecific antibody is a trifunctional antibody or a bispecific microantibody. In some cases, the bispecific antibody is a trifunctional antibody. In some cases, the trifunctional antibody is a full-length monoclonal antibody containing binding sites against two different antigens.

[0128] In some cases, bispecific antibodies are bispecific microantibodies. In some cases, these bispecific microantibodies include bivalent Fab2, F(ab)'3 fragments, biscFv, (scFv)2, biantibodies, microantibodies, triantibodies, tetraantibodies, or bispecific T-cell conjugates (BiTE). In some embodiments, the bispecific T-cell conjugate is a fusion protein comprising two single-chain variable fragments (scFv) that target epitopes of two different antigens.

[0129] In some embodiments, the binding portion A is a bispecific microantibody. In some cases, A is a bispecific Fab2. In some cases, A is a bispecific F(ab)'3 fragment. In some cases, A is a bispecific biscFv. In some cases, A is a bispecific (scFv)2. In some embodiments, A is a bispecific biantibody. In some embodiments, A is a bispecific microantibody. In some embodiments, A is a bispecific triantibody. In other embodiments, A is a bispecific tetraantibody. In other embodiments, A is a bispecific T-cell conjugate (BiTE).

[0130] In some embodiments, the binding portion A is a trispecific antibody. In some cases, the trispecific antibody comprises an F(ab)'3 fragment or a triantibody. In some cases, A is a trispecific F(ab)'3 fragment. In some cases, A is a triantibody. In some embodiments, A is a trispecific antibody, as in Dimas et al., “Development of atrispecific antibody designed to simultaneously and efficiently target threedifferent antigens on tumor cells,” Mol. Pharmaceutics As stated in , 12(9): 3490-3501(2015).

[0131] In some embodiments, binding moiety A is an antibody or an antigen-binding fragment thereof that recognizes a cell surface protein. In some cases, binding moiety A is an antibody or an antigen-binding fragment thereof that recognizes a cell surface protein on muscle cells. In some cases, binding moiety A is an antibody or an antigen-binding fragment thereof that recognizes a cell surface protein on skeletal muscle cells.

[0132] In some implementations, 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 cases, the antibody is an anti-transferrin receptor (anti-CD71) antibody.

[0133] In some embodiments, when the antibody is an anti-transferrin receptor (anti-CD71) antibody, the anti-transferrin receptor antibody specifically binds to the transferrin receptor (TfR), preferably specifically binds to transferrin receptor 1 (TfR1), or more preferably specifically binds to human transferrin receptor 1 (TfR1) (or human CD71).

[0134] In some cases, anti-transferrin receptor antibodies comprise 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, wherein X1 is selected from N or Q and X2 is selected from Q or E; and an HCDR3 sequence comprising SEQ ID NO: 19.

[0135] In some implementations, the VH region of the anti-transfer antibody contains HCDR1, HCDR2 and HCDR3 sequences selected from Table 2.

[0136] Table 2

[0137] *13E4_VH2 shares the same HCDR1, HCDR2, and HCDR3 sequences with the anti-transferrin receptor antibody 13E4_VH4. In some embodiments, the VH region comprises: the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence including SEQ ID NO: 18, 20, or 21; and the HCDR3 sequence including SEQ ID NO: 19. In some cases, the VH region comprises: the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence including SEQ ID NO: 18; and the HCDR3 sequence including SEQ ID NO: 19. In some cases, the VH region comprises: the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence including SEQ ID NO: 20; and the HCDR3 sequence including SEQ ID NO: 19. In some cases, the VH region comprises: the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence including SEQ ID NO: 21; and the HCDR3 sequence including SEQ ID NO: 19.

[0138] In some embodiments, the VL region of the anti-transferrin receptor antibody includes: 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, or if present, is F.

[0139] In some implementations, the VL region of the anti-transferrin receptor antibody contains sequences selected from LCDR1, LCDR2, and LCDR3 in Table 3.

[0140] Table 3

[0141] *13E4_VL1 shares the same LCDR1, LCDR2, and LCDR3 sequences as the anti-transferrin receptor antibody 13E4_VL2. In some cases, the VL region contains: the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence including SEQ ID NO: 23, 25 or 28, and the LCDR3 sequence including SEQ ID NO: 24 or 26, wherein X3 is selected from N or S.

[0142] In some cases, the VL region includes: the LCDR1 sequence comprising SEQ ID NO: 22 or 27, the LCDR2 sequence AX4TNLAX5, and the 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 VL region contains: an LCDR1 sequence including SEQ ID NO: 22 or 27, an LCDR2 sequence including SEQ ID NO: 23, 25 or 28, and an LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and if present, is F.

[0144] In some cases, the VL region contains: the LCDR1 sequence, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6 of SEQ ID NO: 22, wherein X5 is selected from D or E and X6 is present or absent, or F if present.

[0145] In some cases, the VL region includes: the LCDR1 sequence including SEQ ID NO: 22; the LCDR2 sequence including SEQ ID NO: 23; and the LCDR3 sequence including SEQ ID NO: 24.

[0146] In some cases, the VL region includes: the LCDR1 sequence including SEQ ID NO: 22; the LCDR2 sequence including SEQ ID NO: 25; and the LCDR3 sequence including SEQ ID NO: 26.

[0147] In some cases, the VL region includes: the LCDR1 sequence including SEQ ID NO: 27; the LCDR2 sequence including SEQ ID NO: 28; and the LCDR3 sequence including SEQ ID NO: 26.

[0148] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence of SEQ ID NO: 19; and the VL region 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, or if present, is F.

[0149] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence including SEQ ID NO: 23, 25 or 28 and the LCDR3 sequence including SEQ ID NO: 24 or 26, wherein X3 is selected from N or S.

[0150] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence AX4TNLAX5 and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X4 is selected from A or G and X5 is selected from D or E.

[0151] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence including SEQ ID NO: 22 or 27, the LCDR2 sequence SEQ ID NO: 23, 25 or 28, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and if present, is F.

[0152] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of 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, or F if present.

[0153] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence including SEQ ID NO: 22, the LCDR2 sequence including SEQ ID NO: 23, and the LCDR3 sequence including SEQ ID NO: 24.

[0154] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence including SEQ ID NO: 22, the LCDR2 sequence including SEQ ID NO: 25, and the LCDR3 sequence including 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 the HCDR1 sequence including SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence including SEQ ID NO: 27, the LCDR2 sequence including SEQ ID NO: 28, and the LCDR3 sequence including SEQ ID NO: 26.

[0156] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence of SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X3 is selected from N or S.

[0157] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X4 is selected from A or G, and X5 is selected from D or E.

[0158] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence of SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and if present, is F.

[0159] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E and X6 may or may not be present, or if present, it is F.

[0160] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24.

[0161] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 21, and the LCDR3 sequence of SEQ ID NO: 26.

[0162] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 27, the LCDR2 sequence of SEQ ID NO: 28, and the LCDR3 sequence of SEQ ID NO: 26.

[0163] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence of SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X3 is selected from N or S.

[0164] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X4 is selected from A or G, and X5 is selected from D or E.

[0165] In some cases, anti-transferrin receptor antibodies include a VH region and a VL region, wherein the VH region contains the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region contains the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence of SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and if present, it is F.

[0166] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence AATNLAX5, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E and X6 may or may not be present, or if present, it is F.

[0167] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24.

[0168] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 25, and the LCDR3 sequence of SEQ ID NO: 26.

[0169] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 27, the LCDR2 sequence of SEQ ID NO: 28, and the LCDR3 sequence of SEQ ID NO: 26.

[0170] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence RTSENIYX3NLA, the LCDR2 sequence of SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X3 is selected from N or S.

[0171] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence AX4TNLAX5, and the LCDR3 sequence of SEQ ID NO: 24 or 26, wherein X4 is selected from A or G, and X5 is selected from D or E.

[0172] In some cases, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22 or 27, the LCDR2 sequence of SEQ ID NO: 23, 25, or 28, and the LCDR3 sequence QHFWGTPLTX6, wherein X6 is present or absent, and if present, is F.

[0173] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19, and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence AATNLAX, and the LCDR3 sequence QHFWGTPLTX6, wherein X5 is selected from D or E and X6 may or may not be present, or if present, it is F.

[0174] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24.

[0175] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 25, and the LCDR3 sequence of SEQ ID NO: 26.

[0176] In some cases, anti-transferrin receptor antibodies comprise a VH region and a VL region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 27, the LCDR2 sequence of SEQ ID NO: 28, and the LCDR3 sequence of SEQ ID NO: 26.

[0177] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the sequence of the VH region has approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29-33, and the sequence of the VL region has approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 34-38.

[0178] In some implementations, the VH region contains sequences selected from SEQ ID NO: 29-33 (Table 4), and the VL region contains sequences selected from SEQ ID NO: 34-38 (Table 5). The underlined regions in Tables 4 and 5 represent their respective CDR1, CDR2, or CDR3 sequences.

[0179] Table 4

[0180] Table 5

[0181]

[0182] In some implementations, the anti-transferrin receptor antibody includes the VH and VL regions as shown in Table 6.

[0183] Table 6

[0184] In some embodiments, the anti-transferrin receptor antibodies described herein comprise an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some cases, the anti-transferrin receptor antibody comprises an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4). In some cases, the anti-transferrin receptor antibody comprises an IgG1 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2 (e.g., IgG2a or IgG2b) framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2a framework. In some cases, the anti-transferrin receptor antibody comprises an IgG2b framework. In some cases, the anti-transferrin receptor antibody comprises an IgG3 framework. In some cases, the anti-transferrin receptor antibody comprises an IgG4 framework.

[0185] In some cases, anti-transferrin receptor antibodies contain one or more mutations in the framework region (e.g., the CH1 domain, CH2 domain, CH3 domain, hinge region, or combinations thereof). In some cases, the mutations are designed to stabilize the antibody and / or increase its half-life. In some cases, the mutations are designed to modulate Fc receptor interactions to reduce or eliminate Fc effector functions, such as FcγR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In other cases, the mutations are designed to regulate glycosylation.

[0186] In some implementations, one or more mutations are located in the Fc region. In some cases, the Fc region contains a mutation at residue positions L234, L235, or a combination thereof. In some cases, the mutation contains L234 and L235. In some cases, the mutation contains L234A and L235A. In some cases, the residue positions are reference IgG1.

[0187] In some cases, the Fc region contains mutations at residue positions L234, L235, D265, N21, K46, L52, or P53, or combinations thereof. In some cases, the mutations include L234 and L235, and mutations at residue positions K46, L52, or P53. In some cases, the Fc region contains mutations at L234, L235, and K46. In some cases, the Fc region contains mutations at L234, L235, and L52. In some cases, the Fc region contains mutations at L234, L235, and P53. In some cases, the Fc region contains mutations at D265 and N21. In some cases, the residue positions are referenced to IgG1.

[0188] In some cases, the Fc region contains L234A, L235A, D265A, N21G, K46G, L52R, or P53G, or combinations thereof. In some cases, the Fc region contains L234A and L235A, as well as K46G, L52R, or P53G. In some cases, the Fc region contains L234A, L235A, and K46G. In some cases, the Fc region contains L234A, L235A, and L52R. In some cases, the Fc region contains L234A, L235A, and P53G. In some cases, the Fc region contains D265A and N21G. In some cases, the residue positions refer to IgG1.

[0189] In some cases, the Fc region contains mutations at residue positions L235, L236, D265, N21, K46, L52, or P53, or a combination of these mutations. In some cases, the Fc region contains mutations at L235 and L236. In some cases, the Fc region contains mutations at L235 and L236, as well as mutations at residue positions K46, L52, or P53. In some cases, the Fc region contains mutations at L235, L236, and K46. In some cases, the Fc region contains mutations at L235, L236, and L52. In some cases, the Fc region contains mutations at L235, L236, and P53. In some cases, the Fc region contains mutations at D265 and N21. In some cases, the residue positions refer to IgG2b.

[0190] In some implementations, the Fc region comprises L235A, L236A, D265A, N21G, K46G, L52R, or P53G, or a combination thereof. In some cases, the Fc region comprises L235A and L236A. In some cases, the Fc region comprises L235A and L236A, as well as K46G, L52R, or P53G. In some cases, the Fc region comprises L235A, L236A, and K46G. In some cases, the Fc region comprises L235A, L236A, and L52R. In some cases, the Fc region comprises L235A, L236A, and P53G. In some cases, the Fc region comprises D265A and N21G. In some cases, the residue positions refer to IgG2b.

[0191] In some embodiments, the Fc region contains mutations at residue positions L233, L234, D264, N20, K45, L51, or P52, where the residues correspond to positions 233, 234, 264, 20, 45, 51, and 52 of SEQ ID NO: 39. In some cases, the Fc region contains mutations at L233 and L234. In some cases, the Fc region contains mutations at L233 and L234, as well as mutations at residue positions K45, L51, or P52. In some cases, the Fc region contains mutations at L233, L234, and K45. In some cases, the Fc region contains mutations at L233, L234, and L51. In some cases, the Fc region contains mutations at L233, L234, and K45. In some cases, the Fc region contains mutations at L233, L234, and P52. In some cases, the Fc region 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 frames are considered. In some cases, mutations corresponding to residues L233, L234, D264, N20, K45, L51, or P52 in SEQ ID NO: 39 of the IgG1, IgG2, or IgG4 frames are also considered.

[0192] In some embodiments, the Fc region comprises L233A, L234A, D264A, N20G, K45G, L51R, or P52G, wherein the residues correspond to positions 233, 234, 264, 20, 45, 51, and 52 of SEQ ID NO: 39. In some cases, the Fc region comprises L233A and L234A. In some cases, the Fc region comprises L233A and L234A, as well as K45G, L51R, or P52G. In some cases, the Fc region comprises L233A, L234A, and K45G. In some cases, the Fc region comprises L233A, L234A, and L51R. In some cases, the Fc region comprises L233A, L234A, and K45G. In some cases, the Fc region comprises L233A, L234A, and P52G. In some cases, the Fc region contains D264A and N20G.

[0193] In some implementations, the human IgG constant region is modified to alter antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), for example, as described in Natsume et al., 2008. 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., 1992 J Immunol Amino acid modifications described in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11, 148: 3461-3468;

[0194] In some embodiments, the anti-transferrin receptor antibodies described herein are full-length antibodies comprising a heavy chain (HC) and a light chain (LC). In some cases, the heavy chain (HC) comprises sequences selected from Table 7. In some cases, the light chain (LC) comprises sequences selected from Table 8. Underlined regions indicate their respective CDRs.

[0195] Table 7

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] Table 8

[0205] 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 longer than that of the reference anti-transferrin receptor antibody.

[0206] In some embodiments, binding moiety A nonspecifically conjugates to at least one nucleic acid molecule (B). In some cases, binding moiety A conjugates to at least one nucleic acid molecule (B) in a nonsite-specific manner via lysine residues or cysteine ​​residues. In some cases, binding moiety A conjugates to at least one nucleic acid molecule (B) in a nonsite-specific manner via lysine residues (e.g., lysine residues present in binding moiety A). In some cases, binding moiety A conjugates to at least one nucleic acid molecule (B) in a nonsite-specific manner via cysteine ​​residues (e.g., cysteine ​​residues present in binding moiety A).

[0207] In some embodiments, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B). In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via lysine residues, cysteine ​​residues, residues at the 5' end, residues at the 3' end, non-natural amino acids, or residues modified or catalyzed by enzymes. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via lysine residues (e.g., lysine residues present in binding moiety A). In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) at the 5' end. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) at the 3' end. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via non-natural amino acids. In some cases, binding moiety A is site-specifically conjugated to a polynucleotide molecule (B) via enzyme-modified or enzyme-catalyzed residues.

[0208] In some implementations, one or more polynucleotide molecules (B) are conjugated to binding site A. In some cases, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more polynucleotide molecules are conjugated to binding site A. In some cases, approximately 1 polynucleotide molecule is conjugated to binding site A. In some cases, approximately 2 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 3 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 4 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 5 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 6 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 7 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 8 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 9 polynucleotide molecules are conjugated to binding site A. In some cases, approximately 10 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 11 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 12 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 13 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 14 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 15 polynucleotide molecules conjugate to a single binding site A. In some cases, approximately 16 polynucleotide molecules conjugate to a single binding site A. In some cases, one or more polynucleotide molecules are identical. In other cases, one or more polynucleotide molecules are different.

[0209] In some implementations, the number of polynucleotide molecules (B) conjugated to binding site A forms a ratio. In some cases, this ratio is referred to as the DAR (drug-antibody) ratio, where the drug, as mentioned herein, is the polynucleotide molecule (B). In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 1 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 2 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 3 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 4 or greater. In some cases, the DAR ratio of polynucleotide molecule (B) to binding site A is about 5 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 6 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 7 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 8 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 9 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 10 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 11 or greater. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is about 12 or greater.

[0210] In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 1. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 2. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 3. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 4. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 5. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 6. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 7. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 8. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 9. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 10. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 11. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 12. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 13. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 14. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 15. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is approximately 16.

[0211] In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 1. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 2. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 4. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 6. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 8. In some cases, the DAR ratio of the polynucleotide molecule (B) to the binding site A is 12.

[0212] In some cases, conjugates containing both a polynucleotide molecule (B) and a binding moiety A exhibit improved activity compared to conjugates containing a polynucleotide molecule (B) but lacking a binding moiety A. In some cases, this improved activity leads to enhanced biologically relevant functions, such as improved stability, affinity, binding, functional activity, and efficacy in treating or preventing disease states. In some cases, this disease state is a result of one or more mutated exons in a gene. In some cases, conjugates containing both a polynucleotide molecule (B) and a binding moiety A result in increased exon skipping of one or more mutated exons compared to conjugates containing a polynucleotide molecule (B) but lacking a binding moiety A. In some cases, the increase in exon skipping in conjugates containing both a polynucleotide molecule (B) and a binding moiety A is at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% compared to conjugates containing a polynucleotide molecule (B) but lacking a binding moiety A.

[0213] In some embodiments, the antibody or its binding fragment is further modified using conventional techniques known in the art, such as by using, alone or in combination, amino acid deletions, insertions, substitutions, additions, and / or by recombination and / or any other modifications known in the art (e.g., post-translational modifications and chemical modifications, such as glycosylation and phosphorylation). In some cases, the modification further includes modifications for modulating the interaction with the Fc receptor. In some cases, one or more modifications include those described, for example, in International Publication No. WO97 / 34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence below the amino acid sequence of the antibody or its binding fragment are well known to those skilled in the art.

[0214] In some cases, the antibody-binding fragment also includes its derivatives and comprises a polypeptide sequence containing at least one CDR.

[0215] In some cases, as used herein, the term "single-stranded" refers to the covalent connection of the first and second domains of a bispecific single-stranded construct, preferably in the form of a collinear amino acid sequence that can be encoded by a single nucleic acid molecule.

[0216] In some cases, bispecific single-chain antibody constructs involve constructs comprising two antibody-derived binding domains. In such embodiments, the bispecific single-chain antibody construct is a tandem dual scFv or a dual antibody. In some cases, the scFv contains VH and VL domains linked by a linker peptide. In some cases, the linker length and sequence are sufficient to ensure that each of the first and second domains can independently retain its differential binding specificity.

[0217] In some embodiments, as used herein, binding or interaction defines the binding / interaction of at least two antigen-interacting sites with each other. In some cases, antigen-interacting sites define a motif of a polypeptide that exhibits the ability to specifically interact with a particular antigen or a particular set of antigens. In some cases, binding / interaction is also understood to define specific recognition. In this context, specific recognition refers to the ability of an antibody or its binding fragment to specifically interact with and / or bind to at least two amino acids of each target molecule. For example, specific recognition involves the specificity of an antibody molecule or its ability to distinguish specific regions of a target molecule. In other cases, the specific interaction of an antigen-interacting site with its specific antigen results in the initiation of a signal, for example, due to the induction of conformational changes in the antigen, oligomerization of the antigen, etc. In further embodiments, binding is exemplified by the "key-lock principle" of specificity. Thus, in some cases, a specific motif in the amino acid sequence of an antigen-interacting site and the antigen bind to each other as a result of their primary, secondary, or tertiary structures and secondary modifications of said structures. In this case, the specific interaction of an antigen-interacting site with its specific antigen also results in the simple binding of that site to the antigen.

[0218] In some cases, specific interactions also refer to reduced cross-reactivity or off-target effects of antibodies or their binding fragments. For example, antibodies or their binding fragments that bind to a polypeptide / protein of interest but not, or substantially not, any other polypeptide are considered specific to that polypeptide / protein. Examples of specific interactions between antigen-interacting sites and specific antigens include the specificity of a ligand to its receptor, such as the interaction between an antigenic determinant (epitope) and the antigen-binding site of an antibody.

[0219] Therefore, in some cases, polynucleotide conjugates comprise polynucleotide molecules having sense and antisense strands, and an antitransferrin receptor antibody or its antigen-binding fragment conjugated to the polynucleotide, wherein the sense strand has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1, and the antisense strand has a sequence having at least 80% identity with the sequence of SEQ ID NO: 2, such that the polynucleotide conjugate mediates the inhibition of the antigen against the transferrin receptor. DMPK RNA interference.

[0220] In some implementations, the polynucleic acid conjugate includes conjugation to... DMPKThe target sequence hybridization polynucleotide molecule includes an anti-transferrin receptor antibody or its antigen-binding fragment, and a polynucleotide molecule having a sense strand and an antisense strand, wherein the sense strand has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 3, 5, 8, 10, 12, 14, or 16, and the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence including SEQ ID NO: 17, the HCDR2 sequence including SEQ ID NO: 20, and the HCDR3 sequence including SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence including SEQ ID NO: 22, the LCDR2 sequence including SEQ ID NO: 23, and the HCDR3 sequence including SEQ ID NO: 19. The LCDR3 sequence of 24, and the anti-transferrin receptor antibody or its antigen-binding fragment are conjugated to polynucleotide molecules via a linker including 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).

[0221] In some implementations, the polynucleic acid conjugate includes conjugation to... DMPK The target sequence hybridization polynucleotide molecule includes an anti-transferrin receptor antibody or its antigen-binding fragment, and a polynucleotide molecule having a sense strand and an antisense strand, wherein the sense strand has a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% with SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15, and the antisense strand has a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, and the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has a sequence identity of at least 80%, 85%, 90%, 95%, 99%, or 100% with SEQ ID NO: 30, and wherein the VL ... 34 has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity, and the anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0222] In some implementations, polynucleic acid conjugates include those containing conjugates to... DMPKThe target sequence hybridization polynucleotide molecule includes an anti-transferrin receptor antibody or its antigen-binding fragment, and a polynucleotide molecule having a sense strand and an antisense strand. The sense strand has a sequence identity of at least 80%, 85%, 90%, 95%, 99%, or 100% with SEQ ID NO: 1, and the antisense strand has the sequence of SEQ ID NO: 2. The sense strand contains at least 3, 4, 5, or 6 consecutive 2'-O-methyl modified nucleotides at the 5' end and at least 2 or 3 2'-F modified nucleotides. The anti-transferrin receptor antibody or its antigen-binding fragment contains a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has a sequence identity of at least 80%, 85%, 90%, 95%, 99%, or 100% with SEQ ID NO: 30, and wherein the VL region has a sequence identity of at least 80%, 85%, 90%, 95%, 99%, or 100% with SEQ ID NO: 2. NO:34 has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity, and the anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0223] In some implementations, the polynucleic acid conjugate includes conjugation to... DMPK The target sequence hybridization polynucleotide molecule includes an anti-transferrin receptor antibody or its antigen-binding fragment, and a polynucleotide molecule having a sense strand and an antisense strand. The sense strand has a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity with SEQ ID NO: 1, and the antisense strand has the sequence of SEQ ID NO: 2. The antisense strand contains at least two, at least three, at least four, or at least five consecutive 2'-O-methyl modified nucleotides at the 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 its antigen-binding fragment contains a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region contains the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region contains the LCDR1 sequence of SEQ ID NO: 22 and the HCDR3 sequence of SEQ ID NO: 22. The LCDR2 sequence of SEQ ID NO: 23 and the LCDR3 sequence including SEQ ID NO: 24, and the anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0224] In some implementations, the polynucleic acid conjugate includes conjugation to... DMPKThe target sequence hybridization polynucleotide molecule includes an anti-transferrin receptor antibody or its antigen-binding fragment, and a polynucleotide molecule having a sense strand and an antisense strand. The sense strand has a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 1, and the antisense strand has the sequence of SEQ ID NO: 2. The antisense strand contains 2'-O-methyl modified nucleotides at the 5' and 3' ends. The anti-transferrin receptor antibody or its antigen-binding fragment contains a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region contains the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19. The VL region contains the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 3, and the HCDR3 sequence of SEQ ID NO: 19. The LCDR3 sequence of 24, and the anti-transferrin receptor antibody or its antigen-binding fragment are conjugated to polynucleotide molecules via maleimide linkers.

[0225] In some implementations, the polynucleic acid conjugate includes conjugation to... DMPK The target sequence hybridization polynucleotide molecule includes an anti-transferrin receptor antibody or its antigen-binding fragment, and a polynucleotide molecule having a sense strand and an antisense strand. The sense strand has a sequence with at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 1, and the antisense strand has the sequence of SEQ ID NO: 2. The antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides and four 2'-F modified nucleotides at the 3' end, wherein any two of the four 2'-F modified nucleotides are not consecutive. The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, and the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3, and the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. 34 has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity, and the anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a 6-amino-1-hexanol linker.

[0226] Other parts of the combination In some embodiments, the binding moiety is a plasma protein. In some cases, the plasma protein includes albumin. In some cases, binding moiety A is albumin. In some cases, albumin conjugates to polynucleotide molecules via one or more conjugation chemistry methods described herein. In some cases, albumin conjugates to polynucleotide molecules via native linker chemistry. In some cases, albumin conjugates to polynucleotide molecules via lysine conjugation.

[0227] In some cases, the binding moiety is a steroid. Exemplary steroids include cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, saturated, unsaturated, substituted hydrocarbons, or combinations thereof. In some cases, the steroid is cholesterol. In some cases, the binding moiety is cholesterol. In some cases, cholesterol is conjugated to a polynucleotide molecule via one or more conjugation chemistry methods described herein. In some cases, cholesterol is conjugated to a polynucleotide molecule via natural linking chemistry. In some cases, cholesterol is conjugated to a polynucleotide molecule via lysine conjugation.

[0228] In some cases, the binding moiety is a polymer, including but not limited to polynucleotide aptamers that bind to specific surface markers on cells. In this case, the binding moiety is a polynucleotide that does not hybridize with the target gene or mRNA, but rather resembles an antibody that binds to a specific epitope of a cell surface marker, enabling selective binding to the cell surface marker.

[0229] In some cases, the binding moiety is a peptide. In some cases, the peptide has a mass of about 1 to about 3 kDa. In some cases, the peptide has a mass of 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 cases, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some cases, the binding moiety is a bicyclic peptide (e.g., a bicyclic compound from Bicycle Therapeutics).

[0230] In other cases, the binding moiety is a small molecule. In some cases, this small molecule is a small molecule that recruits antibodies. In some cases, the small molecule that recruits antibodies contains a target-binding terminus and an antibody-binding terminus, wherein the target-binding terminus is capable of recognizing and interacting with cell surface receptors. For example, in some cases, a target-binding terminus containing a glutamate compound enables interaction with PSMA, thereby enhancing antibody interaction with cells expressing PSMA. In some cases, 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,” JAm 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).

[0231] Production of antibodies or their antigen-binding fragments In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments, anti-transferrin receptor antibodies or their antigen-binding fragments) are produced using any method known in the art for synthesizing polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression technology.

[0232] In some cases, antibodies or their antigen-binding fragments are recombinantly expressed, and the nucleic acids encoding the antibodies or their binding fragments are assembled from chemically synthesized oligonucleotides (e.g., Kutmeier et al., 1994). BioTechniques As described in 17:242, it involves synthesizing overlapping oligonucleotides containing partial sequences encoding antibodies, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0233] Alternatively, nucleic acid molecules encoding antibodies may be generated from suitable sources (e.g., antibody cDNA libraries, or cDNA libraries generated from any tissue or cell expressing immunoglobulins) by using PCR amplification with synthetic primers that can hybridize to the 3' and 5' ends of the sequence, or by using the cloning of oligonucleotide probes that are specific to a particular gene sequence.

[0234] In some cases, antibodies or antigen-binding fragments are optionally produced by immunizing animals, such as rabbits, to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, as Kohler and Milstein (1975) did. , Nature As described in 256:495-497 or as Kozbor et al. (1983) Immunology Today 4:72) or Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy As described in Alan R. Liss, Inc., pp. 77-96. Alternatively, clones encoding at least the Fab portion of an antibody may be obtained by screening Fab expression libraries for clones of Fab fragments that bind to a specific antigen (e.g., as described in Huse et al., 1989). Science (as described in 246:1275-1281) or screening antibody libraries (see, for example, Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0235] In some implementations, techniques developed for generating "chimeric antibodies" are used by splicing genes from mouse antibody molecules with appropriate antigen specificity to genes from human antibody molecules with appropriate biological activity (Morrison et al., 1984). , Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984 , Nature 312:604-608; Takeda et al., 1985 Nature 314:452-454). Chimeric antibodies are molecules in which different parts are derived from different animal species, such as antibodies with a variable region derived from mouse monoclonal antibodies and a constant region of human immunoglobulins, such as humanized antibodies.

[0236] In some implementations, the technology described for generating single-chain antibodies (US Patent 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) is suitable for producing single-chain antibodies. Single-chain antibodies are formed by generating single-chain polypeptides by linking the heavy and light chain fragments of the Fv region via amino acid bridges. Alternatively, *Escherichia coli* (…) can also be used. E. coli Techniques for assembling functional Fv fragments in (Skerra et al., 1988) Science 242:1038-1041).

[0237] In some embodiments, an expression vector containing the antibody's nucleotide sequence or the antibody's nucleotide sequence is transferred into host cells using conventional techniques (e.g., electroporation, liposome transfection, and / or calcium phosphate precipitation), and the transfected cells are then cultured using conventional techniques to produce the antibody. In specific embodiments, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

[0238] In some embodiments, multiple host expression vector systems are used to express the antibodies or their binding fragments described herein. Such host expression systems represent vectors through which antibody-coding sequences are generated and subsequently purified, and also represent cells that express antibodies or their binding fragments in situ when transformed or transfected with suitable nucleotide-coding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors containing sequences encoding antibodies or their binding fragments (e.g., *Escherichia coli* and *Bacillus subtilis*). B. subtilis Yeast transformed with a recombinant yeast expression vector containing the coding sequence of an antibody or its binding fragment (e.g., Pichia pastoris). Saccharomyces Pichia Insect cell systems infected with recombinant viral expression vectors (e.g., baculoviruses) containing sequences encoding antibodies or their binding fragments; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or with recombinant plasmid expression vectors (e.g., Ti plasmids) containing sequences encoding antibodies or their binding fragments; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) containing a recombinant expression construct containing a promoter derived from the mammalian cell genome (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0239] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines stably expressing antibodies are optionally engineered. Instead of 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 selectable markers. After the introduction of exogenous DNA, the engineered cells are grown in enrichment medium for 1–2 days, then switched to selective medium. Selectable markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are then cloned and amplified into cell lines. This method can be advantageously used to engineer cell lines expressing antibodies or their binding fragments.

[0240] In some cases, multiple selection systems are used, including but not limited to herpes simplex virus thymidine kinase used in tk-, hgprt-, or aprt- cells respectively (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) gene. Furthermore, antimetabolite resistance was used as the basis for selecting the following gene: dhfr, which confers resistance to methotrexate (Wigler et al., 1980). , Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981 , Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981) , Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 ( Clinical Pharmacy 12:488-505; Wu and Wu, 1991 Biotherapy 3:87-95; Tolstoshev, 1993 , Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993 Science 260:926-932; and Morgan and Anderson, 1993 , Ann. Rev. Biochem. 62:191-217; May, 1993 , TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984). Gene30:147). Well-known and usable methods in the field of recombinant DNA technology are 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 Dracopoli et al. (eds.), 1994 , Current Protocols in Human Genetics John Wiley & Sons, NY, Chapters 12 and 13; Colberre-Garapin et al., 1981 , J. Mol. Biol. 150:1.

[0241] In some cases, antibody expression levels can be increased through vector amplification (see Bebbington and Henschel for a review). The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning Volume 3 (Academic Press, New York, 1987). When the marker in an antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody's nucleotide sequence, antibody production will also increase (Crouse et al., 1983). , Mol. Cell Biol. 3:257).

[0242] In some cases, any method known in the art for purifying or analyzing antibodies or antibody conjugates may be used, such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for specific antigens following protein A, and size column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and rapid protein liquid chromatography.

[0243] Conjugation Chemistry In some embodiments, the polynucleotide molecule B is conjugated to the binding moiety. In some embodiments, the polynucleotide molecule B is conjugated to the binding moiety of formula AXB (where X is the linker connecting A and B). In some cases, the binding moiety comprises amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all these classes of substances. Other examples of the binding moiety include steroids, such as cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or containing substitutions), enzyme substrates, biotin, digitalisin, and polysaccharides. In some cases, the binding moiety is an antibody or an antigen-binding fragment thereof. In some cases, the polynucleotide molecule is further conjugated to a polymer and optionally to an endosome dissolution moiety.

[0244] In some implementations, polynucleotide molecules are conjugated to the binding site via a chemical ligation process. In other cases, polynucleotide molecules are conjugated to the binding site via native ligation. In some cases, the conjugation is as described in: Dawson et al., “Synthesis of proteins by native chemical ligation,” Science 1994, 266 , 776–779; Dawson et al., “Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives,” J. Am. Chem. Soc . 1997, 119, 4325–4329; Hackeng et al., “Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.,” Proc. Natl. Acad. Sci. USA 1999, 96, 10068–10073; or Wu et al., “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45 ,4116–4125. In some cases, conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, polynucleotide molecules are conjugated to the binding site specifically or nonspecifically via natural linker chemistry.

[0245] In some cases, polynucleotide molecules are conjugated to the binding site using a site-directed method employing a "seamless" conjugation technique (Philochem). In other cases, this "seamless" conjugation technique utilizes the N-terminal 1,2-aminothiol group on the binding site, which is subsequently conjugated to a polynucleotide molecule containing an aldehyde group (see Casi et al.). , “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmaceutical codelivery,” JACS 134(13): 5887-5892 (2012)).

[0246] In some cases, polynucleotide molecules are conjugated to the conjugating moiety via a site-directed method that incorporates a non-natural amino acid into the conjugating moiety. In some cases, this non-natural amino acid includes p-acetylphenylalanine (pAcPhe). In other cases, the ketone group of pAcPhe is selectively coupled to the conjugating moiety derived from an alkoxy-amine to form an oxime bond (see Axup et al.). , “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

[0247] In some cases, polynucleotide molecules are conjugated to their binding sites using a site-directed approach employing an enzymatic catalytic process. In some cases, this site-directed approach utilizes SMARTag™ technology (Catalent, Inc.). In some cases, SMARTag™ technology involves the oxidation of cysteine ​​to a formylgly (FGly) residue via a formylglyase (FGE) in the presence of an aldehyde tag, followed by conjugation of FGly to an alkylhydrazine-functionalized polynucleotide molecule via hydrazine-Pictet-Spengler (HIPS) linker (see Wu et al., “Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,”). PNAS106(9): 3000-3005 (2009); Agarwal et al., “A Pictet-Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51(2013)).

[0248] In some cases, the enzymatic catalytic process involves microbial transglutaminase (mTG). In others, polynucleotide molecules are conjugated to their binding sites using a process catalyzed by microbial transglutaminase. In still others, mTG catalyzes the formation of a covalent bond between the amide side chain of the recognition sequence and the primary amine of the functionalized polynucleotide molecule. In some cases, mTG is produced by *Streptomyces malnourishes* (…). Streptomyces mobarensis ) produced (see Strop et al.) , “Locationmatters: site of conjugation modulates stability and pharmacokinetics ofantibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013)).

[0249] In some cases, polynucleotide molecules are conjugated to the binding site using a method described in PCT Publication No. WO2014 / 140317, which utilizes a sequence-specific transpeptidase.

[0250] In some cases, polynucleotide molecules are conjugated to the binding site using methods described in U.S. Patent Publications 2015 / 0105539 and 2015 / 0105540.

[0251] Polymer conjugate In some embodiments, polymer moiety C is further conjugated to the polynucleotide molecule described herein, the binding moiety described herein, or a combination thereof. In some cases, polymer moiety C is conjugated to a polynucleotide molecule with the molecular formula A-X1-B-X2-C (X1 and X2 serve as two linkers conjugating A to B and B to C, respectively). In some cases, polymer moiety C is conjugated to the binding moiety. In other cases, polymer moiety C is conjugated to the polynucleotide molecule binding moiety. In still other cases, polymer moiety C is conjugated, as shown above.

[0252] In some cases, polymer part C is a natural or synthetic polymer composed of long chains of branched or unbranched monomers and / or cross-linked networks of two-dimensional or three-dimensional monomers. In some cases, polymer part C includes polysaccharides, lignin, rubber, or polyepoxides (e.g., polyethylene glycol). In some cases, at least one polymer part C includes, but is not limited to, α-dihydroxy polyethylene glycol, ω-dihydroxy polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (also known as poly(ethylene terephthalate), PET, PETG, or PETE), polybutane glycol (PTG), or polyurethanes and mixtures thereof. As used herein, mixtures refer to the use of different polymers within the same compound and involve the use of different polymers in block copolymers. In some cases, a block copolymer is a polymer in which at least a portion of the polymer is composed of monomers of another polymer. In some cases, polymer part C includes polyepoxides. In some cases, polymer part C includes PEG. In other cases, polymer part C includes polyvinylimide (PEI) or hydroxyethyl starch (HES).

[0253] In some cases, C represents the PEG moiety. In some cases, the PEG moiety is conjugated at the 5' end of the polynucleotide molecule, while the binding moiety is conjugated at the 3' end. In some cases, the PEG moiety is conjugated at the 3' end of the polynucleotide molecule, while the binding moiety is conjugated at the 5' end. In some cases, the PEG moiety is conjugated to an internal site of the polynucleotide molecule. In some cases, the PEG moiety, the binding moiety, or a combination thereof is conjugated to an internal site of the polynucleotide molecule. In some cases, the conjugation is direct. In some cases, the conjugation occurs via natural linker.

[0254] In some embodiments, the polyepoxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, the polydisperse material comprises a dispersion of materials with different molecular weights, characterized by average weight (weight-average) size and dispersibility. In some cases, the monodisperse PEG comprises molecules of a single size. In some embodiments, C is a polydisperse or monodisperse polyepoxide (e.g., PEG), and the indicated molecular weight represents the average molecular weight of the polyepoxide (e.g., PEG) molecules.

[0255] In some embodiments, the polyepoxide (e.g., PEG) has a molecular weight of 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, or 270. 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.

[0256] In some embodiments, C is a polyepoxide (e.g., PEG) and has a strength of 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, 27 Molecular weights 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 implementations, C is PEG, and has a strength of approximately 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 28 Molecular weights of 0, 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 cases, the molecular weight of C is approximately 200 Da. In some cases, the molecular weight of C is approximately 300 Da. In some cases, the molecular weight of C is approximately 400 Da. In some cases, the molecular weight of C is approximately 500 Da. In some cases, the molecular weight of C is approximately 600 Da. In some cases, the molecular weight of C is approximately 700 Da. In some cases, the molecular weight of C is approximately 800 Da. In some cases, the molecular weight of C is approximately 900 Da. In some cases, the molecular weight of C is approximately 1000 Da. In some cases, the molecular weight of C is approximately 1100 Da. In some cases, the molecular weight of C is approximately 1200 Da. In some cases, the molecular weight of C is approximately 1300 Da. In some cases, the molecular weight of C is approximately 1400 Da. In some cases, the molecular weight of C is approximately 1450 Da. In some cases, the molecular weight of C is approximately 1500 Da. In some cases, the molecular weight of C is approximately 1600 Da. In some cases, the molecular weight of C is approximately 1700 Da.In some cases, the molecular weight of C is approximately 1800 Da. In some cases, the molecular weight of C is approximately 1900 Da. In some cases, the molecular weight of C is approximately 2000 Da. In some cases, the molecular weight of C is approximately 2100 Da. In some cases, the molecular weight of C is approximately 2200 Da. In some cases, the molecular weight of C is approximately 2300 Da. In some cases, the molecular weight of C is approximately 2400 Da. In some cases, the molecular weight of C is approximately 2500 Da. In some cases, the molecular weight of C is approximately 2600 Da. In some cases, the molecular weight of C is approximately 2700 Da. In some cases, the molecular weight of C is approximately 2800 Da. In some cases, the molecular weight of C is approximately 2900 Da. In some cases, the molecular weight of C is approximately 3000 Da. In some cases, the molecular weight of C is approximately 3250 Da. In some cases, the molecular weight of C is approximately 3350 Da. In some cases, the molecular weight of C is approximately 3500 Da. In some cases, the molecular weight of C is approximately 3750 Da. In some cases, the molecular weight of C is approximately 4000 Da. In some cases, the molecular weight of C is approximately 4250 Da. In some cases, the molecular weight of C is approximately 4500 Da. In some cases, the molecular weight of C is approximately 4600 Da. In some cases, the molecular weight of C is approximately 4750 Da. In some cases, the molecular weight of C is approximately 5000 Da. In some cases, the molecular weight of C is approximately 5500 Da. In some cases, the molecular weight of C is approximately 6000 Da. In some cases, the molecular weight of C is approximately 6500 Da. In some cases, the molecular weight of C is approximately 7000 Da. In some cases, the molecular weight of C is approximately 7500 Da. In some cases, the molecular weight of C is approximately 8000 Da. In some cases, the molecular weight of C is approximately 10,000 Da. In some cases, the molecular weight of C is approximately 12,000 Da. In some cases, the molecular weight of C is approximately 20,000 Da. In some cases, the molecular weight of C is approximately 35,000 Da. In some cases, the molecular weight of C is approximately 40,000 Da. In some cases, the molecular weight of C is approximately 50,000 Da. In some cases, the molecular weight of C is approximately 60,000 Da. In some cases, the molecular weight of C is approximately 100,000 Da.

[0257] In some embodiments, the polyepoxide (e.g., PEG) comprises discrete ethylene oxide units (e.g., 4 to about 48 ethylene oxide units). In some cases, the polyepoxide comprising discrete ethylene oxide units is linear. In other cases, the polyepoxide comprising discrete ethylene oxide units is branched.

[0258] In some cases, polymer portion C is a polyepoxide (e.g., PEG) comprising discrete ethylene oxide units. In some cases, polymer portion C comprises 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.

[0259] In some cases, polymer portion C is a discrete PEG containing, for example, about 4 to about 48 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 4 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 5 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 6 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 7 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 8 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 9 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 10 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 11 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 12 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 13 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 14 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 15 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 16 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 17 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 18 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 19 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 20 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 21 ethylene oxide units. In some cases, the polymer portion C is a discrete PEG that contains, for example, about 22 ethylene oxide units.In some cases, polymer portion C is a discrete PEG containing, for example, about 23 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 24 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 25 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 26 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 27 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 28 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 29 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 30 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 31 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 32 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 33 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 34 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 35 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 36 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 37 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 38 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 39 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 40 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 41 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 42 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 43 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 44 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 45 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 46 ethylene oxide units. In some cases, polymer portion C is a discrete PEG containing, for example, about 47 ethylene oxide units.In some cases, the polymer portion C is a discrete PEG that contains, for example, about 48 ethylene oxide units.

[0260] In some cases, polymer part C is dPEG® (Quanta Biodesign Ltd).

[0261] In some embodiments, polymer portion C comprises a cationic viscosic acid-based polymer (cMAP). In some cases, the cMAP comprises one or more subunits of at least one repeating subunit, and the subunit structure is represented by formula (V):

[0262] Formula V Where m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 each time it appears, preferably 4-6 or 5; and n is independently 1, 2, 3, 4 or 5 each time it appears. In some embodiments, m and n are, for example, about 10.

[0263] In some cases, cMAP is further conjugated to the PEG moiety to form a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the PEG moiety ranges from about 500 Da to about 50,000 Da. In some cases, the PEG moiety ranges from about 500 Da to about 1,000 Da, greater than 1,000 Da to about 5,000 Da, greater than 5,000 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.

[0264] In some cases, polymer moiety C is a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In other cases, polymer moiety C is a cMAP-PEG copolymer. In still other cases, polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

[0265] In some implementations, polymer portion C is conjugated to multiple nucleic acid molecules, a binding portion, and an optional endosome dissolution portion, as described above.

[0266] Endosome dissolution portion or cell membrane penetration portion In some embodiments, the molecule of formula (I) – A-X1-B-X2-C – further comprises additional conjugated portions. In some cases, the additional conjugated portions are endosome-dissolving portions and / or cell membrane-penetrating portions. In some cases, the endosome-dissolving portion is a cell compartment-releasing component, such as a compound capable of being released from any cell compartment known in the art, such as endosomes, lysosomes, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other intracellular vesicles. In some cases, the endosome-dissolving portion comprises an endosome-dissolving polypeptide, an endosome-dissolving polymer, an endosome-dissolving lipid, or an endosome-dissolving small molecule. In some cases, the endosome-dissolving portion comprises an endosome-dissolving polypeptide. In other cases, the endosome-dissolving portion comprises an endosome-dissolving polymer. In some cases, the cell membrane-penetrating portion comprises a cell-penetrating peptide (CPP). In other cases, the cell membrane-penetrating portion comprises a cell-penetrating lipid. In other cases, the cell membrane-penetrating portion comprises a cell-penetrating small molecule.

[0267] Endosome-dissolving peptides and cell membrane-penetrating peptides In some embodiments, the molecule of formula (I) – A-X1-B-X2-C – is further conjugated to an endosomal dissolving peptide. In some cases, the endosomal dissolving peptide is a pH-dependent membrane-active peptide. In some cases, the endosomal dissolving peptide is an amphiphilic peptide. In other cases, the endosomal dissolving peptide is a peptide mimic. In some cases, the endosomal dissolving peptide comprises INF, meliostein, meucin, or derivatives thereof. In some cases, the endosomal dissolving peptide comprises INF or a derivative thereof. In other cases, the endosomal dissolving peptide comprises meliostein or a derivative thereof. In still other cases, the endosomal dissolving peptide comprises meucin or a derivative thereof.

[0268] In some cases, INF7 is a 24-residue polypeptide containing sequences such as CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 67) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 68). In other cases, INF7 or its derivatives contain the 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).

[0269] In some cases, melittin is a 26-residue polypeptide whose sequence contains CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 72) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 73). In some cases, melittin comprises a polypeptide sequence as described in U.S. Patent No. 8,501,930.

[0270] In some cases, meucin is an antimicrobial peptide (AMP) derived from the venom glands of the striped scorpion (Mesobuthus eupeus). In some cases, meucin comprises meucin-13 (those sequences containing IFGAIAGLLKNIF-NH2 (SEQ ID NO: 74)) and meucin-18 (those sequences containing FFGHLFKLATKIIPSLFQ (SEQ ID NO: 75)).

[0271] In some cases, the endosome-dissolving polypeptide includes a polypeptide whose sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity with INF7 or a derivative thereof, melitoxin or a derivative thereof, or meucin or a derivative thereof. In some cases, the endosome-dissolving portion includes INF7 or a derivative thereof, melitoxin or a derivative thereof, or meucin or a derivative thereof.

[0272] In some cases, the endosome dissolution portion is INF7 or a derivative thereof. In some cases, the endosome dissolution portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 67. In some cases, the endosome dissolution portion contains a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 68-71. In some cases, the endosome dissolution portion comprises SEQ ID NO: 67. In some cases, the endosome dissolution portion comprises SEQ ID NO: 68-71. In some cases, the endosome dissolution portion consists of SEQ ID NO: 67. In some cases, the endosome dissolution portion consists of SEQ ID NO: 68-71.

[0273] In some cases, the endosome dissolution portion is a melitoxin or a derivative thereof. In some cases, the endosome dissolution 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 with SEQ ID NO: 72. In some cases, the endosome dissolution 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 with SEQ ID NO: 73. In some cases, the endosome dissolution 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 with SEQ ID NO: 73. In some cases, the endosome dissolution portion comprises SEQ ID NO: 72. In some cases, the endosome dissolution portion comprises SEQ ID NO: 73. In some cases, the endosome dissolution portion consists of SEQ ID NO: 72. In some cases, the endosome dissolution portion consists of SEQ ID NO: 73.

[0274] In some cases, the endosome dissolution portion is meucin or a derivative thereof. In some cases, the endosome dissolution 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 with SEQ ID NO: 74. In some cases, the endosome dissolution portion comprises a polypeptide having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 75. In some cases, the endosome dissolution portion comprises a polypeptide having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 74. In some cases, the endosome dissolution portion comprises SEQ ID NO: 74. In some cases, the endosome dissolution portion comprises SEQ ID NO: 75. In some cases, the endosome dissolution portion comprises SEQ ID NO: 74. In some cases, the endosome dissolution portion comprises SEQ ID NO: 75.

[0275] In some cases, the endosome dissolution portion contains the sequences shown in Table 9.

[0276] Table 9

[0277]

[0278] In some cases, the endosome-dissolving portion contains the Bak BH3 peptide, which antagonizes inhibitory targets such as Bcl-2 and / or Bcl-x. L Inducing apoptosis. In some cases, the endosome lysis fraction contains Albarran et al., “Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier,” Reactive & Functional Polymers The Bak BH3 polypeptide described in 71: 261-265 (2011).

[0279] In some cases, the endosome dissolution portion contains a polypeptide (e.g., a cell-penetrating polypeptide) as described in PCT Publication Nos. WO2013 / 166155 or WO2015 / 069587.

[0280] Endosome-dissolved lipids In some embodiments, the endosome-dissolving portion is a lipid (e.g., a fusion-promoting lipid). In some embodiments, the molecule of formula (I) – A-X1-B-X2-C – is further conjugated with an endosome-dissolving lipid (e.g., a fusion-promoting lipid). Exemplary fusion-promoting lipids include 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadec-9,12-dienyl)-1,3-dioxolane-4-yl)methylamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadec-9,12-dienyl)-1,3-dioxolane-4-yl)ethylamine (XTC).

[0281] In some cases, the endosome-dissolving portion is a lipid (e.g., a fusion-promoting lipid) as described in PCT Publication No. WO09 / 126,933.

[0282] Endosome Dissolution of Small Molecules In some embodiments, the endosome dissolution moiety is a small molecule. In some embodiments, the molecule of formula (I) – A-X1-B-X2-C – is further conjugated with the endosome dissolution small molecule. Exemplary small molecules suitable as the endosome dissolution moiety include, but are not limited to, quinine, chloroquine, hydroxychloroquine, carnoquine, amopiquine, primaquine, mefloquine, chloroquine sulfate, halopanthracene, quinone imines, or combinations thereof. In some cases, the endosome dissolved portion of quinoline includes, but is not limited to, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 4-(4-diethylamino-1-methylbutyl-amino)quinoline; 7-hydroxy-4-(4-diethylamino-1-methylbutyl-amino)quinoline; 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline (demethylchloroquine) Quinoline); 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-butylamino)quinoline; 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))quinoline; 7-Amino-1-methylbutylamino)quinoline; hydroxychloroquine phosphate; 7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline (demethylhydroxychloroquine); 7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-Fluoro-4-(1-Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 4-(1-Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-Hydroxy-4-(1-Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline; 7-Chloro-4-(1-Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-Fluoro-4-(1-Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 4-(1-Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 7-Hydroxy-4-(1- Carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline; 8-[(4-aminopentyl)amino-6-methoxyquinoline dihydrochloride; 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-dimethylaminoquinoline; 4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline; 3-fluoro-4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolidinyl)-2,5-dimethylaminoquinoline, 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 4-(4-hydroxy-α,α'-bis(2-methyl-1-pyrrolyl)-2,5-dimethylaminoquinoline; 4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline; 3,4-dihydro-1-(2H)-quinoline carboxyl aldehyde; 1,1'-pentamethylenediquinoline diiodide; 8-hydroxyquinoline sulfate and their amino, aldehyde, carboxyl, hydroxyl, halogen, ketone, thiol, and vinyl derivatives or analogs. In some cases, the endosome-dissolved moiety is a small molecule as described in Naisbitt et al. (1997, J Pharmacol Exp Therapy 280:884-893) and U.S. Patent No. 5,736,557.

[0283] Cell-penetrating peptides (CPP) In some embodiments, the cell-penetrating polypeptide comprises a positively charged short 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 or combination thereof listed in Table 10.

[0284] Table 10

[0285] connector In some embodiments, the connector described herein is either a cuttable connector or a non-cuttable connector. In some cases, the connector is a cuttable connector. In other cases, the connector is a non-cuttable connector.

[0286] In some cases, the linker is a nonpolymeric linker. A nonpolymeric linker is a linker that does not contain repeating monomer units generated through a polymerization process. Exemplary nonpolymeric linkers include, but are not limited to, C1-C6 alkyl (e.g., C5, C4, C3, C2, or C1 alkyl), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In some cases, the nonpolymeric linker comprises C1-C6 alkyl (e.g., C5, C4, C3, C2, or C1 alkyl), homobifunctional crosslinkers, heterobifunctional crosslinkers, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In other cases, the nonpolymeric linker does not contain more than two linkers of the same type, for example, more than two homobifunctional crosslinkers or more than two peptide linkers. In still other cases, the nonpolymeric linker optionally contains one or more reactive functional groups.

[0287] In some cases, the non-polymerizable joint does not contain the polymers described above. In some cases, the non-polymerizable joint does not contain the polymer contained in polymer portion C. In some cases, the non-polymerizable joint does not contain polyepoxides (e.g., PEG). In some cases, the non-polymerizable joint does not contain PEG.

[0288] In some cases, the connector includes a homodifunctional connector. Exemplary homodifunctional connectors include, but are not limited to, Lomant reagent dithiobis(succinimide propionate). DSP, 3'3'-dithiobis(sulfosuccinimide propionate) (DTSSP), disuccinimide octanoate (DSS), bis(sulfosuccinimide) octanoate (BS), disuccinimide tartrate (DST), disulfosuccinimide tartrate (sulfonated DST), bis(succinimide) ethylene glycol ester (EGS), disuccinimide glutarate (DSG), N,N'-disuccinimide carbonate (DSC), dimethyl adipamide (DMA), dimethyl heptamethimide (DMP), dimethyl octanoide (DMS), dimethyl-3,3'-dithiobispropionide (DTBP), 1,4-di-3'-(2'- Pyridyl dithiopropamidobutane (DPDPB), bismaleimide hexane (BMH), aryl halogenated compounds (DFDNB) such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylic acid)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide).

[0289] In some embodiments, the connector includes a heterodifunctional connector. Exemplary heterodifunctional connectors include, but are not limited to, amine-reactive and thiol crosslinkers, such as N-succinimidyl 3-(2-pyridyl dithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyl dithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyl dithio)propionate (sulfonyl-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl dithio)toluene (sMPT), and sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyl dithio)toluamide]hexyl Sulfosinate-LC-sMPT, succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimide benzoyl-N-hydroxysuccinimide (MBs), m-maleimide benzoyl-N-hydroxysulfosuccinimide (sulfo-MBs), N-succinimide (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimide (4-iodoacetyl)aminobenzoate (Sulfo-sIAB), succinimide-4-(p-maleimide-phenyl)butyrate (sMPB), sulfosuccinimide-4-(p-maleimide-phenyl)butyrate (sulfo-sMPB), N-(γ-maleimide-butyryloxy)succinimide (GMBs), N-(γ-maleimide-butyryloxy)sulfosuccinimide (sulfo-GMBs), succinimide-6-((iodoacetyl)amino)hexanoate (sIAX), succinimide-6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX) ), succinimide-4-((((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimide-6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA); carbonyl reactive and thiol reactive crosslinkers, such as 4-(4-N-maleimide-phenyl)butyric acid hydrazide (MPBH), 4-(N-maleimide-methyl)cyclohexane-1-carboxy-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH);Amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimide-4-azidosalicylic acid (sulfon-NHs-AsA), sulfosuccinimide-(4-azidosalicylamido)hexanoate (sulfon-NHs-LC-AsA), sulfosuccinimide-2-(p(ρ)-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimide-4-azidosalicylate (HsAB), and N-hydroxysulfosuccinimide-4-azidosalicylate (sulfon-NHs-LC-AsA). -HsAB), N-succinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimide-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfon-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimide-2-(m-azido-o-nitrobenzoamide)-ethyl-1,3'-dithiopropionate (sAND), N-succinimide-4-(4-azidophenyl)1,3'-dithiopropionate (sADP), N-sulfosuccinimide Amino(4-azidophenyl)-1,3'-dithiopropionate (sulfon-sADP), sulfosuccinimide-4-(p-azidophenyl)butyrate (sulfon-sAPB), sulfosuccinimide-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimide-7-azido-4-methylcoumarin-3-acetate (sulfon-sAMCA), p-nitrophenyldiazopyruvate (ρNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP); thiol reactivity and photoreactivity Crosslinkers, such as 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide; carbonyl-reactive and photoreactive crosslinkers, such as p-azidobenzoylhydrazide (ABH); carboxylic acid ester-reactive and photoreactive crosslinkers, such as 4-(p-azidosalicylamido)butylamine (AsBA); and arginine-reactive and photoreactive crosslinkers, such as p-azidophenylglyoxal (APG).

[0290] In some cases, the connector contains a reactive functional group. In other cases, the reactive functional group contains a nucleophilic group that is reactive to an electrophilic group present on the bonding moiety. Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, ketenes, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include acylhydrazides, oximes, amino groups, hydrazines, thioureas, hydrazide carboxylates, and aromatic hydrazides.

[0291] In some embodiments, the connector comprises a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer group. In some cases, the maleimide group further comprises hexanoic acid, forming a maleimide hexanoyl (mc). In some cases, the connector comprises a maleimide hexanoyl (mc). In some cases, the connector is a maleimide hexanoyl (mc). In other cases, the maleimide group comprises a maleimide methyl group, such as succinimidyl-4-(N-maleimide methyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimide methyl)cyclohexane-1-carboxylate (sulfon-sMCC) as described above.

[0292] In some embodiments, the maleimide group is a self-stabilized maleimide. In some cases, the self-stabilized maleimide utilizes diaminopropionic acid (DPR) incorporated into the basic amino group adjacent to the maleimide to provide intramolecular catalysis for the thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination reactions via the reverse Michael reaction. In some cases, the self-stabilized maleimide is... (The sentence is incomplete and requires further context to translate accurately.) , “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol The maleimide group described in 32(10):1059-1062 (2014). In some cases, the linker contains a self-stabilized maleimide. In some cases, the linker is a self-stabilized maleimide.

[0293] In some embodiments, the linker comprises a peptide moiety. In some cases, the peptide moiety comprises at least 2, 3, 4, 5, or 6 or more amino acid residues. In some cases, the peptide moiety comprises up to 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some cases, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some cases, the peptide moiety is a cleavable peptide moiety (e.g., ...). ，(Enzymatic or chemical). In some cases, the peptide moiety is an insoluble peptide moiety. In some cases, the peptide moiety contains Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 106), Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 107), or Gly-Phe-Leu-Gly (SEQ ID NO: 108). In some cases, the linker contains 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 contains Val-Cit. In some cases, the linker is Val-Cit.

[0294] In some embodiments, the connector comprises a benzoic acid group or a derivative thereof. In some cases, the benzoic acid group or its derivative comprises p-aminobenzoic acid (PABA). In some cases, the benzoic acid group or its derivative comprises γ-aminobutyric acid (GABA).

[0295] In some embodiments, the connector comprises one or more of any combination of maleimide groups, peptide moieties, and / or benzoic acid groups. In some embodiments, the connector comprises a combination of maleimide groups, peptide moieties, and / or benzoic acid groups. In some cases, the maleimide group is maleimide hexanoyl (mc). In some cases, the peptide group is val-cit. In some cases, the benzoic acid group is PABA. In some cases, the connector comprises an mc-val-cit group. In some cases, the connector comprises a val-cit-PABA group. In other cases, the connector comprises an mc-val-cit-PABA group.

[0296] In some embodiments, the connector is a self-degrading connector or a self-eliminating connector. In some cases, the connector is a self-degrading connector. In other cases, the connector is a self-eliminating connector (e.g., a cyclization self-eliminating connector). In some cases, the connector includes the connector described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.

[0297] In some embodiments, the linker is a dendritic linker. In some cases, the dendritic linker comprises a branched multifunctional linker portion. In some cases, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some cases, the dendritic linker comprises a PAMAM dendritic polymer.

[0298] In some embodiments, the linker is a traceless linker or a linker that leaves no linker portion (e.g., atoms or linker groups) on the binding site A, polynucleotide B, polymer C, or endosome-dissolved site D after cleavage. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazine linkers. In some cases, the linker is as described by Hejesen et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Org Biomol Chem The traceless aryl-triazine linker described in 11(15): 2493-2497 (2013). In some cases, the linker is Blaney et al., “Traceless solid-phase organic synthesis,” Chem. Rev The non-marking connector described in 102:2607-2024 (2002). In some cases, the connector is the non-marking connector described in U.S. Patent No. 6,821,783.

[0299] In some cases, the connector is the connector described in the following documents: U.S. Patent Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014 / 0127239; 2013 / 028919; 2014 / 286970; 2013 / 0309256; 2015 / 037360; or 2014 / 0294851; or PCT Publication Nos. WO2015057699; WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

[0300] In some implementations, X1 and X2 are independently bonded or non-polymerized joints. In some cases, X1 and X2 are independently bonded. In some cases, X1 and X2 are independently non-polymerized joints.

[0301] In some cases, X1 is a bonded or non-polymeric linker. In some cases, X1 is a bonded linker. In some cases, X1 is a non-polymeric linker. In some cases, the linker is a C1-C6 alkyl group. In some cases, X1 is a C1-C6 alkyl group, for example, C5, C4, C3, C2, or C1 alkyl. In some cases, the C1-C6 alkyl group is an unsubstituted C1-C6 alkyl group. As used in the context of linker, particularly in the context of X1, alkyl means a saturated straight-chain or branched hydrocarbon group containing up to six carbon atoms. In some cases, X1 includes the above-described homo-bifunctional or hetero-bifunctional linkers. In some cases, X1 includes a hetero-bifunctional linker. In some cases, X1 includes an sMCC. In other cases, X1 includes a hetero-bifunctional linker optionally conjugated to a C1-C6 alkyl group. In other cases, X1 includes an sMCC optionally conjugated to a C1-C6 alkyl group. In other cases, X1 does not include the same-functional or different-functional connectors described above.

[0302] In some cases, X2 is a bond or connector. In some cases, X2 is a bond. In other cases, X2 is a connector. In some cases, X2 is a non-polymeric connector. In some embodiments, X2 is a C1-C6 alkyl group. In some cases, X2 is a homo- or hetero-bifunctional connector as described above. In some cases, X2 is a homo-bifunctional connector as described above. In some cases, X2 is a hetero-bifunctional connector as described above. In some cases, X2 contains a maleimide group, such as the aforementioned maleimide-hexanoyl (mc) or a self-stabilizing maleimide group. In some cases, X2 contains a peptide moiety, such as Val-Cit. In some cases, X2 contains a benzoic acid group, such as PABA. In other cases, X2 contains a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In other cases, X2 contains an mc group. In other cases, X2 contains an mc-val-cit group. In other cases, X2 contains a val-cit-PABA group. In other cases, X2 contains the mc-val-cit-PABA group.

[0303] How to use Muscular dystrophy refers to loss of muscle mass and / or progressive weakening and degeneration of muscle. In some cases, muscle mass loss and / or progressive weakening and degeneration occur due to high protein degradation rates, low protein synthesis rates, or both. In some cases, high rates of muscle protein degradation are due to muscle protein catabolism (i.e., the breakdown of muscle protein to utilize amino acids as substrates for gluconeogenesis).

[0304] In one embodiment, muscular dystrophy refers to a significant loss of muscle strength. A significant loss of muscle strength is defined as a reduction in the strength of diseased, injured, or unused muscle tissue in the subject relative to the same muscle tissue in a control subject. In one embodiment, a significant loss of muscle strength is a reduction 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, relative to the same muscle tissue in a control subject. In another embodiment, a significant loss of muscle strength is defined as a reduction in the strength of unused muscle tissue relative to the muscle strength of the same muscle tissue in the same subject prior to a period of inactivity. In one embodiment, a significant loss of muscle strength is a reduction in muscle 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, relative to the muscle strength of the same muscle tissue in the same subject prior to a period of inactivity.

[0305] In another embodiment, muscular dystrophy refers to a significant loss of muscle mass. A significant loss of muscle mass means a reduction in the volume of diseased, injured, or unused muscle tissue in the subject relative to the same muscle tissue in a control subject. In one embodiment, a 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, relative to the same muscle tissue in a control subject. In another embodiment, a significant loss of muscle mass means a reduction in the volume of unused muscle tissue relative to the muscle volume of the same muscle tissue in the same subject prior to a period of inactivity. In one embodiment, a 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, relative to the muscle volume of the same muscle tissue in the same subject prior to a period of inactivity. Optionally, muscle volume is measured by assessing the cross-sectional area of ​​the muscle, for example, by magnetic resonance imaging (e.g., by a muscle volume / cross-sectional area (CSA) MRI method).

[0306] Myotonic dystrophy is a multisystem neuromuscular disease that includes two main types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is caused by the gene DM protein kinase (DM2). DMPKThis is caused by the amplification of dominantly inherited "CTG" repeats in the mRNA. When these repeats are transcribed into mRNA, they form hairpins with high affinity for the myoblinoid protein (MBNL) family. MBNL proteins are involved in posttranscriptional splicing and polyadenylate protein site regulation, and loss of MBNL protein function leads to the accumulation of downstream nuclear lesions and an increase in missplicing events, subsequently resulting in myotonia and other clinical symptoms.

[0307] In some embodiments, this document describes a method for treating muscular dystrophy (e.g., DM1) in a subject, comprising providing the polynucleotide molecule or polynucleotide conjugate described herein, and administering an effective amount of the polynucleotide molecule or polynucleotide conjugate to the subject (the subject in need) to treat muscular dystrophy, wherein the polynucleotide conjugate reduces the body's... DMPK The amount of mRNA transcripts. In some implementations, administering polynucleotide conjugates to the subject... DMPK The reduced number of mRNA transcripts in patients not treated with polynucleotide conjugates DMPK The mRNA expression levels are at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, and at least 80%.

[0308] pharmaceutical preparations In some embodiments, the pharmaceutical formulations described herein are administered to the subject via a variety of routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration. In some cases, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intra-arterial, intraperitoneal, intrathecal, intracerebral, intraventricular, or intracranial) administration. In other cases, the pharmaceutical compositions described herein are formulated for oral administration. In still other cases, the pharmaceutical compositions described herein are formulated for intranasal administration.

[0309] In some embodiments, the pharmaceutical formulation includes, but is not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly melting formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsed-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and hybrid formulations of immediate and controlled release.

[0310] In some cases, pharmaceutical formulations include multi-particulate formulations. In some cases, pharmaceutical formulations include nanoparticle formulations. In some cases, nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Other exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendritic polymers (such as metal chelates with covalent bonds), nanofibers, nanohoms, nano-onions, nanorods, nanowires, and quantum dots. In some cases, nanoparticles are metallic 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 thereof, alloys or oxides thereof.

[0311] In some cases, nanoparticles contain a core or a core and a shell, such as in core-shell nanoparticles.

[0312] In some cases, the nanoparticles are further coated with molecules for attaching functional elements (e.g., one or more of the polynucleotide molecules or binding moieties described herein). In some cases, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agar, alginic acid, erythritol, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitosan (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some cases, the nanoparticles include graphene-coated nanoparticles.

[0313] In some cases, nanoparticles have at least one dimension smaller than about 500 nm, 400 nm, 300 nm, 200 nm or 100 nm.

[0314] In some cases, nanoparticle formulations comprise paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendritic polymers (such as metal chelates with covalent bonds), nanofibers, nanohorns, nanoonions, nanorods, nanotethers, or quantum dots. In some cases, the polynucleotide molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles. In some cases, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleotide molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles.

[0315] In some embodiments, the pharmaceutical formulation includes a delivery vector, such as a recombinant vector, to deliver a polynucleotide molecule into cells. In some cases, the recombinant vector is a DNA plasmid. In other cases, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some cases, the recombinant vector capable of expressing the polynucleotide molecule provides stable expression in target cells. In other cases, a viral vector providing transient expression of the polynucleotide molecule is used.

[0316] In some embodiments, the pharmaceutical formulation includes a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile properties of the desired dosage form. Exemplary carrier materials include, for example, binders, suspensions, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, gum arabic, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurine, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sodium saccharide stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, for example, Remington: The Science and Practice of Pharmacy , Nineteenth edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences Mack Publishing Co., Easton, Pennsylvania 1975; edited by Liberman, HA and Lachman, L. Pharmaceutical Dosage Forms Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, 7th Edition (Lippincott Williams & Wilkins1999).

[0317] In some cases, pharmaceutical formulations further comprise pH adjusters or buffers, 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 tris(hydroxymethyl)aminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0318] In some cases, pharmaceutical formulations contain one or more salts in an amount required to bring the weight molar osmolality of the composition within an acceptable range. Such salts include those having 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.

[0319] In some cases, pharmaceutical formulations further include diluents to stabilize the compounds, as they provide a more stable environment. Salts dissolved in buffer solutions (which also provide pH control or maintenance) are used in the art as diluents, including but not limited to phosphate-buffered saline solutions. In some cases, diluents increase the volume of the composition to facilitate compression or to produce sufficient volume for uniform blending for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, and microcrystalline cellulose such as Avicel. ® ; dicalcium phosphate, dicalcium 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, confectionery; basic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, glucose dextrorate; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[0320] In some cases, pharmaceutical formulations contain disintegrants to facilitate the breakdown or disintegration of the substance. The term "disintegration" refers to the dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches like corn starch or potato starch, and pregelatinized starches like National 1551 or Amijel. ® Or sodium glycolate starch such as Promogel® Or Explotab ® Cellulose, such as wood products, methyl crystalline cellulose, such as Avicel ® Avicel ® PH101, Avicel ® PH102, Avicel ® PH105, Elcema ® P100, Emcocel ® Vivacel ® Ming Tia ® and Solka-Floc ® methylcellulose, croscarmellose cellulose, or croscarmellose cellulose such as croscarmellose sodium (Ac-Di-Sol) ® Cross-linked carboxymethyl cellulose or cross-linked carboxymethyl cellulose, cross-linked starch such as sodium glycolate starch, cross-linked polymers such as povidone, cross-linked polyvinylpyrrolidone, alginate such as alginic acid or salts of alginic acid such as sodium alginate, clay such as Veegum ® HV (magnesium aluminum silicate), gums such as agar, guar gum, locust bean gum, ebony gum, pectin or tragacanth gum, sodium glycolate starch, bentonite, natural sponges, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, combinations of sodium lauryl sulfate and starch, etc.

[0321] In some cases, pharmaceutical preparations contain fillers such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, glucose binders, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[0322] Lubricants and flow aids may also be optionally included in the pharmaceutical formulations described herein for preventing, reducing, or inhibiting adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearoyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex). ® Higher fatty acids and their alkali metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, and zinc salts, stearic acid, sodium stearate, glycerol, talc, waxes, Stearotate ® Boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxy polyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl betaine, polyethylene glycol, magnesium dodecyl sulfate or sodium dodecyl sulfate, colloidal silica such as Syloid™, Cab-O-Sil ® Starch, such as corn starch, silicone oil, surfactants, etc.

[0323] Plasticizers are compounds used to soften microencapsulated materials or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. Plasticizers are also used as dispersants or wetting agents.

[0324] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium dodecyl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetrahydrofuran polyethylene glycol ether (glycofurol), diethylene glycol monoethyl ether (transcutol), propylene glycol, and dimethyl isosorbide.

[0325] Stabilizers include compounds such as any antioxidants, buffers, acids, preservatives, etc.

[0326] Suspension agents include polyvinylpyrrolidone such as 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 about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methylcellulose. Compounds include hydroxymethyl cellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, gums such as gum arabic and gum arabic, guar gum, xanthan gum (including xanthan gum), sugars, and celluloses such as sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

[0327] Surfactants include sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin ETPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, and copolymers of ethylene oxide and propylene oxide such as Pluronic. ®Compounds such as (BASF). Other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10 and octoxynol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

[0328] Viscosity enhancers include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose acetate stearate, hydroxypropyl methylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0329] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E, TPGS, and ammonium salts.

[0330] Treatment plan In some embodiments, the pharmaceutical composition described herein is applied for therapeutic use. In some embodiments, the pharmaceutical composition is applied once daily, twice daily, three times daily, or more frequently. The pharmaceutical composition may be applied 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 for a longer period. The pharmaceutical composition may be applied for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen ...

[0331] In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially, or at intervals. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In other cases, one or more pharmaceutical compositions are administered at intervals (e.g., the first administration of the first pharmaceutical composition is on day one, followed by an interval of at least 1, 2, 3, 4, 5, or more days before the administration of at least the second pharmaceutical composition).

[0332] In some embodiments, two or more different pharmaceutical compositions are administered together. In some cases, two or more different pharmaceutical compositions are administered simultaneously. In some cases, two or more different pharmaceutical compositions are administered sequentially without a time interval between administrations. In other cases, two or more different pharmaceutical compositions are administered sequentially with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or longer between administrations.

[0333] If the patient's condition does improve, the medication may continue to be administered based on the physician's judgment; or the dosage of the medication may be temporarily reduced or temporarily suspended for a certain period of time (i.e., a "withdrawal period"). In some cases, the length of the withdrawal period varies from 2 days to 1 year, including, for example, 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. During the withdrawal period, the dose is reduced by 10%-100%, for example only, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0334] Once the patient's condition improves, a maintenance dose may be administered if necessary. Subsequently, depending on changes in symptoms, the dose or frequency, or both, may be reduced to a level where the improvement in the disease, symptom, or condition can be maintained.

[0335] In some embodiments, the amount of a given agent corresponding to such a quantity varies depending on factors such as the specific compound, the severity of the disease, the characteristics of the person or host requiring treatment (e.g., weight), yet it is still conventionally determined in a manner known in the art based on the specific circumstances relevant to that instance, including, for example, the specific agent administered, the route of administration, and the person or host being treated. In some cases, the required dose is conveniently presented as a single dose or in fractions, which are administered simultaneously (or over short periods of time) or at appropriate intervals, such as two, three, four, or more sub-dose per day.

[0336] The aforementioned ranges are merely suggestive, as the number of variables relating to individual treatment regimens is enormous, and considerable deviations from these recommended values ​​are not uncommon. Such dosages vary depending on numerous variables, including but not limited to the activity of the compound used, the disease or condition being treated, the method of administration, the individual's needs, the severity of the disease or condition being treated, and the physician's judgment.

[0337] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined through standard pharmaceutical procedures in cell cultures or laboratory animals, including but not limited to the determination of LD50 (the 50% lethal dose for a population) and ED50 (the 50% therapeutically effective dose for a population). The dose ratio between toxic and therapeutic effects is the therapeutic index and is expressed as the ratio of LD50 to ED50. Compounds exhibiting a high therapeutic index are preferred. Dosage ranges for human use are established using data obtained from cell culture experiments and animal studies. The dosage of such compounds is preferably within the cyclic concentration range containing the ED50 and exhibiting minimal toxicity. The dosage varies within this range depending on the dosage form and route of administration used.

[0338] reagent kits / products In some embodiments, kits and articles thereof are disclosed for use with one or more compositions and methods described herein. Such kits include carriers, packaging, or containers compartmentalized to receive one or more containers such as vials, tubes, etc., each containing a single element to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed of various materials such as glass or plastic.

[0339] The articles described herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, vials, and any packaging materials suitable for the selected formulation and the intended method of administration and treatment.

[0340] For example, the container contains the target nucleic acid molecule described herein. Such a kit may optionally include an identifying description or label or instruction regarding its use in the methods described herein.

[0341] Kits typically include a label listing the contents and / or instructions for use, as well as a packaging insert with usage instructions. A set of instruction manuals will also usually be included.

[0342] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other characters constituting the label are affixed, molded, or etched onto the container itself; the label is associated with the container when it is present within a receiver or carrier that also contains the container (e.g., as a packaging insert). In one embodiment, the label is used to indicate that the contents will be used for a specific therapeutic application. The label also provides guidance regarding the use of the contents, such as in the methods described herein.

[0343] In some embodiments, the pharmaceutical composition is provided in a package or dispenser device containing one or more unit dosage forms of the compound provided herein. For example, the package may contain metal or plastic foil, such as blister packs. In one embodiment, the package or dispenser device is accompanied by instructions for use. In one embodiment, the package or dispenser is also accompanied by a container-associated notice in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting that the agency has approved the form of the drug for human or veterinary administration. Such a notice may be, for example, a label or approved product insert approved by the U.S. Food and Drug Administration for prescription drugs. In one embodiment, a composition containing the compound provided herein formulated in a compatible drug carrier is also prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

[0344] certain terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative only and are not intended to limit any of the claimed subject matter. In this application, the singular is used to include the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the / that” include the plural referents unless the context clearly indicates otherwise. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “having” is non-limiting.

[0345] As used herein, ranges and quantities may be expressed as “about” a specific value or range. The term “about” also includes an exact quantity. Thus, “about 5 μL” means “about 5 μL” and also “5 μL”. Typically, the term “about” includes the quantity expected to be within experimental error.

[0346] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0347] As used herein, the terms “individual,” “object,” and “patient” refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., a physician, registered nurse, nurse practitioner, physician assistant, caregiver, or hospice worker).

[0348] The term "therapeutic effective amount" refers to the amount of a polynucleotide conjugate sufficient to provide the 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) for the purpose of treating, preventing, preventing the onset of, curing, delaying, reducing the severity of, alleviating at least one symptom of, or preventing the recurrence of, a condition, or extending the patient's survival beyond what would be expected without such treatment. Naturally, the dosage level of a specific polynucleotide conjugate used to provide a therapeutically effective amount varies depending on the type of lesion, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the specific inhibitor used. In some cases, as described herein, the therapeutically effective amount of the polynucleotide conjugate is initially estimated from cell cultures and animal models. For example, the IC50 determined in cell culture methods... 50 The value is optionally used as the starting point for the animal model, while the IC determined in the animal model... 50 The value is optionally used to find therapeutically effective doses in humans.

[0349] Skeletal muscles, or voluntary muscles, are typically anchored to bones by tendons and are generally used to achieve skeletal movements, such as motion or posture maintenance. Although some control over skeletal muscles is usually maintained in an involuntary reflex manner (e.g., postural muscles or the diaphragm), skeletal muscles respond to conscious control. Smooth or involuntary muscles are found in the walls of organs and structures such as the esophagus, stomach, intestines, uterus, urethra, and blood vessels.

[0350] Skeletal muscle is further divided into two main categories: Type I (or "slow-twitch muscle") and Type II (or "fast-twitch muscle"). Type I muscle fibers have dense capillaries and are rich in mitochondria and myoglobin, which gives Type I muscle tissue its characteristic red color. In some cases, Type I muscle fibers carry more oxygen and use fat or carbohydrates as fuel to sustain aerobic exercise. Type I muscle fibers can contract for a long time, but with little force. Type II muscle fibers are further subdivided into three main subtypes (IIa, IIx, and IIb), which differ in contraction speed and the force generated. Type II muscle fibers contract rapidly and powerfully, but fatigue quickly, thus producing only a brief burst of anaerobic activity before the muscle contraction becomes painful.

[0351] Unlike skeletal muscles, smooth muscles are not under conscious control.

[0352] Cardiac muscle is also involuntary muscle, but structurally it is more similar to skeletal muscle and exists only in the heart. Cardiac and skeletal muscles are striated because they contain sarcomeres arranged in highly regular bundles. In contrast, myofibrils in smooth muscle cells are not arranged in sarcomeres and therefore lack striation.

[0353] Muscle cells include any cells that contribute to muscle tissue. Exemplary muscle cells include myoblasts, satellite cells, myotubes, and myofibrils.

[0354] As used herein, muscle strength is proportional to cross-sectional area (CSA), while muscle velocity is proportional to muscle fiber length. Therefore, comparing the cross-sectional area and muscle fibers across various muscles can provide an indication of muscle atrophy. Various methods for measuring muscle strength and muscle weight are known in the art; see, for example, Hazel M. Clarkson, “Musculoskeletal assessment: Joint range of motion and manual muscle strength,” Lippincott Williams & Wilkins, 2000. Another method for measuring muscle mass is to evaluate the generation of tomographic images from selected muscle tissue using computed axial computed tomography and ultrasound examination.

[0355] This application provides, but is not limited to, the following implementation methods: 1. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described herein has a sense strand and an antisense strand, wherein the sense strand has a sequence having at least 80% identity with SEQ ID NO: 1, and the antisense strand has a sequence having at least 80% identity with SEQ ID NO: 2; and The polynucleotide conjugates mediated by the above-mentioned polynucleotide molecules target DMPK RNA interference.

[0356] 2. The polynucleotide conjugate as described in Embodiment 1, wherein the anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17; the HCDR2 sequence EINPIX1GRSNYAX2KFQG, wherein X1 is selected from N or Q and X2 is selected from Q or E; and the HCDR3 sequence of SEQ ID NO: 19.

[0357] 3. The polynucleotide conjugate as described in Embodiment 2, wherein the VH region comprises an HCDR1 sequence including SEQ ID NO: 17, an HCDR2 sequence including one of SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 21, and an HCDR3 sequence including SEQ ID NO: 19.

[0358] 4. A polynucleotide conjugate as described in any one of embodiments 2-3, wherein the VL region 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 may or may not be present, or if present, it is F.

[0359] 5. A polynucleotide conjugate as described in any one of embodiments 2-4, wherein the VL region comprises the LCDR1 sequence, the LCDR2 sequence AATNLAX5 and the LCDR3 sequence QHFWGTPLTX6 of SEQ ID NO: 22, wherein X5 is selected from D or E and X6 may or may not be present, or if present, is F.

[0360] 6. The polynucleotide conjugate of any one of embodiments 2-5, wherein the VL region comprises an LCDR1 sequence including SEQ ID NO: 22 or SEQ ID NO: 27, an LCDR2 sequence including SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 28, and an LCDR3 sequence including SEQ ID NO: 24 or SEQ ID NO: 26.

[0361] 7. The polynucleotide conjugate of any one of embodiments 2-6, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24.

[0362] 8. The polynucleotide conjugate of any one of embodiments 2-6, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24.

[0363] 9. The polynucleotide conjugate of any one of embodiments 2-6, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 21, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 25, and the LCDR3 sequence of SEQ ID NO: 26.

[0364] 10. The polynucleotide conjugate of any one of embodiments 2-6, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 27, the LCDR2 sequence of SEQ ID NO: 28, and the LCDR3 sequence of SEQ ID NO: 26.

[0365] 11. The polynucleotide conjugate of any one of embodiments 2-10, wherein the VH region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with a sequence selected from SEQ ID NO: 29-33.

[0366] 12. The polynucleotide conjugate of any one of embodiments 2-11, wherein the VL region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with a sequence selected from SEQ ID NO: 34-38.

[0367] 13. The polynucleotide conjugate as described in any one of embodiments 2-12, wherein the VH region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 34.

[0368] 14. A polynucleic acid conjugate as described in any one of embodiments 1-13, wherein the anti-transferrin receptor antibody comprises a humanized antibody or its antigen-binding fragment, or a chimeric antibody or its antigen-binding fragment, or a multispecific antibody or its antigen-binding fragment.

[0369] 15. A polynucleotide conjugate as described in any one of embodiments 1-14, wherein the anti-transferrin receptor antibody comprises IgG-scFv, nanobody, BiTE, biantibody, DART, TandAb, scDiabody, scDiabody-CH3, triantibody, microantibody, microantibody, TriBi microantibody, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, or intracellular antibody.

[0370] 16. The polynucleotide conjugate of any one of embodiments 1-15, wherein the anti-transferrin receptor antibody comprises an IgG1 framework.

[0371] 17. The polynucleotide conjugate of any one of embodiments 1-15, wherein the anti-transferrin receptor antibody comprises an IgG2 framework.

[0372] 18. The polynucleotide conjugate as described in Embodiment 17, wherein the IgG2 framework is the IgG2b framework.

[0373] 19. The polynucleotide conjugate as described in any one of embodiments 1-15, wherein the anti-transferrin receptor antibody comprises an IgG4 framework.

[0374] 20. A polynucleotide conjugate as described in any one of embodiments 1-19, wherein the anti-transferrin receptor antibody further comprises at least one mutation in the Fc region.

[0375] 21. A polynucleotide molecular conjugate as described in any one of embodiments 1-20, wherein the at least one mutation regulatory effector function is described.

[0376] 22. A polynucleotide conjugate as described in any one of embodiments 20-21, wherein the at least one mutation weakens or eliminates Fc-γ receptor binding.

[0377] 23. A polynucleotide conjugate as described in any one of embodiments 20-22, wherein the at least one mutation is located at residue positions D265, N297, K322, L328 or P329, wherein the residue positions refer to IgG1.

[0378] 24. A polynucleotide conjugate as described in any one of embodiments 20-23, wherein the Fc region comprises two or more, three or more, or four or more mutations.

[0379] 25. A polynucleotide molecular conjugate as described in any one of embodiments 20-24, wherein the Fc region comprises mutations at L233 and L234, wherein the residues correspond to positions 233 and 234 of SEQ ID NO: 39.

[0380] 26. The polynucleotide conjugate of any one of embodiments 20-25, wherein the Fc region comprises mutations at D265 and N297.

[0381] 27. The polynucleotide conjugate of any one of embodiments 1-26, wherein the anti-transferrin receptor antibody comprises a heavy chain (HC) sequence selected from SEQ ID NO: 39-62 and a light chain (LC) sequence selected from SEQ ID NO: 63-66.

[0382] 28. A polynucleic acid conjugate as described in any one of embodiments 1-27, wherein the anti-transferrin receptor antibody specifically binds to the human transferrin receptor (TfR).

[0383] 29. A polynucleotide conjugate as described in any one of embodiments 1-28, wherein the sense strand and the antisense strand each independently comprise at least one 2' modified nucleotide, at least one modified internucleotide linker, or at least one reverse abase-free moiety.

[0384] 30. A polynucleotide conjugate as described in any one of embodiments 1-29, wherein the sense strand comprises a nucleotide modified with a 2'-O-methyl at its 5' end.

[0385] 31. A polynucleotide molecular conjugate as described in any one of embodiments 1-30, wherein the sense strand comprises at least two consecutive 2'-O-methyl modified nucleotides at the 5' end.

[0386] 32. A polynucleotide conjugate as described in any one of embodiments 1-31, wherein the sense strand comprises at least three, four, five or six consecutive 2'-O-methyl modified nucleotides at its 5' end.

[0387] 33. The polynucleotide conjugate of any one of embodiments 1-32, wherein the sense strand comprises six consecutive 2'-O-methyl modified nucleotides at the 5' end.

[0388] 34. A polynucleotide conjugate as described in any one of embodiments 1-33, wherein the sense strand comprises at least one 2'-F modified nucleotide.

[0389] 35. A polynucleotide conjugate as described in any one of embodiments 1-33, wherein the sense strand comprises at least two or at least three 2'-F modified nucleotides.

[0390] 36. A polynucleotide conjugate as described in any one of embodiments 1-33, wherein the sense strand comprises at least two or at least three consecutive 2'-F modified nucleotides.

[0391] 37. A polynucleotide conjugate as described in any one of embodiments 1-36, wherein the sense strand comprises a nucleotide modified with a 2'-O-methyl at its 3' end.

[0392] 38. A polynucleotide conjugate as described in any one of embodiments 1-37, wherein the sense strand comprises at least two consecutive 2'-O-methyl modified nucleotides at the 3' end.

[0393] 39. A polynucleotide conjugate as described in any one of embodiments 1-38, wherein the sense strand comprises at least three, four, five, six, seven, eight, nine, or ten consecutive 2'-O-methyl modified nucleotides at its 3' end.

[0394] 40. A polynucleotide conjugate as described in any one of embodiments 1-39, wherein the sense strand comprises ten consecutive 2'-O-methyl modified nucleotides at the 3' end.

[0395] 41. A polynucleotide molecular conjugate as described in any one of embodiments 1-40, wherein the sense strand comprises at least two phosphate thioester nucleotides linked together.

[0396] 42. A polynucleotide molecular conjugate as described in any one of embodiments 1-41, wherein the sense strand has a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15.

[0397] 43. The polynucleotide conjugate of any one of embodiments 1-42, wherein the antisense strand comprises a nucleotide modified with a 2'-O-methyl at its 5' end.

[0398] 44. The polynucleotide conjugate of any one of embodiments 1-43, wherein the antisense strand comprises a nucleotide modified with a 2'-O-methyl at its 3' end.

[0399] 45. A polynucleotide conjugate as described in any one of embodiments 1-44, 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.

[0400] 46. ​​The polynucleotide conjugate of any one of embodiments 1-45, wherein the antisense strand comprises five consecutive 2'-O-methyl modified nucleotides at the 3' end.

[0401] 47. A polynucleotide conjugate as described in any one of embodiments 1-46, wherein the antisense strand comprises at least one, at least two, at least three, or at least four 2'-F modified nucleotides.

[0402] 48. A polynucleotide conjugate as described in any one of embodiments 1-47, wherein the antisense strand comprises four 2'-F modified nucleotides, wherein any two of the four 2'-F modified nucleotides are not consecutive.

[0403] 49. A polynucleotide conjugate as described in any one of embodiments 1-48, wherein the antisense strand comprises two overhanging nucleotides at the 3' end.

[0404] 50. A polynucleotide molecular conjugate as described in any one of embodiments 1-49, wherein the antisense strand comprises at least two or at least three phosphate thioester nucleotides linked together.

[0405] 51. The polynucleotide conjugate of any one of embodiments 1-50, wherein the antisense strand has the sequence SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16.

[0406] 52. A polynucleotide conjugate as described in any one of embodiments 1-51, wherein the polynucleotide conjugate comprises a linker connecting an anti-transferrin receptor antibody or an antigen-binding fragment thereof to the polynucleotide molecule.

[0407] 53. The polynucleic acid molecular conjugate as described in embodiment 52, wherein the adapter is a C6 adapter.

[0408] 54. The polynucleic acid molecular conjugate as described in Embodiment 53, wherein the C6 linker is a 6-amino-1-hexanol linker.

[0409] 55. The polynucleic acid molecular conjugate as described in Embodiment 52, wherein the linker is a homo-bifunctional linker or a hetero-bifunctional linker, a maleimide group, a dipeptide moiety, a benzoic acid group or a derivative thereof.

[0410] 56. The polynucleotide molecular conjugate of any one of embodiments 52-55, wherein the linker comprises 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).

[0411] 57. A polynucleic acid molecular conjugate as described in any one of embodiments 52-56, wherein the linker is coupled to the 5' end of the sense strand.

[0412] 58. A polynucleotide conjugate as described in any one of embodiments 52-57, wherein the polynucleotide molecule is conjugated to a cysteine ​​residue of the anti-transferrin receptor antibody or its antigen-binding fragment.

[0413] 59. The polynucleotide conjugate as described in Embodiment 58, wherein the cysteine ​​residue is located in the Fc domain of the anti-transferrin receptor antibody or its antigen-binding fragment.

[0414] 60. A polynucleotide conjugate as described in any one of embodiments 1-59, wherein the ratio of the polynucleotide molecule to the anti-transferrin receptor antibody or its antigen-binding fragment is about 1:1, 2:1, 3:1 or 4:1.

[0415] 61. A polynucleotide molecular conjugate as described in any one of embodiments 1-60, wherein the polynucleotide portion mediates RNA interference against the human DMPK gene to regulate muscle atrophy in the subject.

[0416] 62. The polynucleotide conjugate of embodiment 61, wherein the RNA interference comprises reducing the expression of the mRNA transcript of the DMPK gene by at least 50%, at least 60%, or at least 70% compared to the amount of the mRNA transcript of the DMPK gene in cells affected by muscular dystrophy.

[0417] 63. The polynucleic acid conjugate as described in any one of embodiments 61-62, wherein the muscular dystrophy is myotonic dystrophy type 1 (DM1).

[0418] 64. A polynucleic acid conjugate comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, wherein the sense strand has a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15, and the antisense strand has a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; and The anti-transferrin receptor antibody or its antigen-binding fragment is coupled to the polynucleotide molecule via a linker comprising 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).

[0419] 65. A polynucleic acid conjugate comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, wherein the sense strand has a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15, and the antisense strand has a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0420] 66. A polynucleic acid conjugate comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; The sense strand comprises at least three, four, five or six consecutive 2'-O-methyl modified nucleotides at the 5'-end and at least two or three 2'-F modified nucleotides; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0421] 67. A polynucleic acid conjugate comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; 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 and at least one, at least two, at least three, or at least four 2'-F modified nucleotides; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0422] 68. A polynucleic acid conjugate comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; The antisense strand contains nucleotides modified with 2'-O-methyl at the 5' and 3' ends; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

[0423] 69. A polynucleic acid conjugate comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; The antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides at the 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 its antigen-binding fragment 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 with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a 6-amino-1-hexanol linker.

[0424] 70. A pharmaceutical composition comprising: The polynucleic acid molecular conjugate as described in any one of embodiments 1-69; and Pharmaceutically acceptable excipients.

[0425] 71. The pharmaceutical composition of embodiment 70, wherein the pharmaceutical composition is formulated as a nanoparticle formulation.

[0426] 72. The pharmaceutical composition of any one of embodiments 70-71, wherein the pharmaceutical composition is formulated for parenteral, oral, intranasal, oral, rectal or transdermal administration.

[0427] 73. A method for treating muscular dystrophy in patients of need, comprising: Provide a polynucleic acid conjugate as described in any one of embodiments 1-69 or a pharmaceutical composition as described in any one of embodiments 70-72; and The polynucleotide conjugate is administered to the recipient to treat the muscular dystrophy, wherein the polynucleotide conjugate reduces the body's... DMP The number of K mRNA transcripts.

[0428] 74. The method as described in embodiment 73, wherein the polynucleotide moiety mediates targeting of humans DMPK RNA interference regulates muscle atrophy in the target organism.

[0429] 75. The method of any one of embodiments 73-74, wherein the muscular dystrophy is myotonic dystrophy type 1 (DM1).

[0430] 76. Use of the polynucleic acid molecular conjugate as described in any one of Embodiments 1-69 or the pharmaceutical composition as described in any one of Embodiments 70-72 for the treatment of a subject diagnosed with or suspected of having myotonic dystrophy type 1 (DM1).

[0431] 77. Use of the polynucleic acid molecular conjugate as described in any one of Embodiments 1-69 or the pharmaceutical composition as described in any one of Embodiments 70-72 for the manufacture of a medicament for the treatment of a subject diagnosed with or suspected of having myotonic dystrophy type 1 (DM1).

[0432] 78. A kit comprising a polynucleotide molecular conjugate as described in Examples 1-69 or a pharmaceutical composition as described in any one of Examples 70-72.

[0433] Example These embodiments are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0434] Example 1. Antibody-siRNA conjugate DMPK-AOC is an antibody-siRNA conjugate drug product, formed by conjugating a humanized IgG1 antibody targeting human transferrin receptor 1 (anti-human transferrin receptor antibody) with a double-stranded siRNA oligonucleotide (DMPK siRNA) targeting DMPK mRNA. Figure 1 The SMCC maleimide linker is located at the 5' end of the transpass chain and conjugates to the antibody via a cysteine ​​residue in the antibody's amino acid sequence. The conjugate binds to the human transferrin receptor on the cell surface, is internalized into the cell, and delivers the siRNA oligonucleotide to the intracellular compartment. After being taken up by the cell, the siRNA is loaded into the RISC and hydrolyzes the target pathogenic DMPK mRNA.

[0435] The anti-human transferrin receptor antibody and DMPK siRNA used to create DMPK-AOC were manufactured by a GMP-compliant commercial contract research and manufacturing organization (CDMO) using a sophisticated manufacturing process. The anti-human transferrin receptor antibody was produced in CHO cells using recombinant protein expression technology, while the DMPK siRNA was produced using a standard phosphoramide solid-phase synthetic chemistry method. In this paper, DMPK siRNA is a double-stranded siRNA oligonucleotide targeting DMPK mRNA, which is also conjugated to an SMCC linker attached to the 5' end of the transferase chain. Each of these has been fully characterized and formally released. The production of DMPK-AOC involved a standard random cysteine ​​bioconjugation reaction of the anti-human transferrin receptor antibody with maleimide of DMPK siRNA, followed by anion exchange chromatography purification to separate the bulk conjugate, which was then converted to the finished product DMPK-AOC. The finished product DMPK-AOC was formally released using standard methods for protein therapeutics. After completion of manufacturing, testing, and release, each antibody and DMPK siRNA were bioconjugated together to form the drug substance.

[0436] Example 2. Preparation of antibody AV01mAb The stable research cell bank (RCB) for stable cell lines is constructed using CHOK1SV host working cells and has been confirmed to be free from contamination by mycoplasma, bacteria, fungi, and yeast. A master cell bank (MCB) of 200 vials has been prepared using research cell bank vials.

[0437] Antibody generation from the master cell bank – Cells from ampoules of the master cell bank are gradually increased in volume using a protein-free culture medium before being inoculated into the production bioreactor. Downstream processing – After cell culture is complete, cells and cell debris are removed by filtration of the culture.

[0438] Example 3. Structural characterization of anti-human transferrin receptor antibody Structure - The amino acid sequences of the heavy and light chains have been determined by translating the nucleotide sequence of the anti-human transferrin receptor antibody.

[0439] Heavy chain sequence of anti-human transferrin receptor antibody - SEQ ID NO: 48 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Light chain sequence of anti-human transferrin receptor antibody - SEQ ID NO: 63 DIQMTQSPSSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGKSPKLLIYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 4: DMPK siRNA DMPK siRNA is a synthetic double-stranded oligonucleotide comprising a 19-mer transit strand and a complementary 21-mer guide strand with a dinucleotide overhang at the 3' end of the guide strand. A C6-SMCC linker {4-(N-maleimidemethyl)cyclohexane-1-carboxamide} is attached to the 5' end of the transit strand, enabling conjugation to antibody intermediates. The nucleotide sequence and internucleotide linkages are shown in Table 11.

[0440] Table 11

[0441] RNA single strands (guide strand and guest strand) were individually produced via solid-phase synthesis using a well-established phosphoramide solid-phase synthesis method. The purified and lyophilized single strands were then double-stranded in equimolar ratios to generate double-stranded siRNA. The SMCC adapter was conjugated to the primary amine stalk at the 5' end of the sense strand of the siRNA using standard N-hydroxysuccinimide chemistry. Excess unreacted SMCC adapters were removed using a UF / DF step, releasing the resulting SMCC-siRNA.

[0442] Example 5. Antibody Selection Antibody screening criteria Several antibodies against human transferrin receptor 1 (TfR1) were tested by ELISA and found to have high affinity for the receptor. Binding of these antibodies to cynomolgus monkey TfR1 was also tested to ensure species cross-reactivity. The specificity of mouse IgG2a monoclonal antibodies (mAbs) binding to human and cynomolgus monkey TfR1 was assessed by ELISA to demonstrate the lack of binding to the closely related transferrin receptor 2 (TfR2). Figure 3 By ELISA, the commercially available anti-TfR2 antibody B-6 showed a clear binding to TfR2, while mouse anti-human TfR1 mAb did not bind to TfR2 at concentrations up to 10 µM. The binding of mouse anti-human TfR1 mAb in the presence of the TfR1 binding ligand transferrin (Tf) and homeostatic iron regulator (HFE) was also evaluated; even in the presence of the TfR1 ligand, TfR1 mAb maintained a strong binding to TfR1. Figure 4 ).like Figure 4As shown, the antibody binds directly to TfR1, or TfR1 is pre-bound to the cofactor transferrin (Tf) or HFE. AF2474 is a commercially available antibody known to bind to the same TfR1 epitope as transferrin or HFE. Some loss of binding was observed between direct TfR1 interaction and the cofactor complex with mouse anti-human TfR1 mAb, but the change in affinity was minor compared to AF2474. Importantly, since TfR1 mAb competing with the natural ligand for TfR1 is expected to be toxic due to the potential to hinder iron entry into cells, the identified TfR1 mAb needs to bind to an epitope on TfR1 that minimizes competition with the natural ligand. Given that it met all these screening criteria, mouse IgG2a anti-human TfR1 mAb was moved to the humanization project to develop an antibody suitable for clinical development.

[0443] Example 6. In vivo activity DMPK-AOC was used in in vivo studies in mice. In vivo mouse studies of AOC used an alternative anti-TfR1 antibody that binds to mouse TfR1 because the lead human antibody AV01Ab does not cross-react with mouse TfR1 (human and monkey only). Mouse cross-reactive siDMPK.36 was conjugated to an anti-mouse TfR1 mAb, and the conjugate was administered intravenously to wild-type female CD1 mice (n=4 per group). Tissue samples were collected 7 days after administration, and DMPK mRNA knockdown was assessed. AOC was administered at doses of 3, 1, 0.3, and 0.1 mg / kg (based on the weight of siRNA), with a 3 mg / kg dose resulting in an 80% reduction in DMPK expression in skeletal muscle. Figure 5 DMPK-AOC exhibited potent activity, with an ED50 < 1 mg / kg and an EC50 of approximately 3 nM. The negative control scrambled siRNA sequence showed no DMPK mRNA knockdown, demonstrating the specificity of DMPK-AOC activity.

[0444] The mouse cross-reactive DMPK-AOC was conjugated with anti-mouse TfR1 mAb and administered intravenously to wild-type female CD-1 mice (n=4 per group) at 3 mg / kg (based on siRNA weight). Tissues were collected, and DMPK mRNA knockdown was assessed weekly until 5 weeks post-administration. Figure 6 The greatest DMPK knockdown (approximately 75%) was achieved in skeletal muscle between days 7 and 35 post-dose. Slightly lower DMPK knockdown (approximately 65%) was achieved in the heart, while no DMPK knockdown was observed in the liver despite the presence of DMPK siRNA. The long duration of activity following a single AOC dose reduces the need for frequent dosing.

[0445] In vivo pharmacological data in non-human primatesDMPK-AOC was administered intravenously over 30 minutes to wild-type male cynomolgus macaques (n=3 per group), and tissues were collected over 12 weeks post-administration. Skeletal muscle was surgically biopsied under ketamine / toluidine anesthesia. At 12 weeks post-administration, after final blood and muscle biopsies, the animals were euthanized with an overdose of euthanasia solution and administered a sedative. Terminal perforated biopsies were then collected from multiple additional tissue samples. A 75% reduction in DMPK expression following a single intravenous administration of 2 mg / kg AOC (based on siRNA weight) was durable and persisted until 12 weeks post-administration. Figure 7 ).

[0446] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the scope of this disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practice. The scope of this disclosure is intended to be defined by the appended claims, thereby covering the methods and structures within the scope of those claims and their equivalents.

Claims

1. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described herein has a sense strand and an antisense strand, wherein the sense strand has a sequence having at least 80% identity with SEQ ID NO: 1, and the antisense strand has a sequence having at least 80% identity with SEQ ID NO: 2; and The polynucleotide conjugates mediated by the above-mentioned polynucleotide molecules target DMPK RNA interference.

2. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, wherein the sense strand has a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15, and the antisense strand has a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; and The anti-transferrin receptor antibody or its antigen-binding fragment is coupled to the polynucleotide molecule via a linker comprising 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).

3. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, wherein the sense strand has a sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13 or 15, and the antisense strand has a sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14 or 16; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

4. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; The sense strand comprises at least three, four, five or six consecutive 2'-O-methyl modified nucleotides at the 5'-end and at least two or three 2'-F modified nucleotides; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

5. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; 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 and at least one, at least two, at least three, or at least four 2'-F modified nucleotides; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 20, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

6. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; The antisense strand contains nucleotides modified with 2'-O-methyl at the 5' and 3' ends; The anti-transferrin receptor antibody or its antigen-binding fragment comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the HCDR1 sequence of SEQ ID NO: 17, the HCDR2 sequence of SEQ ID NO: 18, and the HCDR3 sequence of SEQ ID NO: 19; and the VL region comprises the LCDR1 sequence of SEQ ID NO: 22, the LCDR2 sequence of SEQ ID NO: 23, and the LCDR3 sequence of SEQ ID NO: 24; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a maleimide linker.

7. A polynucleic acid conjugate, comprising: Anti-transferrin receptor antibodies or their antigen-binding fragments conjugated to polynucleotide molecules, wherein the polynucleotide molecules are conjugated to... DMPK Hybridization with the target sequence; The polynucleotide molecule described therein has a sense strand and an antisense strand, the sense strand having the sequence of SEQ ID NO: 1 and the antisense strand having the sequence of SEQ ID NO: 2; The antisense strand comprises at least five consecutive 2'-O-methyl modified nucleotides at the 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 its antigen-binding fragment 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 with SEQ ID NO: 30, and wherein the VL region has at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 34; and The anti-transferrin receptor antibody or its antigen-binding fragment is conjugated to the polynucleotide molecule via a 6-amino-1-hexanol linker.

8. A pharmaceutical composition comprising: Polynucleic acid molecular conjugates as described in any one of claims 1-7; and Pharmaceutically acceptable excipients.

9. A method for treating muscular dystrophy in a subject of need, comprising: Provide a polynucleic acid conjugate as described in any one of claims 1-7 or a pharmaceutical composition as described in claim 8; as well as The polynucleotide conjugate is administered to the recipient to treat the muscular dystrophy, wherein the polynucleotide conjugate reduces the body's... DMP The number of K mRNA transcripts.

10. Use of the polynucleic acid molecular conjugate of any one of claims 1-7 or the pharmaceutical composition of claim 8 for the treatment of subjects diagnosed with or suspected of having myotonic dystrophy type 1 (DM1).

11. Use of the polynucleic acid molecular conjugate of any one of claims 1-7 or the pharmaceutical composition of claim 8 for the manufacture of a medicament for the treatment of a subject diagnosed with or suspected of having myotonic dystrophy type 1 (DM1).

12. A kit comprising the polynucleotide molecular conjugates as described in claims 1-7 or the pharmaceutical composition as described in claim 8.

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