Drug for treating duchenne muscular dystrophy and becker muscular dystrophy

Abstract

The present invention is applicable to the technical field of molecular biology, and particularly provides an oligonucleotide and a conjugate comprising the oligonucleotide. The oligonucleotide is configured to bind to a precursor mRNA (pre-mRNA) of a DMD gene, thereby promoting the increase or restoration of the expression or activity of dystrophin. A pharmaceutical composition comprising the oligonucleotide or the conjugate can be used for treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

Description

TECHNICAL FIELD

[0001] The present invention relates to the field of biomedicine, and in particular to a medicament for treating Duchenne muscular dystrophy and Becker muscular dystrophy and the use thereof. BACKGROUND

[0002] Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are both X-linked recessive monogenic disorders that predominantly affect male children. The underlying cause is a reduction in dystrophin levels in muscle cells, which leads to muscle inflammation, fibrosis, and decreased motor function. Without intervention, the majority of patients die before the age of 30.

[0003] Dystrophin is expressed in the human body by the DMD gene, which is a large gene comprising 79 exons and over 2 million total base pairs. Mutations in the DMD gene, including frameshift, deletion, substitution, and duplication mutations, often result in premature termination of mRNA or nonsense-mediated mRNA decay (NMD), thereby reducing the expression of functional dystrophin.

[0004] The use of oligonucleotides to induce exon skipping of the mutated DMD gene, thereby restoring the expression of functional dystrophin, represents one of the therapeutic strategies for DMD. Currently, four drugs have been approved by the U.S. Food and Drug Administration (FDA), and their long-term efficacy remains under evaluation.

[0005] Existing oligonucleotide therapies have limited efficacy due to their short plasma half-life and poor distribution to muscle tissue. For example, in the pivotal registration clinical trial of eteplirsen, which was approved in 2016, administration at a dose of at least 30 mg / kg weekly for 48 weeks increased dystrophin levels in DMD patients from 0.16% before treatment to only 0.44% after treatment.

[0006] Therefore, there is a need to improve the efficacy of existing therapeutics. SUMMARY

[0007] One object of the present application is to provide an oligonucleotide that can increase or restore the expression or activity of dystrophin, thereby treating Duchenne muscular dystrophy and / or Becker muscular dystrophy in a subject.

[0008] Another object of the present application is to provide a conjugate comprising the above-mentioned oligonucleotide and a muscle-targeting agent.

[0009] A further object of the present application is to provide a use of the oligonucleotide or the conjugate for preventing or treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

[0010] To solve the above problems, the present application provides a conjugate that targets muscle cells for delivering an active ingredient to these cells.

[0011] In a first aspect, the present application provides an oligonucleotide, wherein the oligonucleotide is used for promoting exon skipping of dystrophin pre- mRNA and / or hybridizing to a target sequence of human DMD mRNA and mediating RNA interference against human DMD mRNA in muscle cells of a human subject, thereby promoting an increase or restoration of dystrophin expression or activity, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or a phosphorodiamidate morpholino oligomer (PMO) sequence, and the oligonucleotide binds to at least a portion of an exon and / or a non-exon region of dystrophin, or binds to the DMD gene pre-mRNA.

[0012] Specifically, the PMO sequence refers to a sequence in which the five membered furanosyl ring in all nucleotides is replaced by a six-membered morpholine ring.

[0013] In some embodiments, the oligonucleotide comprises or consists of a sequence that is capable of at least partially binding to any one of dystrophin pre-mRNA exons 44, 45, 50, 51, 52 and 53, and the sequence has 10 to 33 nucleotides.

[0014] In some embodiments, the oligonucleotide has a sequence of SEQ ID NOs: 001-121, or a sequence having at least 90% homology thereto, preferably at least 92%, 95%, or 98% homology thereto, and most preferably at least 99% homology thereto; or for RNA-form sequences, T in the above SEQ ID NOs: 001121 is replaced with U.

[0015] In a second aspect, the present application provides a conjugate comprising the above-mentioned oligonucleotide and a muscle-targeting agent, wherein the muscle-targeting agent is an antibody or an antigen-binding fragment.

[0016] In other embodiments, the conjugate comprises a ribonucleoprotein (RNP) or an active compound for binding to DMD gene pre-mRNA in the muscle cell nucleus, thereby promoting an increase or restoration of dystrophin expression or activity.

[0017] In some embodiments, the conjugate further comprises a muscle targeting agent.

[0018] In some embodiments, the muscle-targeting agent is an antibody, an antigen-binding fragment, or a small molecule compound.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab’, a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

[0020] In some embodiments, the muscle-targeting agent is an antibody or polypeptide capable of specifically binding to human transferrin receptor 1 (TfR1) on the surface of muscle cells, thereby achieving muscle targeting and enrichment.

[0021] In some embodiments, the muscle-targeting agent has 1 or 2 binding sites for human TfR1, with an affinity to human TfR1 between 0.01 nM and 100 nM.

[0022] In some embodiments, the binding of the muscle-targeting agent to human TfR1 does not affect the function of TfR1 in binding transferrin.

[0023] In some embodiments, the antibody comprises a light chain variable (VL) region and a heavy chain variable (VH) region.

[0024] In other embodiments, the muscle-targeting agent can also deliver the

[0025] In some embodiments, the muscle-targeting agent is covalently linked to the active ingredient via a non-cleavable or cleavable linker.

[0026] In some embodiments, the linker is N-succinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or other chemical linkers that remain stable in plasma.

[0027] In some embodiments, the molar ratio of the active ingredient to the muscle-targeting agent (or drug-to-antibody ratio, DAR) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, or 16:1, preferably 1:1 to 8:1.

[0028] In some embodiments, the antibody or polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 285.

[0029] In some embodiments, the conjugate enters the endosomes of muscle cells via receptor-mediated endocytosis, and there is a certain correlation between endocytosis efficiency and affinity.

[0030] In some embodiments, the conjugate or the active ingredient thereof is released into the cytoplasm through endosomal escape, and the release of the conjugate or the active ingredient thereof may not depend on endosomal cleavage of the linker.

[0031] In some embodiments, the conjugate is used for the treatment of Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD).

[0032] In a third aspect, the present application provides a pharmaceutical composition comprising the oligonucleotide as described above or the conjugate as described above, wherein the pharmaceutical composition is used for treating Duchenne muscular dystrophy and Becker muscular dystrophy.

[0033] In some embodiments, the pharmaceutical composition is for intravenous, subcutaneous, parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

[0034] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier, or excipient.

[0035] In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation.

[0036] In a fourth aspect, the present application provides a method of delivering the above-mentioned oligonucleotide to a cell, the method comprising conjugating the above-mentioned oligonucleotide with an antibody, an antibody fragment, a polypeptide, an antigen-binding sequence, or a small molecule compound.

[0037] In a fifth aspect, the present application provides a method of increasing or restoring dystrophin expression or activity in a cell, the method comprising contacting the cell with the above-mentioned oligonucleotide or conjugate.

[0038] In a sixth aspect, the present application provides a method of treating Duchenne muscular dystrophy and / or Becker muscular dystrophy in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of the above-mentioned oligonucleotide, conjugate, or pharmaceutical composition, to treat Duchenne muscular dystrophy and / or Becker muscular dystrophy in the subject.

[0039] Preferably, the subject is a human.

[0040] In a seventh aspect, the present application provides a use of the above mentioned oligonucleotide or the above-mentioned conjugate in the manufacture of a medicament for increasing or restoring dystrophin expression or activity, or for preventing and / or treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] The embodiments of the present invention provide a series of oligonucleotides, including but not limited to antisense oligonucleotides (ASOs) or phosphorodiamidate morpholino oligomers (PMOs), wherein the oligonucleotides bind to at least a portion of an exon and / or a non-exon region of dystrophin pre-mRNA and promote exon skipping, thereby yielding a protein that is near full-length but retains partial dystrophin function. The exon-skipping efficiency of the PMO drug of the present invention is significantly better than that of PMO drugs disclosed in other patents (Example 2).

[0043] The antibody-oligonucleotide conjugate provided in the embodiments of the present invention significantly alters the drug clearance pathway compared to existing oligonucleotide drugs. Existing oligonucleotide drugs, including peptide-conjugated oligonucleotides, are primarily cleared via the kidneys. After conjugation with an antibody, the molecular weight of the oligonucleotide is greatly increased, no longer available for the renal clearance route, thereby significantly prolonging plasma half-life and achieving dose-dependent muscle-targeted enrichment. At the same dose, the skeletal muscle PMO concentration of the antibody-PMO conjugate of the present invention is two orders of magnitude higher than that of existing PMO drugs.

[0044] The antibody-oligonucleotide conjugate also exhibits efficacy advantages over existing oligonucleotide drugs. The antibody-PMO conjugate significantly increases the exon-skipping ratio compared to existing PMO drugs, in a dose-dependent manner. The antibody-PMO conjugate significantly increases the full-length dystrophin levels in skeletal muscle, cardiac muscle, and diaphragm compared to existing PMO drugs, also in a dose-dependent manner. The antibody-PMO conjugate consistently reduces the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) in mdx mice, demonstrating a reparative effect on DMD-induced liver and muscle damage. At a single dose of 10 mg / kg, the antibody-PMO conjugate is sufficient to restore 100% dystrophin levels in mdx mice and maintain this for at least 28 days, reducing the dosing frequency to one-quarter and the total administered dose to one-twelfth of that required for existing PMO drugs, while the relative dystrophin level is significantly increased to more than 20-fold. Thus, the antibody-oligonucleotide conjugate has the potential to provide superior therapeutic benefits for DMD patients while reducing both the total administered dose and dosing frequency.

[0045] Second, the embodiments of the present invention also conducted mouse pharmacokinetic studies, cynomolgus monkey pharmacokinetic studies, in vitro activity assays, and affinity tests on the antibody-siRNA conjugate. The results showed that the antibody-siRNA conjugate prolonged the plasma half-life of the siRNA and increased siRNA distribution in muscle, with muscle siRNA levels exceeding or approaching those in the liver, demonstrating the muscle-targeting capability of the TfR1 antibody. No significant changes in body weight or behavioral observations were noted in either mice or cynomolgus monkeys during the experiments, indicating good tolerability of the conjugate. The antibody-siRNA conjugate was able to enter RD cells under in vitro co-incubation conditions without the aid of lipofectamine and exerted dose-dependent target gene knockdown. Conjugation of siRNA had an adverse effect on antibody-antigen affinity, and the greater the number of conjugations, the more pronounced the decline in affinity. These findings lay a foundation for further development of antibody-oligonucleotide conjugates.

[0046] Third, the results of affinity and in vitro activity assays on the peptide oligonucleotide conjugates provided in the embodiments of the present invention showed that peptide-oligonucleotide conjugation similarly reduced the peptide-antigen affinity; and that without the aid of lipofectamine, the conjugates could enter RD cells under in vitro co-incubation conditions and exert dose-dependent target gene knockdown.

[0047] All the above results indicate that conjugates comprising an oligonucleotide and a muscle-targeting agent (including antibodies and peptides) can prolong the plasma half-life of small nucleic acid drugs, increase distribution of such drugs in muscle, significantly improve the therapeutic efficacy of existing small nucleic acid drugs for the treatment of muscle-related diseases, thereby exhibiting enhanced drug development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Fig. 1 shows a gel electrophoresis image showing exon skipping achieved by PMO in patient-derived cardiomyocytes.

[0049] Fig 2 shows a capillary electrophoresis image showing exon skipping achieved by PMO in patient-derived cardiomyocytes.

[0050] Fig. 3 shows a schematic diagrams of Linker-PMO and SMCC-linker- PMO.

[0051] Fig. 4 shows SDS-PAGE of TIB-219 and TIB-219-S245C.

[0052] Fig. 5 shows an SEC chromatogram of TIB-219.

[0053] Fig. 6 shows an SEC chromatogram of TIB-219-S245C.

[0054] Fig. 7 shows purification profile of the crude antibody-PMO conjugate after reaction, with fractions P2-P4 collected.

[0055] Fig. 8 shows IEX result of the crude reaction product.

[0056] Fig. 9 shows mass spectrometry results of the collected fractions from the crude reaction product.

[0057] Fig. 10 shows an IEX chromatogram of the purified antibody-PMO conjugate.

[0058] Fig. 11 shows an SEC chromatogram of the purified antibody-PMO conjugate.

[0059] Fig. 12 shows a schematic diagram of the hELISA principle and primer design.

[0060] Fig. 13 shows tissue distribution of CGBC-1011 in mdx mice after a single dose.

[0061] Fig. 14 shows a muscle-to-liver ratio of CGBC-1011 in mdx mice after a single dose.

[0062] Fig. 15 shows exon skipping efficiency (nested PCR) in muscle tissues of mdx mice after a single dose of CGBC-1011.

[0063] Fig. 16 shows exon skipping efficiency (nested PCR) in non-muscle tissues of mdx mice after a single dose of CGBC-1011.

[0064] Fig. 17 shows exon skipping efficiency (ddPCR) in muscle tissues of mdx mice after a single dose of CGBC-1011.

[0065] Fig. 18 shows blood biochemistry parameters in mdx mice after a single dose of CGBC-1011.

[0066] Fig. 19 shows exon skipping efficiency (nested PCR) in muscle tissues of mdx mice after a single dose (low dose) of CGBC-1011.

[0067] Fig. 20 shows dystrophin restoration levels in muscle tissues of mdx mice after a single dose (low dose) of CGBC-1011.

[0068] Fig. 21 shows a molecular chromatogram of CGBC-1004-1 (A280 top, A260 bottom).

[0069] Fig. 22 shows a molecular chromatogram of CGBC-1004-2 (A280 top, A260 bottom).

[0070] Fig. 23 shows a molecular chromatogram of CGBC-1005-1 (A280 top, A260 bottom).

[0071] Fig. 24 shows a molecular chromatogram of CGBC-1005-2 (A280 top, A260 bottom).

[0072] Fig. 25 shows a molecular chromatogram of CGBC-1012-1 (A280 top, A260 bottom).

[0073] Fig. 26 shows a molecular chromatogram of CGBC-1012-2 (A280 top, A260 bottom).

[0074] Fig. 27 shows siRNA distribution of CGBC-1004 (DAR1 and DAR2) in plasma, muscle, and liver of wild-type CD-1 mice.

[0075] Fig 28 shows siRNA distribution of CGBC-1005 (DAR1 and DAR2) in plasma, muscle, and liver of wild-type CD-1 mice.

[0076] Fig. 29 shows muscle-to-liver AUC ratios for different antibody-siRNA conjugate designs.

[0077] Fig 30 shows the PK curve of CGBC-1012 (DAR1) in cynomolgus monkey plasma.

[0078] Fig 31 shows the PK curve of CGBC-1012 (DAR1) in cynomolgus monkey gastrocnemius muscle.

[0079] Fig 32 shows in vitro activity of CGBC-1012 (DAR1 and DAR2) in human rhabdomyosarcoma cells.

[0080] Fig 33 shows antigen affinity of CGBC-1012 (DAR1 and DAR2) and unconjugated antibody.

[0081] Fig 34 shows antigen affinity of CGBC-1004 (DAR1 and DAR2), CGBC-1005 (DAR1 and DAR2), and unconjugated antibody.

[0082] Fig 35 shows conjugation of CGBC-1018. The conjugation process was verified by HPLC and SDS-PAGE, and the molecular weight of DAR1 conjugate was verified by mass spectrometry.

[0083] Fig 36 shows in vitro activity of CGBC-1018 in human rhabdomyosarcoma cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0084] The embodiments of the present application describe various conjugates for delivering oligonucleotide drugs to muscle cells and uses thereof. In order to make the technical problems, technical solutions, and beneficial effects of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0085]     Definition of Terms

[0086] In the present application, "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by an antisense compound. The target nucleic acid can be DNA or RNA; specifically, the target RNA can be mRNA, pre-mRNA, non-coding RNA, pre-microRNA, mature microRNA, promoter-directed RNA, or natural antisense transcripts. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a specific disease or condition, or a nucleic acid molecule from an infectious agent.

[0087] In the present application, "oligonucleotide" refers to a compound comprising multiple linked nucleosides. Thus, although the term "oligonucleotide" generally refers to a polymer of nucleotides in which the nucleosides and their linkages are naturally occurring, it should be understood that the term also includes various analogs in which one or more of the nucleosides are modified. Oligonucleotides typically have about 10-30 nucleoside residues.

[0088] In the present application, the term "antisense oligonucleotide (ASO)" refers to a single-stranded oligonucleotide molecule having a nucleobase sequence that is complementary to a corresponding segment of a target nucleic acid (e.g., a target genomic sequence, pre-mRNA, or mRNA molecule), i.e., it hybridizes to the target nucleic acid and modulates the activity, processing, or expression of the target nucleic acid. The antisense oligonucleotide can be 12 to 30 nucleobases in length and may include unmodified or modified nucleic acids. ASOs first bind to the target RNA and then cleave the target RNA via RNase H, thereby preventing protein expression. Since RNase H is present both inside and outside the nucleus, ASOs can act not only on mRNA but also on certain non-coding RNAs. Among them, "phosphorodiamidate morpholino oligomers (PMOs)" belong to the third-generation antisense oligonucleotides and are synthetic oligonucleotide analogs that primarily inhibit target gene function by blocking mRNA splicing. PMOs contain a sixmembered morpholine ring in place of the five-membered furanosyl ring found in natural DNA and RNA, and each morpholine ring is connected through neutral phosphorodiamidate linkages instead of the negatively charged phosphodiester linkages in natural DNA and RNA. Each phosphorodiamidate morpholino subunit contains one of the heterocyclic bases found in DNA (adenine, cytosine, guanine, or thymine).

[0089] In the embodiments of the present application, PMOs represented by DNA sequences (e.g., the sequences in Table 5), after replacing T with U, can be used to represent RNA sequences capable of binding to the same target sequence, such as ASO sequences.

[0090] In the present application, "antisense activity" refers to any detectable and / or measurable activity based on the hybridization of an antisense compound to its target nucleic acid. Such activity may be an increase or decrease in the amount of a nucleic acid or protein, or a change in the ratio of splice variants of a nucleic acid or protein.

[0091] In the present application, "small interfering RNA (siRNA)" (also known as short interfering RNA or silencing RNA) targets nucleic acids (e.g., mRNA) for degradation through the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule can be determined by the binding of its antisense strand to its target RNA. Typically, about 14 to about 50 of these nucleotides are complementary to the target RNA sequence, constituting the specific antisense sequence of the siRNA molecule. After selecting an appropriate target RNA sequence, siRNA molecules comprising nucleotide sequences complementary to all or part of the target sequences (i.e., an antisense sequences) can be designed and prepared using appropriate methods. siRNA molecules may comprise a duplex with self-complementary sense and antisense strands, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure.

[0092] In the present application, "antisense strand" refers to the strand of a polynucleotide molecule (e.g., dsRNA) that has a region substantially complementary to the target sequence. As used herein, the term "complementary region" generally refers to the region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence). When the complementary region is not fully complementary to the target sequence, mismatches may be present in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[0093] In the present application, "sense strand" (S) refers to a region that is substantially complementary to the region of the antisense strand as defined above. The "sense strand" is sometimes referred to as the "coding strand." By virtue of their sequences, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by introducing modifications into the sense strand or by using 5' caps.

[0094] In the present application, "targeting" or "targeted to" refers to the binding of an antisense oligonucleotide to a specific target nucleic acid molecule or to a specific region of nucleotides within a target nucleic acid molecule. An antisense oligonucleotide targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid molecule to allow hybridization under physiological conditions.

[0095] In the present application, "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at corresponding positions after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has an identity percentage to SEQ ID NO: Y, and is stated as X% of the residues in the amino acid sequence being identical to the residues disclosed in SEQ ID NO: Y.

[0096] In the present application, "modified oligonucleotide" or "chemically modified oligonucleotide" refers to an oligonucleotide comprising at least one modified sugar, modified base, and / or modified internucleoside linkage or backbone.

[0097] In the present application, "internucleoside linkage" or "backbone" refers to the covalent bond between adjacent nucleosides.

[0098] In the present application, the term "expression" generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of a 5'-cap), and translation.

[0099] In the present application, "conjugate" refers to an atom or a group of atoms that is attached to an oligonucleotide or oligomeric compound. Typically, conjugate groups alter a variety of properties of the compounds to which they are attached, such as pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and clearance. Conjugate groups are commonly used in the chemical arts and are attached directly or via optional linking moieties or linking groups (i.e., linkers) to parent compounds such as oligomeric compounds.

[0100] In the present application, "linker" refers to any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. Linking groups or bifunctional linking moieties, such as those known in the art, are amenable to the present invention.

[0101] In the present application, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3.

[0102] In the present application, the term "framework region" or "FR" refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0103] In the present application, the term "specific binding" refers to a non random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of that interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between two molecules can be determined using methods well known in the art, for example, using surface plasmon resonance (SPR) on a BIACORE instrument.

[0104] The antibodies and antigen-binding fragments provided herein also encompass various variants of the antibody sequences provided herein. The term "variant" refers to an antibody that retains the binding specificity of its parent antibody for SARS-CoV-2, but has one or more desirable properties conferred by mutations. Antibody variants may include one or more mutations in one or more of the above CDR sequences, in one or more non-CDR sequences of the heavy chain variable region or light chain variable region, and / or in the constant region (e.g., Fc region). For example, antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamidation, reduced or ablated effector function, improved FcRn receptor binding, prolonged pharmacokinetic half-life, pH sensitivity, and / or compatibility with conjugation.

[0105] In the present application, the term "effective amount" refers to an amount of a compound of the present invention sufficient to treat muscular dystrophy, slow or minimize the progression of muscular dystrophy, or provide a therapeutic benefit in the treatment or management of muscular dystrophy.

[0106] In the present application, the term "subject" includes any human or non-human animal. The term "non-human animal" may be a vertebrate (e.g., nonhuman primate, sheep, dog) and a rodent (e.g., mouse, rat, and guinea pig). The subject is preferably a human. The term "subject" is used interchangeably herein with "individual" and "patient."

[0107] Oligonucleotide

[0108] The oligonucleotide described in the present application can be used to bind / target / hybridize to the DMD gene pre-mRNA, thereby promoting an increase or restoration of dystrophin expression or activity.

[0109] In some embodiments, the oligonucleotide comprises an antisense oligonucleotide (ASO) or a small interfering RNA (siRNA) sequence.

[0110] In some embodiments, the oligonucleotide is based on an antisense oligonucleotide (ASO), and the oligonucleotide is used to induce exon skipping of DMD or dystrophin pre-mRNA in a cell, organ, tissue, and / or individual. Exon skipping results in a mature DMD or dystrophin mRNA that does not contain the skipped exon; therefore, when the exon encodes amino acids, it can lead to the expression of a shorter protein product. Preferably, exon skipping is induced by binding the ASO to specific exon-internal sequences comprising splicing regulatory elements, splice sites, and / or intronic branch point sequences.

[0111] In some embodiments, the oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO) backbone, and the oligonucleotide binds to at least a portion of a dystrophin exon and / or non-exon region sequence, wherein the bound portion has 10 to 33 nucleotides.

[0112] In some embodiments, the oligonucleotide comprises or consists of a sequence that binds to at least a portion of dystrophin pre-mRNA exons 44 to 55, and the sequence has 10 to 33 nucleotides, and the oligonucleotide functions to promote exon skipping of dystrophin pre-mRNA.

[0113] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 001-014, wherein the target sequence of which is SEQ ID NO: 122-135, and the oligonucleotide is capable of promoting skipping of exon 44 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NO: 001-014 or a sequence differing therefrom by no more than 3 nucleotides.

[0114] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 015-035, wherein the target sequence of which is SEQ ID NO: 136-156, and the oligonucleotide is capable of promoting skipping of exon 45 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NO: 015-035 or a sequence differing therefrom by no more than 3 nucleotides.

[0115] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 036-052, wherein the target sequence of which is SEQ ID NO: 157-173, and the oligonucleotide is capable of promoting skipping of exon 50 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NO: 036-052 or a sequence differing therefrom by no more than 3 nucleotides.

[0116] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 053-073, wherein the target sequence of which is SEQ ID NO: 174-194, and the oligonucleotide is capable of promoting skipping of exon 51 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NO: 053-073 or a sequence differing therefrom by no more than 3 nucleotides.

[0117] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 074-092, wherein the target sequence of which is SEQ ID NO: 195-213, and the oligonucleotide is capable of promoting skipping of exon 52 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NO: 074-092 or a sequence differing therefrom by no more than 3 nucleotides.

[0118] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 093-121, wherein the target sequence of which is SEQ ID NO: 214-242, and the oligonucleotide is capable of promoting skipping of exon 53 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NO: 093-121 or a sequence differing therefrom by no more than 3 nucleotides.

[0119] In some embodiments, the oligonucleotide has a small interfering RNA (siRNA) sequence, wherein the siRNA molecule hybridizes to a target sequence of human DMD mRNA and mediates RNA interference against human DMD mRNA in muscle cells of a human subject.

[0120] In some embodiments, the siRNA comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 267- 272. In some embodiments, the siRNA has a sequence selected from any one of SEQ ID NO: 267-272 or a sequence differing therefrom by no more than 3 nucleotides.

[0121] In some embodiments, the siRNA is a double-stranded structure comprising a sense strand and an antisense strand. In some embodiments, the antisense strand comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 268, SEQ ID NO: 270, and SEQ ID NO: 272. In some embodiments, the antisense strand has a sequence selected from any one of SEQ ID NO: 268, SEQ ID NO: 270, or SEQ ID NO: 272, or a sequence differing therefrom by no more than 3 nucleotides. In some embodiments, the sense strand comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of the sequences set forth in SEQ ID NO: 267, SEQ ID NO: 269, and SEQ ID NO: 271. In some embodiments, the sense strand has a sequence selected from any one of SEQ ID NO: 267, SEQ ID NO: 269, or SEQ ID NO: 271, or a sequence differing therefrom by no more than 3 nucleotides.

[0122] In some embodiments, the nucleic acid monomers in the PMO backbone and the siRNA sequence are chemically modified. Specifically, the chemical modification comprises modification of the sugar moiety, modification of the internucleoside linkage, modification of the nucleotide itself, and combinations thereof.

[0123] Muscle-Targeting Agent

[0124] In some embodiments, the muscle-targeting agent is an antibody, a polypeptide, an antigen-binding fragment, or a small molecule compound.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab’, a bivalent Fab2, an F(ab)’3 fragment, a single-chain variable fragment (scFv), a dual scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an IgNAR, or a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or the chemically modified derivatives thereof.

[0126] In some embodiments, the muscle-targeting agent is an antibody or antigen-binding fragment that specifically binds to human transferrin receptor 1 (TfR1) on the surface of muscle cells, thereby achieving muscle targeting and enrichment.

[0127] In some embodiments, the binding of the muscle-targeting agent to human TfR1 does not affect the function of TfR1 in binding transferrin.

[0128] In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is an anti-human TfR1 antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an anti-mouse TfR1 antibody.

[0129] In some embodiments, the antibody comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the antibody comprises an IgG framework. In some embodiments, the antibody comprises an IgG1 framework. In some embodiments, the Fc region of the antibody is derived from a human IgG1 subclass monoclonal antibody.

[0130] In some embodiments, the antibody comprises one or more mutations in the framework regions, for example, in the CH1 domain, CH2 domain, CH3 domain, hinge region, or combinations thereof. In some embodiments, the one or more mutations are for stabilizing the antibody and / or prolonging half-life. In some embodiments, the one or more mutations are for modulating Fc receptor interactions to reduce or eliminate Fc effector functions, such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In other cases, the one or more mutations are for modulating glycosylation.

[0131] In some embodiments, the one or more mutations are located in the Fc region. In some embodiments, the mutation is an LALA-LR mutation, which can reduce immunogenicity of the Fc region. In some embodiments, the mutation is an S239C, S238C, or S245C mutation, which can create a site suitable for site-specific conjugation of nucleic acids.

[0132] In some embodiments, the antibody is conjugated to the oligonucleotide by random (non-specific) or site-specific conjugation. In some embodiments, the conjugation process is generally applicable to different antibodies and sequences, and differences in antibody targets or sequences, as well as nucleic acid sequences, generally do not result in significant changes to the conjugation process.

[0133] In some embodiments, the antibody is non-specifically conjugated to the oligonucleotide. In some embodiments, the antibody is non-specifically conjugated to the siRNA. In some embodiments, the antibody is conjugated to the oligonucleotide via lysine residues or cysteine residues in a non-site-specific manner.

[0134] In some embodiments, the antibody is conjugated to the oligonucleotide in a site-specific manner. In some embodiments, the binding moiety A is conjugated to the polynucleotide molecule (B) via lysine residues, cysteine residues, at the 5' end, 3' end, non-natural amino acids, or enzyme-modified or enzyme-catalyzed residues in a site-specific manner.

[0135] In some embodiments, the muscle-targeting agent has 1 or 2 binding sites for human TfR1, with an affinity between 0.01 nM and 100 nM.

[0136] In some embodiments, one or more of the oligonucleotides are conjugated to the muscle-targeting agent. In some embodiments, the DAR (or molar ratio) of the oligonucleotide to the muscle-targeting agent is between 1 and 8.

[0137] In some embodiments, the antibody comprises a light chain variable (VL) region and a heavy chain variable (VH) region.

[0138] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 245, 247, 249, or 251, and the VL region comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 246, 248, 250, or 252, wherein SEQ ID NO: 245 corresponds to SEQ ID NO: 246, SEQ ID NO: 247 corresponds to SEQ ID NO: 248, SEQ ID NO: 249 corresponds to SEQ ID NO: 250, and SEQ ID NO: 251 corresponds to SEQ ID NO: 252.

[0139] In some embodiments, the anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region has the sequence of SEQ ID NO: 245, 247, 249, or 251, and the VL region has the sequence of SEQ ID NO: 246, 248, 250, or 252, wherein SEQ ID NO: 245 corresponds to SEQ ID NO: 246, SEQ ID NO: 247 corresponds to SEQ ID NO: 248, SEQ ID NO: 249 corresponds to SEQ ID NO: 250, and SEQ ID NO: 251 corresponds to SEQ ID NO: 252.

[0140] In some embodiments, the antibody or binding fragment thereof is further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, alone or in combination, and / or by recombinant and / or any other modifications known in the art (e.g., post-translational and chemical modifications such as glycosylation and phosphorylation). In some embodiments, the modification further includes a modification for modulating interactions with receptors.

[0141] In some embodiments, the muscle-targeting agent is a polypeptide. In some embodiments, the polypeptide comprises a polypeptide sequence containing at least one CDR. The polypeptide sequence comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 285. In some embodiments, the polypeptide has the sequence of SEQ ID NO: 285.

[0142] Conjugate

[0143] The conjugate described in the present application increases or restores the expression or activity of dystrophin, and the active ingredient of the conjugate is based on the oligonucleotide described above.

[0144] In other embodiments, the active ingredient of the conjugate is based on a ribonucleoprotein (RNP) or a small molecule, and is capable of binding to the DMD gene pre-mRNA in the muscle cell nucleus, thereby promoting an increase or restoration of dystrophin expression or activity.

[0145] In some embodiments, the conjugate comprises an oligonucleotide and a muscle-targeting agent. In some embodiments, the muscle-targeting agent also has the effect of delivering the active ingredient to muscle. In some embodiments, the muscle is heart, diaphragm, quadriceps, gastrocnemius, etc. In other embodiments, the muscle-targeting agent also has the effect of delivering the active ingredient to the nervous system.

[0146] In some embodiments, the conjugate enters the endosomes of muscle cells via receptor-mediated endocytosis, and there is a certain correlation between endocytosis efficiency and affinity. In some embodiments, the conjugate or the active ingredient thereof is released into the cytoplasm through endosomal escape, and the release of the conjugate or the active ingredient thereof may not depend on endosomal cleavage of the linker. In some embodiments, the conjugate is used for the treatment of Duchenne muscular dystrophy and Becker muscular dystrophy.

[0147] In some embodiments, the muscle-targeting agent has the effect of prolonging serum half-life. In some embodiments, the 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, or 30 days.

[0148] In some embodiments, the conjugate comprising an oligonucleotide and a muscle-targeting agent exhibits greater activity than the oligonucleotide without the muscle-targeting agent. In some embodiments, the higher activity can enhance biologically relevant functions, for example, improved stability, affinity, binding, functional activity, and efficacy in the treatment or prevention of disease states. In some embodiments, the disease state is the result of one or more mutated exons of a gene. In some embodiments, the conjugate comprising an oligonucleotide and a muscle-targeting agent, compared to the oligonucleotide without the muscletargeting agent, is capable of increasing exon skipping of one or more mutated exons. In some embodiments, the exon skipping is increased by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0149] In some embodiments, the muscle-targeting agent is covalently linked to the active ingredient via a linker, and the conjugate comprises a linker connecting the muscle-targeting agent to the active ingredient. In some embodiments, the linker is a cleavable linker or a non-cleavable linker.

[0150] In some embodiments, the linker is a non-polymeric linker. A non- polymeric linker refers to a linker that does not contain repeating units of monomers produced by a polymerization process. The non-polymeric linker includes, but is not limited to, C1-C6 alkyl groups (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 non-polymeric 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 other cases, the non-polymeric linker optionally contains one or more reactive functional groups. crosslinker. The homobifunctional crosslinker includes, but is not limited to, Lomant's reagent dithiobis(succinimidyl propionate) (DSP), 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycol bis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1,4-di-[3'-(2'-pyridyldithio) propionamido] butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing 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-[P-(4-azidosalicylamido)ethyl]disulfide       (BASED),       formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-tolidine, 3,3'-dimethylbenzidine, benzidine, a,a‘-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylenebis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide), and the like.

[0152] In some embodiments, the linker comprises a heterobifunctional crosslinker. The heterobifunctional crosslinker includes, but is not limited to, aminereactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl    3-(2- pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene        (sMPT),        N-succinimidyl        4-(N- maleimidomethyl)cyclohexane-1-carboxylate       (SMCC),       sulfo-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide      ester      (MBs),      m-maleimidobenzoyl-N- hydroxysulfosuccinimide     ester     (sulfo-MBs),     N-succinimidyl     (4- iodoacetyl)aminobenzoate (sIAB), N-(Y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl   6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate   (sIAXX), succinimidyl 4-((((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl                   6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1- carbonyl)amino)hexanoate (sIACX), and the like.

[0153] In some embodiments, the linker is N-succinimidyl 4-(N- maleimidomethyl) cyclohexane-1-carboxylate (SMCC) or other chemical linkers that remain stable in plasma.

[0154] In some embodiments, the linker is conjugated to the 3' end of the PMO. In some embodiments, the 3' end of the PMO is activated with C3-NH2 and then coupled to the linker under aqueous conditions.

[0155] In some embodiments, the linker is conjugated to the sense strand of the siRNA. In some embodiments, the linker is conjugated to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the 5' end of the sense strand of the siRNA contains a C6-NH2 conjugation handle, and the linker is coupled to the conjugation handle under aqueous conditions. In some embodiments, the conjugation handle is linked to the sense strand of the siRNA via a phosphodiester bond on the terminal base.

[0156] In some embodiments, the muscle-targeting agent and the oligonucleotide are conjugated via random or site-specific conjugation to form the conjugate.

[0157] In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent via a chemical conjugation process. In some embodiments, the oligonucleotide is conjugated to the muscle-targeting agent via native ligation. In some embodiments, the oligonucleotide is conjugated to the muscle-targeting agent via native ligation chemistry in a site-specific or non-specific manner.

[0158] In some embodiments, the oligonucleotide is mixed and incubated with the reduced muscle-targeting agent for conjugation in a random manner. In some embodiments, the oligonucleotide is a PMO conjugated to SMCC at its 3' end, or an siRNA conjugated to SMCC at its 5' end. In some embodiments, the 3' end of the PMO is activated with C3-NH2 and then coupled to SMCC under aqueous conditions. In some embodiments, the 5' end of the sense strand of the siRNA contains a C6-NH2 conjugation handle, and the linker is coupled to the conjugation handle under aqueous conditions. In some embodiments, the muscle-targeting agent is an antibody. In some embodiments, the muscle-targeting agent is an antibody or antigen-binding fragment that specifically binds to TfR1. In some embodiments, the muscle-targeting agent is reduced with TCEP (a thiol-based reducing agent). In some embodiments, the interchain disulfide bonds of the antibody are reduced.

[0159] In some embodiments, the oligonucleotide is mixed and incubated with the muscle-targeting agent that has been reduced and then re-oxidized, for conjugation in a site-specific manner. In some embodiments, the oligonucleotide is an siRNA conjugated to SMCC at its 5' end. In some embodiments, the 5' end of the sense strand of the siRNA contains a C6-NH2 conjugation handle, and the linker is coupled to the conjugation handle under aqueous conditions. In some embodiments, the muscle-targeting agent is an antibody. In some embodiments, the muscletargeting agent is an antibody or antigen-binding fragment that specifically binds to TfR1. In some embodiments, the muscle-targeting agent is a cysteine-mutated antibody. The muscle-targeting agent is reduced with TCEP (a thiol-based reducing agent). In some embodiments, the site-specific cysteine conjugation sites of the antibody (depending on the specific antibody sequence, including but not limited to S239C, S238C, S245C, etc.) and the interchain disulfide bonds are reduced. In some embodiments, the interchain disulfide bonds are oxidized.

[0160] In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent via a site-specific method utilizing "traceless" conjugation technology (Philochem). In some embodiments, the "traceless" conjugation technology utilizes the 1,2-aminothiol group at the N-terminus of the muscletargeting agent to conjugate with an aldehyde-containing oligonucleotide.

[0161] In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent via a site-specific method that utilizes an unnatural amino acid incorporated into the muscle-targeting agent. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the ketone group of pAcPhe selectively couples to an alkoxy-amine-derived conjugate moiety to form an oxime bond.

[0162] Pharmaceutical Composition

[0163] The present application provides a pharmaceutical composition comprising the oligonucleotide as described above or the conjugate as described above. In some embodiments, the pharmaceutical composition further optionally comprises a pharmaceutically acceptable salt, carrier, or excipient.

[0164] In some embodiments, the pharmaceutical composition or medicament may comprise a pharmacologically effective amount of at least one of the oligonucleotides or conjugates described herein and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance, other than the active pharmaceutical ingredient (API, therapeutic product, such as an oligonucleotide), that has been properly evaluated for safety and is intentionally included in a drug delivery system. Excipients do not exert or are not intended to exert therapeutic effects at the intended dosage. Excipients may be used for a) aiding in the processing of the drug delivery system during manufacture, b) protecting, supporting, or enhancing the stability, bioavailability, or patient acceptability of the API, c) assisting in product identification, and / or d) enhancing any other attribute that contributes to the overall safety and effectiveness of the API delivery during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0165] Excipients include: absorption enhancers, anti-adherents, antifoaming agents, antioxidants, binders, buffering agents, carriers, coating agents, pigments, delivery enhancers, delivery polymers, dextrans, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained- release matrices, sweeteners, thickeners, tonicity agents, vehicles, hydrophobic agents, and wetting agents.

[0166] In some embodiments, the pharmaceutical composition or medicament may comprise a pharmacologically effective amount of at least one of the oligonucleotides or conjugates described herein and one or more pharmaceutically acceptable carriers. As pharmaceutically acceptable carriers, for example, carriers for oral administration or carriers for parenteral administration may be additionally included. Carriers for oral administration include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Also, carriers for parenteral administration include water, suitable oils, saline, aqueous glucose, and ethylene glycols, and may further include stabilizers and preservatives. Suitable stabilizers may be antioxidants, for example, sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives may be benzalkonium chloride, methylparaben, propylparaben, and chlorobutanol. Other pharmaceutically acceptable carriers known in the art may also be used.

[0167] In some embodiments, the oligonucleotide, the conjugate, and the pharmaceutical composition comprising the oligonucleotide or conjugate disclosed herein may be packaged or comprised in a kit, container, package, or dispenser. In some embodiments, the oligonucleotide, the conjugate, or the pharmaceutical composition comprising the oligonucleotide or conjugate disclosed herein may be packaged in a pre-filled syringe or vial.

[0168] In some embodiments, the pharmaceutical composition may be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form may be nebulized or lyophilized. In some embodiments, the nebulized or lyophilized form may be reconstituted with an aqueous solution or liquid solution. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation.

[0169] In some embodiments, the pharmaceutical composition is lyophilized for extending its shelf life, and subsequently reconstituted into a solution before use (e.g., administration to a subject). Thus, the excipient may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, Ficoll, or gelatin).

[0170] In some embodiments, the pharmaceutical composition may be formulated in a buffer solution such as phosphate-buffered saline, liposomes, micellar structures, and capsids.

[0171] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Some examples of routes of administration include parenteral administration, such as intravenous, intradermal, and subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

[0172] The pharmaceutical composition of the present application can bind to the DMD gene pre-mRNA, thereby promoting an increase or restoration of dystrophin expression or activity. In some embodiments, the pharmaceutical composition is used for treating a subject suffering from a disease, disorder, or condition resulting from DMD gene mutations (including exon frameshift, deletion, substitution, and duplication mutations) that lead to premature termination of mRNA or nonsense-mediated mRNA decay. In some embodiments, the pharmaceutical composition is used for treating a subject suffering from a disease, disorder, or condition caused by reduced or inhibited dystrophin expression. In some embodiments, the pharmaceutical composition is used for treating a subject suffering from a disease, disorder, or condition caused by reduced or inhibited dystrophin expression resulting from premature termination of mRNA or nonsense-mediated mRNA decay. In some embodiments, diseases, disorders, or conditions that would benefit from restoration of dystrophin expression include, but are not limited to, (progressive) muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD). In some embodiments, the subject is a mammal, including but not limited to a human.

[0173] Use / Method of Treatment

[0174] The present application provides a method for preventing and / or treating a disease or condition, the method comprising administering to a target subject an effective amount of the aforementioned oligonucleotide and / or the aforementioned conjugate and / or the aforementioned pharmaceutical composition.

[0175] In some embodiments, the oligonucleotide and / or conjugate and / or pharmaceutical composition described herein can be used for treating Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD) by increasing or restoring dystrophin expression or activity.

[0176] In some embodiments, a therapeutically effective amount of one or more of the aforementioned oligonucleotides and / or the aforementioned conjugates and / or the aforementioned pharmaceutical compositions is administered to a subject, thereby increasing dystrophin expression in the subject (e.g., effectively increasing or restoring dystrophin expression or activity). In some embodiments, the subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject carries a disease-associated DMD gene mutation (including exon frameshift, deletion, substitution, and duplication mutations) that may optionally be present.

[0177] Generally, any of the oligonucleotides and / or conjugates and / or pharmaceutical compositions described herein may be administered to a patient as a single dose, or may also be administered in a divided treatment regimen via multiple doses over a long-term period. The amount of the active ingredient of the pharmaceutical composition of the present application may vary according to the symptoms of the disease. Preferably, the total dose of the composition of the present application may be from about 0.01 pg to 1000 mg, and most preferably from 0.1 gg / kg / day to 100 mg / kg / day of patient body weight. In some embodiments, treatment will be administered once. In some embodiments, treatment will be administered daily, bi-weekly, weekly, bi-monthly, monthly, or at any time interval that provides maximum efficacy while minimizing safety risk to the subject. Generally, efficacy and treatment as well as safety risk may be monitored throughout the course of treatment. An appropriate effective dose of the pharmaceutical composition of the present application may be determined by a person of ordinary skill in the art based on the route of administration and number of treatments, as well as various factors such as the patient's age, body weight, health status, sex, disease severity, diet, and excretion rate. The pharmaceutical composition according to the present application is not particularly limited in dosage form, route of administration, or method of administration, as long as it exhibits the effects of the present application.

[0178] In some embodiments, a single administration or application of the pharmaceutical composition to the subject is sufficient to increase or restore dystrophin expression or activity for at least 1 to 5 days, 1 to 10 days, 5 to 15 days, 10 to 20 days, 15 to 30 days, 20 to 40 days, 25 to 50 days, or more days. In some embodiments, a single administration or application of the pharmaceutical composition to the subject is sufficient to increase or restore dystrophin expression or activity for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, a single administration or application of the pharmaceutical composition to the subject is sufficient to increase or restore dystrophin expression or activity for at least 1, 2, 3, 4, 5, or 6 months.

[0179] Any suitable method may be used to assess the efficacy of the treatment. In some embodiments, the efficacy of the treatment may be assessed by observation of symptoms associated with DMD and / or BMD, by measuring patient-reported outcomes (e.g., mobility, self-care, daily activities, pain / discomfort, and anxiety / depression), or by quality-of-life indicators (e.g., lifespan).

[0180] In some embodiments, the pharmaceutical composition is administered to the subject at an effective concentration sufficient to increase target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% relative to a control (e.g., baseline level of gene expression prior to treatment).

[0181] In some embodiments, the oligonucleotides or compositions described herein may be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery techniques known in the art. Generally, any suitable method recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for the oligonucleotides described herein. For example, delivery may be by local administration (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intracerebroventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), intranasal, rectal, or topical (including buccal and sublingual) administration. In some embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0182] Furthermore, the pharmaceutical composition of the present application may be administered as a sole therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the composition of the present application and the other therapeutic agents may be administered simultaneously, separately, or sequentially. In such cases, the other therapeutic agents may be substances known to have therapeutic or ameliorative effects on muscular dystrophy. Other therapeutic agents also include all other treatments other than drug therapy, such as radiation therapy.

[0183] In other embodiments, the oligonucleotide may be combined with lipids, nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art. The oligonucleotide may also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art. The oligonucleotide may be conjugated to a delivery polymer. In some embodiments, the delivery polymer is a reversibly masked / modified amphipathic membrane-active polyamine.

[0184] Use

[0185] The present application provides use of the aforementioned oligonucleotide and / or the aforementioned pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a disease, disorder, or condition including, but not limited to, (progressive) muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD).

[0186] Antibody-oligonucleotide conjugate (AOC) drugs have significantly prolonged the plasma half-life of oligonucleotides in preclinical studies and clinical practice. Antibodies or polypeptides targeting transferrin receptor (TfR) enhance muscle enrichment of oligonucleotides, while receptor-mediated endocytosis also facilitates cell penetration and release of oligonucleotides in muscle cells, thereby promoting the expression of functional dystrophin in muscle cells. Therefore, antibody-oligonucleotide conjugate drugs have the potential to significantly improve the efficacy of existing DMD and BMD therapies.

[0187] The present application is further described below with reference to specific examples.

[0188] Example 1: Expression and Affinity Validation of Anti-TfR1 Antibodies

[0189] The inventors selected and expanded stable cell pools, and inoculated them in shake flasks or cell bags using Serum-free Expression medium (GenScript) at 37OC and 5% CO2. On the day of transfection, the medium was warmed to 25-37OC. One hour before transfection, 50 aM Swainsonine (MedChemExpress) was added to the medium. Appropriate amounts of plasmids and reagents were added to the transfection mixture according to the manufacturer's instructions, and mixed thoroughly. The mixture was incubated at room temperature. Appropriate feed was added to the cell culture based on cell growth conditions. Culture supernatants were harvested on day 7 for purification. The supernatant was loaded onto MabSelect™ PrismA Crude at an appropriate flow rate. After washing and elution with suitable buffers, the eluate buffer was exchanged into the final formulation buffer. The buffer of the eluted protein was exchanged into PBS (pH 7.2), and the purified protein was analyzed by SDS-PAGE and SEC-HPLC (column: TSKgel G3000SWxl, TOSOH) to determine molecular weight and purity. The final product had a protein purity >98% as determined by analysis.

[0190] The anti-human TfR1 antibody sequences used in the experiments are shown in Table 1. The Fab region was derived from 13E4-Variant 2iii disclosed in U.S. Patent No. US10913800B2, and the Fc region was derived from a human IgG1 subclass monoclonal antibody. The inventors introduced mutations in the Fc region, such as LALA-LR mutations or the S238C mutation. The affinities of the anti-human TfR1 antibody for human TfR1 / cynomolgus monkey TfR1 were confirmed by Octet BLI, and the results are shown in Table 2.

[0191] The anti-mouse TfR1 antibody sequences used in the experiments are shown in Table 3. The variable regions were derived from the prior art rat anti-mouse TfR1 antibody TIB-219 (light chain GenBank: ABV48920.1; heavy chain GenBank: ABV48917.1), and the Fc region was derived from a human IgG1 subclass monoclonal antibody. The inventors introduced mutations in the Fc region, such as LALA-LR mutations or the S245C mutation. Considering that the expressed antibodies were human-mouse chimeric antibodies, quality control of the antibodies was performed during the experiments. SDS-PAGE results showed that both TIB-219 and TIB-219-S245C achieved 99% purity under both reducing and non-reducing conditions (see Figure 4, where M: Marker; Lane 1: reduced TIB-219, 99% purity; Lane 2: reduced TIB-219-S245C, 99% purity; Lane 3: non-reduced TIB-219, 99% purity; Lane 4: non-reduced TIB-219-S245C, 99% purity; Lane 5: non-reduced human IgG). SEC results showed that the main peak purity of TIB-219 was 97.32%, while that of TIB-219-S245C was 97.71% (Figures 5-6). The affinity of the antimouse TfR1 antibody for mouse TfR1 was confirmed by Octet BLI, and the results are shown in Table 4.

[0192] Table 1: Anti-human TfR1 antibody sequences Antibody Name Antibody Sequence Var2iii >VH, 445aa, LALA-LR QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQG LEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYC ARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 245) >VL, 214aa DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGKSPKLLI YAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFG GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 246) Var2iii- S238C >VH, 445aa, LALA-LR + S238C QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQAPGQG LEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYC ARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPCVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 247) >VL, 214aa DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGKSPKLLI YAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFG GGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 248)

[0193] Table 2: Affinity assay results of anti-human TfR1 antibodies Antibody Antigen Chi2 (RU2) ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU) hIgG1 TfR1-Var2iii Human TfR1 5.88E-01 4.36E+05 7.42E-05 1.70E-10 96.3 hIgG1 TfR1-Var2iii Cyno TfR1 6.30E-01 3.31E+05 1.33E-03 4.02E-09 69.1

[0194] Table 3: Anti-mouse TfR1 antibody sequences Antibody Name Antibody Sequence TIB-219 >VH, 452aa, LALA-LR EVQLVESGGGLVQSGRSLKLSCVASGFTFSSYGMNWIRQAPGKGLEWVAYISSAG KYIYYAETMKGRFTISRDNARNTLYLQMTSLRSENTALYYCARLGTGSEGHYWYFDF WGPGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 249) >VL, 214aa DVQMTQSPYNLAASPGESISISCKASKSISKYLAWYQQKPGKANKLLIYDGSTLQS GIPSRFSGSGSGTNFTLTIRSLEPEDFGLYYCQQHDESPPTFGAGTKLELKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 250) TIB-219- S245C >VH, 452aa, LALA-LR + S245C EVQLVESGGGLVQSGRSLKLSCVASGFTFSSYGMNWIRQAPGKGLEWVAYISSAG KYIYYAETMKGRFTISRDNARNTLYLQMTSLRSENTALYYCARLGTGSEGHYWYFDF WGPGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGGPCVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 251) >VL, 214aa DVQMTQSPYNLAASPGESISISCKASKSISKYLAWYQQKPGKANKLLIYDGSTLQS GIPSRFSGSGSGTNFTLTIRSLEPEDFGLYYCQQHDESPPTFGAGTKLELKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 252)

[0195] Table 4: Affinity assay results of anti-mouse TfR1 antibodies Antibody Antigen Chi2 (RU2) ka (1 / Ms) kd (1 / s) KD (M) Rmax (RU) TIB-219 Mouse TfR1 3.73E-02 1.34E+05 1.06E-04 7.87E-10 67.2

[0196] Example 2: Design and Activity Evaluation of PMO

[0197] The inventors designed PMO sequences targeting a total of six exons of the human DMD gene, namely exons 44, 45, 50, 51, 52, and 53. The transcript sequence of the DMD gene used in the design was derived from ENST00000357033.9. All designed PMO sequences and their target sequences are shown in Table 5, wherein SEQ ID NO: 001-014 are used for skipping of exon 44, with target sequences SEQ ID NO: 122-135; SEQ ID NO: 015-035 are used for skipping of exon 45, with target sequences SEQ ID NO: 136-156; SEQ ID NO: 036052 are used for skipping of exon 50, with target sequences SEQ ID NO: 157-173; SEQ ID NO: 053-073 are used for skipping of exon 51, with target sequences SEQ ID NO: 174-194; SEQ ID NO: 074-092 are used for skipping of exon 52, with target sequences SEQ ID NO: 195-213; and SEQ ID NO: 093-121 are used for skipping of exon 53, with target sequences SEQ ID NO: 214-242.

[0198] Four factors were primarily considered for the drug activity of PMOs: 1) sequence length between 25-30 nt, 2) distance from the splicing acceptor no more than 100 nt, 3) GC% no less than 40%, and 4) binding energy to the target sequence no higher than-40. The validation results showed that most of the PMOs with good activity had lengths of 25-30 nt, with only 12 out of 121 sequences being shorter than 25 nt; PMOs with good activity generally had a distance from the splicing acceptor of no more than 100 nt, with only 17 out of 121 sequences exceeding 100 nt. After design, GC% and binding energy were tested using the RNAeval algorithm and RNAstructure v5.3. The calculated GC% and binding energy results are shown in Table 5.

[0199] Table 5: PMO sequences and activity Name Sequence (5'to 3') SEQ ID NO GC % Length Target sequence binding energy Target sequence (5'to 3') SEQ ID NO Target exon H44A(-08+16) AGATCTGTCA AATCGCCTGC AGGT 001 50 24 -45.5 ACCTGCAGGC GATTTGACAG ATCT 122 44 H44A(-13+12) CTGTCAAATC GCCTGCAGGT AAAAG 002 48 25 -44.6 CTTTTACCTG CAGGCGATTT GACAG 123 44 H44A(+ 15+39) TGAAAACGCC GCCATTTCTCA ACAG 003 48 25 -44.4 CTGTTGAGAA ATGGCGGCGT TTTCA 124 44 H44A(-20+10) GTCAAATCGC CTGCAGGTAA AAGCATATGG 004 47 30 -54.8 CCATATGCTTT TACCTGCAGG CGATTTGAC 125 44 H44A(+ 62+91) GAGAAACTGT TCAGCTTCTG TTAGCCACTG 005 47 30 -55.4 CAGTGGCTAA CAGAAGCTG AACAGTTTCT C 126 44 H44A(+ 66+90) AGAAACTGTT CAGCTTCTGT TAGCC 006 44 25 -44.0 GGCTAACAGA AGCTGAACA GTTTCT 127 44 H44A(+ 17+41) AATGAAAACG CCGCCATTTCT CAAC 007 44 25 -42.1 GTTGAGAAAT GGCGGCGTTT TCATT 128 44 H44A(+ 58+85) CTGTTCAGCT TCTGTTAGCC ACTGATTA 008 43 28 -49.7 TAATCAGTGG CTAACAGAAG CTGAACAG 129 44 H44A(+ 58+80) CAGCTTCTGT TAGCCACTGA TTA 009 43 23 -40.0 TAATCAGTGG CTAACAGAAG CTG 130 44 H44A(+ 93+119) CCCAATTCTC AGGAATTTGT GTCTTTC 010 41 27 -45.4 GAAAGACAC AAATTCCTGA GAATTGGG 131 44 H44A(+ 83+112) CTCAGGAATT TGTGTCTTTCT GAGAAACTG 011 40 30 -50.7 CAGTTTCTCA GAAAGACAC AAATTCCTGA 132 44 G H44A(+ 117+14 1) TACCATTTGTA TTTAGCATGTT CCC 012 36 25 -39.9 GGGAACATGC TAAATACAAA TGGTA 133 44 H44D(+ 11-11) CAAAGACTTA CCTTAAGATAC C 013 36 22 -33.9 GGTATCTTAA GGTAAGTCTT TG 134 44 H44A(+ 114+13 9) CCATTTGTATT TAGCATGTTCC CAAT 014 35 26 -40.5 ATTGGGAACA TGCTAAATAC AAATGG 135 44 H45A(+ 01+22) GCCCAATGCC ATCCTGGAGT TC 015 59 22 -45.2 GAACTCCAGG ATGGCATTGG GC 136 45 H45A(+ 08+31) TTTGCCGCTG CCCAATGCCA TCCT 016 58 24 -49.5 AGGATGGCAT TGGGCAGCG GCAAA 137 45 H45A(+ 120+14 3) TCCTGCCACC GCAGATTCAG GCTT 017 58 24 -49.8 AAGCCTGAAT CTGCGGTGGC AGGA 138 45 H45A(-04+21) CCCAATGCCA TCCTGGAGTT CCTGT 018 56 25 -51.0 ACAGGAACTC CAGGATGGCA TTGGG 139 45 H45A(+ 115+13 6) ACCGCAGATT CAGGCTTCCC AA 019 55 22 -43.2 TTGGGAAGCC TGAATCTGCG GT 140 45 H45A(-05+19) CAATGCCATC CTGGAGTTCC TGTA 020 50 24 -45.7 TACAGGAACT CCAGGATGGC ATTG 141 45 H45D(+ 18-7) GCCCTACCTC TTTTTTCTGTC TGAC 021 48 25 -45.9 GTCAGACAG AAAAAAGAG GTAGGGC 142 45 H45D(+ 06-22) CCTATTAGATC TGTCGCCCTA CCTCTTT 022 46 28 -51.9 AAAGAGGTA GGGCGACAG ATCTAATAGG 143 45 H45D(+ 04-24) TTCCTATTAGA TCTGTCGCCC TACCTCT 023 46 28 -53.0 AGAGGTAGG GCGACAGATC TAATAGGAA 144 45 H45A(+ 40+64) CTTCCCCAGT TGCATTCAATG TTCT 024 44 25 -43.8 AGAACATTGA ATGCAACTGG GGAAG 145 45 H45A(+ 74+98) CTGGCATCTG TTTTTGAGGAT TGCT 025 44 25 -43.8 AGCAATCCTC AAAAACAGAT GCCAG 146 45 H45A(+ 102+12 6) CAGGCTTCCC AATTTTTCCTG TAGA 026 44 25 -43.9 TCTACAGGAA AAATTGGGAA GCCTG 147 45 H45D(+ 23-02) ACCTCTTTTTT CTGTCTGACA GCTG 027 44 25 -43.8 CAGCTGTCAG ACAGAAAAA AGAGGT 148 45 H45A(-15+10) CCTGGAGTTC CTGTAAGATA CCAAAA 028 42 26 -45.4 TTTTGGTATCT TACAGGAACT CCAGG 149 45 H45A(+ 94+119) CCCAATTTTTC CTGTAGAATA 029 42 26 -44.5 GCCAGTATTC TACAGGAAA 150 45 CTGGC AATTGGG H45A(+ 52+78) TTGCTGAATTA TTTCTTCCCCA GTTGC 030 41 27 -46.1 GCAACTGGG GAAGAAATAA TTCAGCAA 151 45 H45A(+ 80+106) GTAGAATACT GGCATCTGTTT TTGAGG 031 41 27 -45.8 CCTCAAAAAC AGATGCCAGT ATTCTAC 152 45 H45A(+ 87+111) TTCCTGTAGA ATACTGGCATC TGTT 032 40 25 -43.1 AACAGATGCC AGTATTCTAC AGGAA 153 45 H45A(+ 28+53) GCATTCAATGT TCTGACAACA GTTTG 033 38 26 -41.8 CAAACTGTTG TCAGAACATT GAATGC 154 45 H45A(+ 44+70) TTATTTCTTCC CCAGTTGCAT TCAATG 034 37 27 -43.8 CATTGAATGC AACTGGGGA AGAAATAA 155 45 H45A(+ 67+96) GGCATCTGTTT TTGAGGATTG CTGAATTAT 035 37 30 -49.4 ATAATTCAGC AATCCTCAAA AACAGATGCC 156 45 H50A(+ 56+80) AGGTCAGGCT GCTTTGCCCT CAGCT 036 60 25 -53.6 AGCTGAGGG CAAAGCAGC CTGACCT 157 50 H50A(+ 71+96) GTCAGTCCAG GAGCTAGGTC AGGCT 037 60 25 -53.9 AGCCTGACCT AGCTCCTGGA CTGAC 158 50 H50A(+ 60+90) TCCAGGAGCT AGGTCAGGCT GCTTT 038 56 25 -51.8 AAAGCAGCCT GACCTAGCTC CTGGA 159 50 H50D(+ 75+99) AGTGGTCAGT CCAGGAGCTA GGTCA 039 56 25 -52.3 TGACCTAGCT CCTGGACTGA CCACT 160 50 H50A(+ 10+34) CCTTCCACTC AGAGCTCAGA TCTTC 040 52 25 -48.8 GAAGATCTGA GCTCTGAGTG GAAGG 161 50 H50D(+ 07-19) TGGGATCCAG TATACTTACAG GCTCC 041 50 26 -50.9 GGAGCCTGTA AGTATACTGG ATCCCA 162 50 H50A(+ 47+71) TGCTTTGCCCT CAGCTCTTGA AGTA 042 48 25 -47.1 TACTTCAAGA GCTGAGGGC AAAGCA 163 50 H50A(-01+25) CAGAGCTCAG ATCTTCTAACT TCCTC 043 46 26 -47.7 GAGGAAGTTA GAAGATCTGA GCTCTG 164 50 H50A(+ 02+26) TCAGAGCTCA GATCTTCTAAC TTCC 044 44 25 -45.1 GGAAGTTAGA AGATCTGAGC TCTGA 165 50 H50A(+ 45+69) CTTTGCCCTC AGCTCTTGAA GTAAA 045 44 25 -43.9 TTTACTTCAA GAGCTGAGG GCAAAG 166 50 H50D(+ 15-10) GTATACTTACA GGCTCCAATA GTGG 046 44 25 -44.3 CCACTATTGG AGCCTGTAAG TATAC 167 50 H50D(+ 09-16) GATCCAGTATA CTTACAGGCT 047 44 25 -45.2 TTGGAGCCTG TAAGTATACT 168 50 CCAA GGATC H50D(+ 15-10) GTATACTTACA GGCTCCAATA GTGGT 048 42 26 -46 ACCACTATTG GAGCCTGTAA GTATAC 169 50 H50A(-07+23) GAGCTCAGAT CTTCTAACTTC CTCTTTAAC 049 40 30 -51.8 GTTAAAGAGG AAGTTAGAAG ATCTGAGCTC 170 50 H50A(-21+06) CTTCCTCTTTA ACAGAAAAGC ATACAC 050 37 27 -43.2 GTGTATGCTT TTCTGTTAAA GAGGAAG 171 50 H50A(-15+10) CTAACTTCCTC TTTAACAGAA AAGC 051 36 25 -38.7 GCTTTTCTGT TAAAGAGGA AGTTAG 172 50 H50A(-07+18) CAGATCTTCTA ACTTCCTCTTT AAC 052 36 25 -39.4 GTTAAAGAGG AAGTTAGAAG ATCTG 173 50 H51A(+ 99+125) AGCCCGGTTG AAATCTGCCA GAGCAGG 053 59 27 -56.6 CCTGCTCTGG CAGATTTCAA CCGGGCT 174 51 H51A(+ 82+108) CCAGAGCAGG TACCTCCAAC ATCAAGG 054 56 27 -54.8 CCTTGATGTT GGAGGTACCT GCTCTGG 175 51 H51A(+ 79+103) GCAGGTACCT CCAACATCAA GGAAG 055 52 25 -48.2 CTTCCTTGAT GTTGGAGGTA CCTGC 176 51 H51A(+ 97+121) CGGTTGAAAT CTGCCAGAGC AGGTA 056 52 25 -48.0 TACCTGCTCT GGCAGATTTC AACCG 177 51 H51A(+ 123+14 4) AGTCGGTAAG TTCTGTCCAA GC 057 50 22 -40.4 GCTTGGACAG AACTTACCGA CT 178 51 H51A(+ 188+21 7) ATCTCGTTGAT ATCCTCAAGG TCACCCACC 058 50 30 -58.6 GGTGGGTGAC CTTGAGGATA TCAACGAGAT 179 51 H51A(+ 12+36) CAGGTTGTGT CACCAGAGTA ACAGT 059 48 25 -46.3 ACTGTTACTC TGGTGACACA ACCTG 180 51 H51A(+ 20+44) AGTAACCACA GGTTGTGTCA CCAGA 060 48 25 -47.0 TCTGGTGACA CAACCTGTGG TTACT 181 51 H51A(+ 128+15 2) AGAAAGCCAG TCGGTAAGTT CTGTC 061 48 25 -45.8 GACAGAACTT ACCGACTGGC TTTCT 182 51 H51A(+ 170+19 4) ACCCACCATC ACCCTCTGTG ATTTT 062 48 25 -47.0 AAAATCACAG AGGGTGATGG TGGGT 183 51 H51A(+ 02+26) TCACCAGAGT AACAGTCTGA GTAGGA 063 46 26 -47.2 TCCTACTCAG ACTGTTACTC TGGTG 184 51 H51A(+ 05+33) GTTGTGTCAC CAGAGTAACA GTCTGAGTA 064 45 29 -53.4 TACTCAGACT GTTACTCTGG TGACACAAC 185 51 H51A(+ 69+93) CCAACATCAA GGAAGATGGC 065 44 25 -43.5 GAAATGCCAT CTTCCTTGAT 186 51 ATTTC GTTGG H51A(- 06+24) CCAGAGTAAC AGTCTGAGTA GGAGCTAAAA 066 43 30 -54.5 TTTTAGCTCC TACTCAGACT GTTACTCTGG 187 51 H51A(+ 66+90) ACATCAAGGA AGATGGCATT TCTAG 067 40 25 -42.2 CTAGAAATGC CATCTTCCTT GATGT 188 51 H51D(+ 16-09) TTCTCATACCT TCTGCTTGATG ATC 068 40 25 -42.9 GATCATCAAG CAGAAGGTAT GAGAA 189 51 H51A(-15+15) CAGTCTGAGT AGGAGCTAAA ATATTTTGGG 069 40 30 -51.2 CCCAAAATAT TTTAGCTCCT ACTCAGACTG 190 51 H51A(-15+13) GTCTGAGTAG GAGCTAAAAT ATTTTGGG 070 39 28 -47 CCCAAAATAT TTTAGCTCCT ACTCAGAC 191 51 H51A(-15+10) TGAGTAGGAG CTAAAATATTT TGGG 071 36 25 -39.9 CCCAAAATAT TTTAGCTCCT ACTCA 192 51 H51D(+ 07-23) ACTTTTATCAT TTTTTCTCATA CCTTCTGC 072 30 30 -44.8 GCAGAAGGT ATGAGAAAA AATGATAAAA GT 193 51 H51A(+ 66+95) CTCCAACATC AAGGAAGATG GCATTTCTAG 073 43 30 nd CTAGAAATGC CATCTTCCTT GATGTTGGAG 194 51 H52A(+ 11+35) TGGGGACGCC TCTGTTCCAA ATCCT 074 56 25 -51.0 AGGATTTGGA ACAGAGGCG TCCCCA 195 52 H52A(+ 15+39) CAACTGGGGA CGCCTCTGTT CCAAA 075 56 25 -49.9 TTTGGAACAG AGGCGTCCCC AGTTG 196 52 H52A(+ 31+55) CGGTAATGAG TTCTTCCAACT GGGG 076 52 25 -47.2 CCCCAGTTGG AAGAACTCAT TACCG 197 52 H52A(-03+22) GTTCCAAATC CTGCATTGTTG CCTG 077 48 25 -45.3 CAGGCAACA ATGCAGGATT TGGAAC 198 52 H52A(+ 40+65) TTTTGGGCAG CGGTAATGAG TTCTTC 078 46 26 -46.6 GAAGAACTC ATTACCGCTG CCCAAAA 199 52 H52A(+ 75+100) TTCTAGCCTCT TGATTGCTGG TCTTG 079 46 26 -47.7 CAAGACCAG CAATCAAGAG GCTAGAA 200 52 H52A(-05+20) TCCAAATCCT GCATTGTTGC CTGTA 080 44 25 -44.7 TACAGGCAAC AATGCAGGAT TTGGA 201 52 H52A(+ 01+25) TCTGTTCCAA ATCCTGCATTG TTGC 081 44 25 -43.9 GCAACAATGC AGGATTTGGA ACAGA 202 52 H52A(+ 53+80) GTCTTGTTTTT CAAATTTTGG GCAGCGG 082 43 28 -46.9 CCGCTGCCCA AAATTTGAAA AACAAGAC 203 52 H52A(+ CCTCTTGATTG 083 42 26 -44.1 GAAAAACAA 204 52 69+94) CTGGTCTTGTT TTTC GACCAGCAAT CAAGAGG H52A(+ 81+106) TGATTGTTCTA GCCTCTTGATT GCTG 084 42 26 -45.5 CAGCAATCAA GAGGCTAGA ACAATCA 205 52 H52A(+ 90+115) GATCCGTAAT GATTGTTCTAG CCTCT 085 42 26 -45.4 AGAGGCTAG AACAATCATT ACGGATC 206 52 H52D(+ 25-02) ACTTCGATCC GTAATGATTGT TCTAGC 086 41 27 -45.5 GCTAGAACAA TCATTACGGA TCGAAGT 207 52 H52A(+ 44+68) AAATTTTGGG CAGCGGTAAT GAGTT 087 40 25 -41.1 AACTCATTAC CGCTGCCCAA AATTT 208 52 H52D(+ 18-02) ACTTCGATCC GTAATGATTG 088 40 20 -31.1 CAATCATTAC GGATCGAAGT 209 52 H52A(+ 48+74) TTTTTCAAATT TTGGGCAGCG GTAATG 089 37 27 -42.2 CATTACCGCT GCCCAAAATT TGAAAAA 210 52 H52A(+ 59+88) GATTGCTGGT CTTGTTTTTCA AATTTTGGG 090 37 30 -47.6 CCCAAAATTT GAAAAACAA GACCAGCAAT C 211 52 H52D(+ 04-23) CCCATGCTTGT TAAAAAACTT ACTTCG 091 37 27 -41.7 CGAAGTAAGT TTTTTAACAA GCATGGG 212 52 H52D(+ 14-14) GTTAAAAAAC TTACTTCGATC CGTAATG 092 32 28 -40.5 CATTACGGAT CGAAGTAAGT TTTTTAAC 213 52 H53A(+ 114+13 8) TGGCCTGTCC TAAGACCTGC TCAGC 093 60 25 -53.7 GCTGAGCAG GTCTTAGGAC AGGCCA 214 53 H53A(+ 39+62) CTGTTGCCTC CGGTTCTGAA GGTG 094 58 24 -48.2 CACCTTCAGA ACCGGAGGC AACAG 215 53 H53A(+ 126+15 0) CAAGCTTGGC TCTGGCCTGT CCTAA 095 56 25 -50.9 TTAGGACAGG CCAGAGCCA AGCTTG 216 53 H53A(+ 133+15 7) CATGACTCAA GCTTGGCTCT GGCCT 096 56 25 -50.9 AGGCCAGAG CCAAGCTTGA GTCATG 217 53 H53A(+ 32+56) CCTCCGGTTC TGAAGGTGTT CTTGT 097 52 25 -47.7 ACAAGAACA CCTTCAGAAC CGGAGG 218 53 H53A(+ 34+56) CCTCCGGTTC TGAAGGTGTT CTT 098 52 23 -43.4 AAGAACACCT TCAGAACCGG AGG 219 53 H53A(+ 99+123) CCTGCTCAGC TTCTTCCTTAG CTTC 099 52 25 -48.2 GAAGCTAAG GAAGAAGCT GAGCAGG 220 53 H53A(+ 46+70) TCATTCAACT GTTGCCTCCG GTTCT 100 48 25 -46.2 AGAACCGGA GGCAACAGTT GAATGA 221 53 H53A(+ 88+112) TCTTCCTTAGC TTCCAGCCATT 101 48 25 -46.4 CACAATGGCT GGAAGCTAA 222 53 GTG GGAAGA H53A(+ 91+115) GCTTCTTCCTT AGCTTCCAGC CATT 102 48 25 -46.4 AATGGCTGGA AGCTAAGGA AGAAGC 223 53 H53A(+ 109+13 3) TGTCCTAAGA CCTGCTCAGC TTCTT 103 48 25 -47.4 AAGAAGCTG AGCAGGTCTT AGGACA 224 53 H53A(+ 164+18 8) GATTGCATCTA CTGTATAGGG ACCC 104 48 25 -47.0 GGGTCCCTAT ACAGTAGATG CAATC 225 53 H53A(+ 27+51) GGTTCTGAAG GTGTTCTTGTA CTTC 105 44 25 -43.6 GAAGTACAA GAACACCTTC AGAACC 226 53 H53A(+ 51+75) ACATTTCATTC AACTGTTGCC TCCG 106 44 25 -43.0 CGGAGGCAA CAGTTGAATG AAATGT 227 53 H53A(+ 14+39) GTTCTTGTACT TCATCCCACT GATTC 107 42 26 -45.1 GAATCAGTGG GATGAAGTAC AAGAAC 228 53 H53A(+ 74+99) CCAGCCATTG TGTTGAATCCT TTAAC 108 42 26 -44.2 GTTAAAGGAT TCAACACAAT GGCTGG 229 53 H53A(+ 06+30) CTTCATCCCAC TGATTCTGAAT TCT 109 40 25 -42.3 AGAATTCAGA ATCAGTGGGA TGAAG 230 53 H53A(+ 10+39) GTTCTTGTACT TCATCCCACT GATTCTGAA 110 40 30 -52.2 TTCAGAATCA GTGGGATGAA GTACAAGAAC 231 53 H53A(+ 52+81) CCTTTAACATT TCATTCAACT GTTGCCTCC 111 40 30 -50.5 GGAGGCAAC AGTTGAATGA AATGTTAAAG G 232 53 H53A(+ 167+19 1) TTGGATTGCAT CTACTGTATAG GGA 112 40 25 -43.6 TCCCTATACA GTAGATGCAA TCCAA 233 53 H53A(-05+24) CCCACTGATT CTGAATTCTTT CAACTAGA 113 38 29 -48.6 TCTAGTTGAA AGAATTCAGA ATCAGTGGG 234 53 H53D(+ 13-13) CTTTGATACTA ACCTTGGTTT CTGTG 114 38 26 -42.0 CACAGAAAC CAAGGTTAGT ATCAAAG 235 53 H53A(+ 01+27) CATCCCACTG ATTCTGAATTC TTTCAA 115 37 27 -44.2 TTGAAAGAAT TCAGAATCAG TGGGATG 236 53 H53A(+ 183+20 9) GGTTTCTGTG ATTTTCTTTTG GATTGC 116 37 27 -42.8 GCAATCCAAA AGAAAATCAC AGAAACC 237 53 H53A(+ 183+21 2) CTTGGTTTCTG TGATTTTCTTT TGGATTGC 117 37 30 -47.9 GCAATCCAAA AGAAAATCAC AGAAACCAA G 238 53 H53D(+ 06-19) AGGTATCTTTG ATACTAACCTT GGT 118 36 25 -40.7 ACCAAGGTTA GTATCAAAGA TACCT 239 53 H53A(+ 69+95) CCATTGTGTTG AATCCTTTAAC ATTTC 119 33 27 -40.7 GAAATGTTAA AGGATTCAAC ACAATGG 240 53 H53A(-05+20) CTGATTCTGA ATTCTTTCAAC TAGA 120 32 25 -37.7 TCTAGTTGAA AGAATTCAGA ATCAG 241 53 H53A(+ 57+85) GAATCCTTTA ACATTTCATTC AACTGTTG 121 31 29 -42.8 CAACAGTTGA ATGAAATGTT AAAGGATTC 242 53

[0200] The inventors selected a subset of PMOs for further validation of cellular activity. Nineteen sequences targeting skipping of exon 52 (SEQ ID NOs: 074-092) were selected, along with the positive control sequence H52A(-01+24) from Patent Application Publication No. US20230110479A1. Normal saline was set as a negative control. In vitro transfection was performed using primary cardiomyocytes derived from DMD patients with exon 51 deletion. The PCR fragment length after skipping was 278 bp, while the fragment length before skipping was 396 bp. The cellular activity of PMOs was assessed by two-step PCR-gel electrophoresis and capillary electrophoresis to evaluate the skipping efficiency of exon 52 (primers shown in Table 6).

[0201] Table 6: Primer sequences for PMO cellular activity studies qDMD47-F TGAAAATAAGCTCAAGCAGACAAATC (SEQ ID NO: 253) First-step    nested   PCR, product   length   unskip: 1138bp, skip:1020bp qDMD56-R TGAGCTTCAATTTCACCTTGGAG (SEQ ID NO: 254) qDMD48-F CAACCAAACCAAGAAGGACCAT (SEQ ID NO: 255) Second-step PCR, product length unskip: 396bp, skip: 278bp qDMD53-R TGTACTTCATCCCACTGATTCTGAA ( SEQ ID NO: 256)

[0202] The gel electrophoresis results of the experiment are shown in Figure 1. Gel electrophoresis can semi-quantitatively assess the skipping efficiency of PMOs. The PCR fragments corresponding to unskipped and skipped products are located near the 400 bp and 300 bp markers, respectively. The skipping efficiency (skip%) for each PMO was calculated by grayscale analysis as: skip% = skip / (skip+unskip) X 100%. The calculated results are shown in Table 7, wherein SEQ ID NOs: 89 and 90 exhibited better skipping efficiency than the positive control.

[0203] Table 7: Results of gel electrophoresis experiments Sample / Lane No. Name SEQ ID NO: 396bp Area 278bp Area Skip% 1 H52A(+11+35) 74 41704.72 37315.75 47% 2 H52A(+15+39) 75 50759.87 34581.68 41% 3 H52A(+31+55) 76 36070.46 44320.27 55% 4 H52A(-03+22) 77 45844.51 34346.02 43% 5 H52A(+40+65) 78 41481.51 41149.68 50% 6 H52A(+75+100) 79 41197.68 42591.05 51% 7 H52A(-05+20) 80 45524.68 16359.31 26% 8 H52A(+01+25) 81 39682.87 19368.55 33% 9 H52A(+53+80) 82 34578.95 18681.72 35% 10 H52A(+69+94) 83 34905.65 48401.48 58% 11 H52A(+81+106) 84 43579.8 8511.045 16% 12 H52A(+90+115) 85 26287.84 43563.74 62% 13 H52D(+25-02) 86 46434.7 32628.14 41% 14 H52A(+44+68) 87 49418.7 32604.67 40% 15 H52D(+18-02) 88 47851.55 24252.74 34% 16 H52A(+48+74) 89 13073.05 39763.61 75% 17 H52A(+59+88) 90 6861.803 43161.66 86% 18 H52D(+04-23) 91 27254 15532.6 36% 19 H52D(+14-14) 92 38209.02 29746.05 44% 20 H52A(-01+24) Positive control 20523.17 42617.32 67% NEG Normal Saline Negative control 40901.02 1639.113 4%

[0204] Capillary electrophoresis can quantitatively assess the skipping efficiency of PMOs. The raw data of the experimental results are shown in Figure 2. The nucleic acid concentrations of the PCR fragments corresponding to unskipped and skipped products were calculated by integrating the peak areas of the electropherograms. The skipping efficiency (skip%) for each PMO was calculated using the following formula: skip% = skip / (skip+unskip) X 100%. The calculated results are shown in Table 8, which again demonstrated that SEQ ID NOs: 89 and 90 exhibited better skipping efficiency than the positive control.

[0205] Table 8: Results of capillary electrophoresis experiments Sample / Lane No. Name SEQ ID NO: 396bp Conc. Cng / gl) 278bp   Conc. Cng / gl) Skip% 1 H52A(+11+35) 74 5.53 5.39 49% 2 H52A(+15+39) 75 5.37 3.38 39% 3 H52A(+31+55) 76 3.92 5.34 58% 4 H52A(-03+22) 77 5.97 3.97 40% 5 H52A(+40+65) 78 4.81 5.49 53% 6 H52A(+75+100) 79 5.61 4.99 47% 7 H52A(-05+20) 80 7.35 4.57 38% 8 H52A(+01+25) 81 5.43 4.16 43% 9 H52A(+53+80) 82 3.13 2.64 46% 10 H52A(+69+94) 83 5.69 6.86 55% 11 H52A(+81+106) 84 4.7 3.58 43% 12 H52A(+90+115) 85 12.01 9.06 43% 13 H52D(+25-02) 86 7.42 4.79 39% 14 H52A(+44+68) 87 6.36 4.28 40% 15 H52D(+18-02) 88 8.12 3.94 33% 16 H52A(+48+74) 89 2.55 5.91 70% 17 H52A(+59+88) 90 1.19 7.26 86% 18 H52D(+04-23) 91 5.46 3.19 37% 19 H52D(+14-14) 92 8.01 4.51 36% 20 H52A(-01+24) Positive control 3.73 5.82 61% NEG Normal Saline Negative control 7.27 0.58 7%

[0206] Example 3: Synthesis of Antibody-PMO Conjugates

[0207] A: Synthesis of Linker-PMO and SMCC-linker-PMO

[0208] The 3'-C3-NH2-activated M23D tool PMO (Linker-PMO) (EG3- GGCCAAACCTCGGCTTACCTGAAAT-C3-NH2, SEQ ID NO: 243) was obtained using standard solid-phase synthesis methods and purified by HPLC. The 3'-C3-NH2-activated M23D PMO was coupled with the N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker under aqueous conditions to form the 3'-SMCC-activated M23D PMO (SMCC-linker-PMO) (EG3-GGCCAAACCTCGGCTTACCTGAAAT-C3-NH-SMCC, SEQ ID NO: 244, the nucleic acid sequence portion of which is identical to SEQ ID NO: 243). After purification by HPLC, the product was used for subsequent antibody conjugation reactions. The specific structures of Linker-PMO and SMCC-linker-PMO are shown in Figure 3.

[0209] B: Synthesis of Antibody-PMO Conjugates

[0210] TIB-219 antibody (10 mg / mL) was stored in PBS solution, and then 2.1 equivalents of TCEP (a thiol-based reducing agent) aqueous solution was added, followed by incubation at 25 C for 2.5 hours to reduce the interchain disulfide bonds. The reduced antibody was mixed with 8 equivalents of 3'-SMCC-activated M23D PMO (SEQ ID NO: 244) and incubated at 25C for 3 hours. Then 12 equivalents of L-cysteine were added to the mixture at room temperature for 30 minutes to quench unreacted maleimide groups.

[0211] The reaction mixture was purified using an AKTA Explorer FPLC system with HIC Method 1 (see Table 9). Fractions P2, P3, and P4, which were expected to contain conjugates with different drug-to-antibody ratios (DARs), were collected, and the fractions as well as the reaction mixture were analyzed by HIC Method 2 (see Table 10) and mass spectrometry. Figure 7 shows the purification profile of the crude product after reaction, wherein P2, P3, and P4 were collected for confirmation of DAR and molecular weight.

[0212] Because antibodies and nucleic acids have different absorbances at 260 nm and 280 nm, the A260 / A280 absorbance ratio of a single fraction can be used to confirm that the main components of P2, P3, and P4 correspond to DAR2, DAR4, and DAR6 molecules, respectively (validation results shown in Figure 8). Mass spectrometry was used to confirm molecular weight and DAR values (see Figure 9), wherein B260T01-E01-TFF-02 corresponds to fraction P2, B260T01-E01-TFF-03 to fraction P3, and B260T01-E01-TFF-04 to fraction P4. Based on the differences in main peak molecular weights, it was determined that P2 was the DAR2 conjugate, P3 the DAR4 conjugate, and P4 the DAR6 conjugate. Thus, the DAR and purity of the reaction mixture and final purified product were established. Figure 10 shows the chromatogram of the purified product analyzed by HIC Method 2, indicating DAR2 (15.274), DAR4 (16.582), and DAR6 (two peaks at 17.226 and 17.735), with the numbers in parentheses representing retention times (minutes). This result ultimately determined the final product to have a DAR of 4.6 (see Table 11). Figure 11 shows the SEC chromatogram of the purified product, with a product purity of 96.43%.

[0213] Table 9: HIC Method 1 Column Sepax FPLC (0.66cm) Resin Capto Butyl ImpRes (Cytiva) volume5.8 mL Flow rate 150cm / h, 5 mL / min Buffer A 50 nM PB, 0.8M ammonium sulfate, pH 7.0 Buffer B 50 nM PB, pH 7.0

[0214] Table 10: HIC Method 2 Column Thermo, MAbPacTM HIC-20, 5 ^ m, 1000 A, 4.6*100 mm Column temperature 25’C Sample temperature 7’C Injection volume 80 ^ g Flow rate 0.7 mL / min Mobile phase Mobile phase A: 100 mM PB-1.8 M (NH4)2SO4 (pH 7.0); Mobile phase B: 100 mM PB (pH 7.0) / IPA=(80: 20); Seal wash solution 10% IPA Needle wash solution 10% IPA Detector VWD      wavelength: 280nm

[0215] Table 11: Analytical results of antibody-PMO conjugates Conjugate DAR % Purity (by peak area) CGBC-1011 4.6 96.43

[0216] Example 4: In Vivo Pharmacodynamic Study of Antibody-PMO Conjugate in mdx Mice (Multiple-Dose)

[0217] The experimental design and dosing regimen are shown in Table 12. The antibody-PMO conjugate CGBC-1011 was prepared according to the method of Example 3, wherein the antibody was TIB-219 from Example 2, and the PMO was the 3'-SMCC-activated M23D PMO (SEQ ID NO: 244) from Example 1.

[0218] PBS control and the antibody-PMO conjugate were administered to mdx mice by intravenous injection, and the animals were euthanized at 14 and 28 days post-dosing. Submandibular blood was collected, and plasma samples were obtained using EDTA-K2 as anticoagulant. Tissue samples including heart, diaphragm, gastrocnemius, and liver were harvested and snap-frozen in liquid nitrogen for subsequent pharmacokinetic, exon-skipping efficiency, and blood biochemistry studies.

[0219] Table 12: Pharmacodynamic study of antibody-PMO conjugate in mdx mice (multiple-dose) Group Compound N DAR PMO Equivalent Dose (mg / kg) Injection Volume (mL / kg) End Point (days) 1 PBS 4 / / 5 14 2 CGBC-1011 4 4.6 10 5 14 3 CGBC-1011 4 4.6 30 5 14 4 CGBC-1011 4 4.6 60 5 14 5 CGBC-1011 4 4.6 10 5 28 6 CGBC-1011 4 4.6 30 5 28 7 CGBC-1011 4 4.6 60 5 28

[0220] A: Pharmacokinetic study of antibody-PMO conjugate in mdx mice

[0221] In the experiment, a hybrid ELISA method was used, and the experimental method and primer design principle are shown in Figure 12. A probe complementary to the M23D sequence was used to quantify PMO accumulation in plasma and tissues. The results are shown in Figures 13 and 14. The antibody-PMO conjugate prolonged the plasma half-life of conventional PMO drugs; the plasma PMO concentration in mdx mice treated with the conjugate remained detectable within 28 days, much longer than the conventional PMO plasma half-life of approximately 2 hours (Figure 13). Meanwhile, the antibody-PMO conjugate achieved dose-dependent muscle-targeted PMO enrichment. As the dose increased, the PMO concentration in muscle increased proportionally and declined slowly over time (Figure 13). The antibody-PMO conjugate dose-dependently increased liver enrichment, and as the dose increased, the PMO concentration ratio of gastrocnemius to liver gradually decreased. This tissue distribution pattern differs from that of antibody-siRNA conjugates, suggesting that PMO may have an antibody-independent liver enrichment pathway (Figures 13-14). Therefore, continuously increasing the dose may lead to PMO enrichment in non-target tissues such as the liver, and the 10 mg / kg dose is more conducive to PMO enrichment in muscle tissue.

[0222] B: Exon skipping efficiency of antibody-PMO conjugate in mdx mice

[0223] Two methods were used to study exon skipping efficiency. Nested PCR was first used to evaluate exon skipping efficiency (Table 13). Animal tissues were lysed for RNA isolation. In a 20 pL reaction system, 2 pg of RNA was reverse-transcribed into cDNA, diluted 4-fold, and then subjected to two-step nested PCR (Table 13). The final amplified products were analyzed for fragment length on 4% TAE agarose gels. The results showed that wild-type DMD product had 788 base pairs, while the exon-skipped product DMD A 23 had 575 base pairs. Finally, the product content was quantified by densitometry, and the exon skipping efficiency was calculated using the formula: skip rate = skip / (skip+unskip) X 100%.

[0224] Table 13: Nested PCR primer design and experimental protocol First-step   nested PCR hot start at 95°C for 2 minutes, followed by 10 cycles of 95°C for 0.5 minutes (denaturation), 50°C for 0.5 minutes (annealing), and 72°C for 1 minute (extension), with a final extension at 72°C for 5 minutes EXON 20 F1 5’-CAGAATTCTGCCAATTGCTGAG - 3’ (SEQ ID NO: 257) EXON 26 R1 5’-TTCTTCAGCTTGTGTCATCC - 3’ (SEQ ID NO: 258) Second-step nested PCR hot start at 95°C for 2 minutes, followed by 35 cycles of 95°C for 0.5 minutes (denaturation), 50°C for 0.5 minutes (annealing), and 72°C for 1 minute (extension), with a final extension at 72°C for 5 minutes EXON 20 F2 5’-ACCCAGTCTACCACCCTATC - 3’ (SEQ ID NO: 259) EXON 25 R2 5’-CTCTTTATCTTCTGCCCACCTT - 3’ (SEQ ID NO: 260)

[0225] In addition to nested PCR, ddPCR can also be used to evaluate exon skipping efficiency (Table 14). The method includes RNA extraction and ddPCR reaction using the QX200 Droplet Digital PCR system. The reaction system, primer design, and detailed procedures are as follows, and the exon skipping efficiency is finally calculated using the built-in software of the QX200 system.

[0226] Table 14: ddPCR primer design and experimental protocol ddPCR protocol 95°C for 10 minutes for enzyme activation, 40 cycles of 95°C for 0.5 minutes (denaturation) and 60°C for 1 minute (annealing / extension)*, followed by 98°C for 10 minutes for enzyme inactivation. *Temperature ramp rate set to 2°C / second. Unskip23 F2 TCGGGAAATTACAGGCTCTGC (SEQ ID NO: 261) Unskip23 R2 AGCTTTCCACCAACTGGGAG (SEQ ID NO: 262) Unskip23 Probe2 CTGAGTGACACTGTGAAGGAGATGGCC (SEQ ID NO: 263) Skip23 F4 GCGCTATCAGGAGACAATGAG (SEQ ID NO: 264) Skip23 R4 GTTTTTATGTGATTCTGTAATTTCCC (SEQ ID NO: 265) Skip23 probe4 CTCTCTGTACCTTATCTTAGTGTT (SEQ ID NO: 266)

[0227] The nested PCR results showed that the antibody-PMO conjugate achieved maximum skipping efficiencies of 51%, 23%, and 55% in gastrocnemius, heart, and diaphragm, respectively, in mdx mice after a single dose, with efficacy lasting at least 28 days and showing clear dose dependence (Figure 15). Among nonmuscle tissues, only the liver exhibited a certain level of exon skipping efficiency higher than that of PBS, generally below 10% (Figure 16). Combined with the tissue distribution data (Figure 13), it can be inferred that antibody-receptor specific interaction-mediated endocytosis plays a critical role in exerting the pharmacodynamic effect of PMO, rather than non-specific uptake of the antibody by the liver. In muscle tissues, the results from ddPCR (Figure 17) and nested PCR (Figure 15) showed no significant difference, but ddPCR significantly reduced the background noise in the PBS group.

[0228] C: Effect of antibody-PMO conjugate on blood biochemistry in mdx mice

[0229] Mdx mice often exhibit elevated creatine kinase (CK) and transaminase levels due to chronic muscle damage and inflammation. Plasma samples were collected, and changes in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) were measured using a blood biochemical analyzer. The results showed that the antibody-PMO conjugate significantly and persistently reduced ALT, AST, and CK levels in mdx mice, with efficacy lasting at least 28 days after a single dose (Figure 18).

[0230] Example 5: In Vivo Pharmacodynamic Study of Antibody-PMO Conjugate in mdx Mice (Low Dose)

[0231] The results from Example 4 above indicated that the antibody-PMO conjugate dose-dependently increased PMO concentration in muscle, but also led to PMO enrichment in the liver at a faster rate. Moreover, as the dose increased, the PMO concentration ratio of gastrocnemius to liver gradually decreased, suggesting that PMO may have an antibody-independent liver enrichment pathway. In clinical practice, PMO is often administered weekly at recommended doses of 30 mg / kg or higher. Based on the above, it is concluded that dose optimization of the antibody-PMO conjugate will help maximize muscle enrichment of PMO and restoration of dystrophin at lower dosing frequencies and doses. The purpose of this experiment was to evaluate the in vivo efficacy of the antibody-PMO conjugate in mdx mice at a dose lower than the clinically recommended dose, for example 10 mg / kg.

[0232] The experimental design and dosing regimen are shown in Table 15. The antibody-PMO conjugate CGBC-1011 was prepared according to the method of Example 3, wherein the antibody was TIB-219 from Example 2, and the PMO was the 3'-SMCC-activated M23D PMO (SEQ ID NO: 244) from Example 1.

[0233] PBS control and the antibody-PMO conjugate were administered to mdx mice by intravenous injection, and the animals were euthanized at 14 and 28 days post-dosing according to the experimental design. Submandibular blood was collected, and plasma samples were obtained using EDTA-K2 as anticoagulant. Tissue samples including heart, diaphragm, gastrocnemius, and liver were harvested and snap-frozen in liquid nitrogen for subsequent exon-skipping efficiency and Western blot studies.

[0234] Table 15: Pharmacodynamic study of antibody-PMO conjugate in mdx mice (single dose) Group Test Compound N DAR PMO Equivalent Dose (mg / kg) Injection Volume (mL / kg) End  Point (days) 1 PBS 4 / / 5 14 2 CGBC-1011 4 4.6 10 5 28

[0235] A: Exon Skipping Efficiency of Antibody-PMO Conjugate in mdx Mice

[0236] The procedure steps of nested PCR was described in Example 4. The results showed that at 28 days after a single administration of 10 mg / kg antibody-PMO conjugate, the exon skipping efficiencies in gastrocnemius, heart, and diaphragm of mdx mice were 24%, 8%, and 27%, respectively (Figure 19).

[0237] B: Ability of Antibody-PMO Conjugates to Restore Dystrophin Levels in Muscle Tissues of mdx Mice

[0238] The dystrophin content was determined by western blot analysis. The detailed protocol was as follows: The collected muscle tissues were minced using scissors. The samples were homogenized using steel beads, and the supernatants were collected, followed by a protein concentration determination using a BCA protein assay. The total protein (24 pg) was loaded onto 3% to 8% NuPAGE™ Trisacetate protein gels. Electrophoresis was performed at 75 V for 1 hour, followed by continued electrophoresis at 150 V for 1 hour, with the entire electrophoresis process performed on ice. Proteins were then transferred from the gel onto polyvinylidene difluoride membranes using an iBlot™2 transfer system operated at 25 V for 10 minutes. The membranes were cut and incubated overnight at 4OC with a 1:2000 dilution of anti-dystrophin antibody (Abcam, Cat. No. ab154168), followed by incubation with goat anti-rabbit IgG (1:5000 dilution) at room temperature for 1 hour. As a control, the remaining blot membrane were incubated overnight at 4OC with anti-alpha-actinin antibody (Abcam, Cat. No. ab9465; 1:2000 dilution), followed by incubation with goat anti-mouse IgG (1:10000 dilution) at room temperature for 1 hour. The blots were developed using an ECL Western detection kit (Cytiva) and quantified using iBright FL1500 (Thermo Fisher Scientific).

[0239] In each western blot experiment, fixed ratios of muscle tissues from mdx mice and corresponding muscles from healthy C57BL / 6J mice were weighed, mixed, and homogenized to establish a western blot standard curve. The percentages of wild-type protein in the standard samples of the standard curve were, from high to low: 64%, 32%, 16%, 8%, 4%, 2%, and 0%. The standard curve was fitted using a 4PL nonlinear regression model.

[0240] The results showed that at 28 days after a single administration of 10 mg / kg antibody-PMO conjugate, dystrophin levels in the gastrocnemius, heart, and diaphragm of mdx mice were restored to 100%, 39%, and 88%, respectively, relative to healthy animal tissues, and the pharmacodynamic effect can last for at least 28 days (Figure 20). These results indicate that antibody-conjugated PMO has the potential to achieve improved exon skipping and dystrophin restoration while reducing the administration dose and dosing frequency.

[0241] Example 6: Synthesis of Antibody-siRNA Conjugates

[0242] In addition to PMOs, chemically modified antisense oligonucleotides also have the ability to achieve exon skipping. These nucleic acid drugs are more similar to siRNA in physicochemical properties and metabolism. Therefore, to systematically study the tissue distribution of antibody-oligonucleotide conjugate drugs, antibody-siRNA conjugates with higher detection sensitivity were used for in vivo pharmacokinetic studies to support subsequent drug optimization and in vivo studies.

[0243] A: Synthesis of Linker-siRNA and SMCC-linker-siRNA

[0244] For the linker-siRNA used in this experiment, single strands were fully assembled on solid phase using standard phosphoramidite chemistry and purified by HPLC. Base, sugar, and phosphate modifications well described in the RNAi field were used to optimize duplex stability. The 5' end of the siRNA sense strand contained a C6-NH2 conjugation handle, which was linked to the siRNA sense strand via a phosphodiester bond on the terminal base.

[0245] The 5'-C6-NH2-activated siRNA was coupled with the N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker under aqueous conditions to form the 5'-SMCC-activated linker-siRNA, which was purified by HPLC and used for subsequent antibody conjugation reactions.

[0246] B: Conjugation of Wild-Type Antibody and siRNA

[0247] Conjugation of wild-type antibody and siRNA was performed using a random conjugation process. Specifically, the interchain disulfide bonds of the antibody were first reduced, and then conjugated with the linker-siRNA whose 5' end of the sense strand was activated with the SMCC linker, ultimately forming the antibody-siRNA conjugate.

[0248] Step 1: Reduction of antibody with TCEP

[0249] The antibody was buffer-exchanged into 25 mM Tris buffer (pH 8) and the concentration of which was adjusted to 10 mg / mL. To this solution, 0.8 equivalents of TCEP in the same buffer was added and incubated at room temperature for 2 hours. The reaction solution was then buffer-exchanged by ultrafiltration centrifugation into 25 mM Tris buffer (pH 8) containing 2 mM EDTA, and SMCC-siRNA (3 equivalents) was added and mixed, followed by reaction at 22OC for 3 hours. Analysis of the reaction mixture by analytical SAX column chromatography showed the presence of antibody-siRNA conjugate along with unreacted antibody and siRNA.

[0250] Step 2: Purification and Analysis

[0251] The crude reaction mixture was purified by AKTA Pure FPLC using anion exchange chromatography (SAX) Method 1 (Table 16). Fractions containing the antibody-siRNA conjugate were collected, concentrated, and buffer-exchanged into PBS (pH 7.4). The purity of the conjugate was assessed by analytical HPLC using SAX Method 2 (Table 17).

[0252] Table 16: Anion exchange chromatography (SAX) Method 1 Column: Cytiva™ HiTrap Capto Q 5 mL Solvent A: 10 mM Tris buffer, pH 8.0 Solvent B: 10 mM Tris, 1.5 M NaCl, pH 8.0 Flow rate: 6.0 mL / min Gradient Time (min) A(%) B(%) 0 100 0 20 70 30 28 70 30 48 34 66 56 34 66 76 0 100 77 100 0 87 100 0

[0253] Table 17: Anion exchange chromatography (SAX) Method 2 Column:Thermo Scientific, ProPacTM SAX-10, Bio LCTM, 4X 250mm Solvent A: 80% 10 mM Tris (pH 8.0), 20% ethanol Solvent B: 80% 10 mM Tris (pH 8.0), 20% ethanol, 1.5 M NaCl Flow rate: 0.6 mL / min Gradient Time (min) A(%) B(%) 0 100 0 1 100 0 3 92 8 12 0 100 15 0 100 17 100 0 20 100 0

[0254] C: Conjugation of cysteine-mutant antibody and siRNA

[0255] Conjugation of cysteine-mutant antibody and siRNA was performed using a site-specific conjugation process. Specifically, the site-specific cysteine conjugation sites of the antibody (depending on the specific antibody sequence, including but not limited to S239C, S238C, S245C, etc.) as well as the interchain disulfide bonds were first reduced. The interchain disulfide bonds were then reoxidized while maintaining the reduced state of the cysteine sites, and finally conjugated with the linker-siRNA whose 5' end of the sense strand was activated with the SMCC linker, forming the antibody-siRNA conjugate.

[0256] Step 1: Reduction of antibody with TCEP

[0257] The antibody was buffer-exchanged into 25 mM Tris buffer (pH 8) and the concentration of which was adjusted to 10 mg / mL. To this solution, 100 equivalents of DTT in the same buffer was added and incubated at room temperature for 16 hours. The reaction solution was then buffer-exchanged by ultrafiltration centrifugation into 25 mM Tris buffer (pH 8) containing 2 mM EDTA, and 20 equivalents of DHAA were added and reacted at room temperature for 2 hours. The resulting reaction mixture was passed through a desalting column to remove excess DHAA, and then mixed with SMCC-siRNA (0.8 equivalents) and reacted at 22OC for 2 hours. Analysis of the reaction mixture by analytical SAX column chromatography showed the presence of antibody-siRNA conjugate along with unreacted antibody and siRNA.

[0258] Step 2: Purification and Analysis

[0259] The crude reaction mixture was purified by AKTA Pure FPLC using anion exchange chromatography (SAX) Method 1 (Table 16). Fractions containing the antibody-siRNA conjugate were collected, concentrated, and buffer-exchanged into PBS (pH 7.4). The purity of the conjugate was assessed by analytical HPLC using SAX Method 2 (Table 17).

[0260] The analytical data for the conjugates synthesized in this example, including HPLC retention time (RT) in minutes and percent purity determined by chromatographic peak area, are shown in Table 19 for representative purified antibody-siRNA conjugate molecules, all with purity above 97%. The corresponding chromatograms are shown in Figures 21-26.

[0261] The siRNAs used for the synthesis of antibody-siRNA conjugates in the experiments are shown in Table 18, wherein siRNA ID#001 was used for synthesis of CGBC-1004-1 and CGBC-1004-2, siRNA ID#002 was used for synthesis of CGBC-1005-1 and CGBC-1005-2, and siRNA ID#003 was used for synthesis of CGBC-1012-1 and CGBC-1012-2. The specific sequences are shown in Table 18 below. Regarding the antibodies, CGBC-1004-1, CGBC-1004-2, CGBC-1005-1, and CGBC-1005-2 used the antibody TIB-219-S245C. CGBC-1012-1 and CGBC-1012-2 used the antibody Var2iii-S238C, both synthesized by the site-specific conjugation process described above.

[0262] Table 18: siRNA sequences siRNA ID# Sense strand sequence (5'-3')  — modified SEQ ID NO: Antisense strand sequence (5'-3') — modified SEQ ID NO: 001 SMCC-NH-C6-5'- / mG* / mA* / mC / mA / i2FU / mU / i2F C / i2FC / i2FU / mC / mG / mG / mU / m A / mU / mU / mU* / mA* / mA / -3' 267 5'- / mU* / i2FU* / mA / mA / mA / i2FU / mA / i2FC / i2FC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 268 002 SMCC-NH-C6-5'- / mG* / mA* / mC / mA / i2FU / mU / i2F C / i2FC / i2FU / mC / mG / mG / mU / m A / mU / mU / mU* / mA* / mA / -3' 269 5'- / VPmU* / i2FU* / mA / mA / mA / i2FU / mA / i2FC / i2FC / mG / mA / mG / mG / i2F A / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 270 003 SMCC-NH-C6-5'- / mG* / mA* / mC / mA / mU / mU / i2FC / i2FC / i2FU / mC / mG / mG / mU / mA / mU / mU / mU* / mA* / mA / -3' 271 5'- / mU* / i2FU* / mA / mA / mA / i2FU / mA / mC / mC / mG / mA / mG / mG / i2FA / mA / i2FU / mG / mU / mC* / mU* / mU / -3' 272

[0263] Modification description: m = 2'-O-methyl; i2F = 2'-fluoro; gn = ethylene glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage; VP = 5-(E)-vinylphosphonate modified nucleic acid

[0264] Table 19: Design and analytical results of antibody-siRNA conjugates Conjugate DAR SAX retention time (min) % Purity (by peak area) CGBC-1004-1 1 7.903 99 CGBC-1004-2 2 8.333 97 CGBC-1005-1 1 7.931 99 CGBC-1005-2 2 8.341 98 CGBC-1012-1 1 7.982 100 CGBC-1012-2 1 8.413 99

[0265] Example 7: Pharmacokinetic study of antibody-siRNA conjugates in mice

[0266] The purpose of the experiment was to investigate the ability of antibody-siRNA conjugates (using the above sequences) to muscle-targeted deliver nucleic acids to muscle tissue in mice. The experimental design and dosing regimen are shown in Table 20. The siRNAs used for synthesis of antibody-siRNA conjugates in the experiments are shown in Table 18.

[0267] Mice were administered PBS control and antibody-siRNA conjugates by intravenous injection, and animals were euthanized at 7 and 14 days post-dosing. Plasma and various tissue samples including quadriceps and liver were harvested and snap-frozen in liquid nitrogen. After tissue homogenization and proteinase K digestion, supernatant samples were collected, diluted, and analyzed by SL-PCR. The pharmacokinetic experimental method mainly used SL-PCR to quantify siRNA concentrations in plasma and tissues. SL-PCR consisted of a first step of reverse transcription using a stem-loop primer sequence and a second step of qPCR quantification. The reaction systems are shown in Tables 21-22, and the primer sequences are shown in Table 23.

[0268] Table 20: Study of antibody-siRNA conjugates for target gene knockdown in muscle of wild-type CD-1 mice Group Test Compound N siRNA Equivalent Dose (mg / kg) Injection Volume (mL / kg) End Point (days) 1 PBS 4 5 5 7,  14 2 CGBC-1004-1 4 5 5 7,  14 3 CGBC-1004-2 4 5 5 7,  14 4 CGBC-1005-1 4 5 5 7,  14 5 CGBC-1005-2 4 5 5 7,  14

[0269] Table 21: SL-PCR reverse transcription reaction system PCR reaction solution component Volume required per reaction (pL) 100mM dNTPs (with dTTP) 0.15 MultiScribe Reverse Transcriptase, 50 U / pL 1 10 X Reverse Transcription Buffer 1.5 RNase Inhibitor, 20 U / pL 0.19 Nuclease-free Water 7.085 5 X RT primer (4pM) 0.1875 siRNA / miRNA 5 Total 15

[0270] Table 22: qPCR reaction system for SL-PCR PCR reaction solution component Volume required per reaction (pL) Universal PCR Master Mix 2 x 5 Forward primer (10pM) 1.5 Reverse primer (10pM) 0.7 Probe (10pM) 0.2 cDNA 2 H2O 0.6 Total 10

[0271] Table 23: Primer sequences for SL-PCR used in mouse experiments Primer Sequences (5’-3’) Stem-loop primer GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAGACAT (SEQ ID NO: 273) Forward GCGCTTAAATACCGAGGAA (SEQ ID NO: 274) Reverse GTGCAGGGTCCGAGGT (SEQ ID NO: 275) Probe FAM-CGCACTGGATACGACAAGAC-MGB (SEQ ID NO: 276)

[0272] A standard curve of Ct-log [concentration] was generated during the experiment. The pharmacokinetic standard curve was prepared by diluting the antibody-siRNA conjugate compound and adding the diluted conjugate into blank mouse tissue homogenates. Except for the above steps, the sample preparation procedures for the actual samples were identical to those for the standard curve samples. The amplification efficiency of the standard curve was required to be within the range of 90%-110%.

[0273] The antibody-siRNA conjugates exhibited a substantially lower plasma clearance rate and higher muscle-specific siRNA delivery efficiency compared with conventional siRNA drugs. At 14 days after administration, siRNA remained detectable in plasma by SL-PCR analysis (Figures 27-28), which was significantly different from the plasma half-life of conventional siRNA drugs, which is typically only several hours. These results indicate that the antibody-siRNA conjugates prolonged the plasma half-life of siRNA. Regarding tissue distribution, the antibody-siRNA conjugates achieved increased siRNA distribution in muscle tissues. The siRNA levels in muscle were higher than or comparable to those in liver, demonstrating the muscle-targeting effect mediated by the TfR1 antibody. Regarding molecular design, molecules having DAR1 exhibited higher plasma conccentration and greater muscle accumulation than molecules having DAR2. In addition, the 5-(E)-VP design significantly enhanced muscle enrichment of the antibody-siRNA conjugates (Figure 29).

[0274] During the experiment, no significant changes in body weight or behavioral observations were observed in mice, indicating good tolerability of the conjugates.

[0275] Example 8: Pharmacokinetic study of antibody-siRNA conjugates in cynomolgus monkeys

[0276] The purpose of the experiment was to investigate the ability of antibody-siRNA conjugates to muscle-targeted deliver nucleic acids to muscle tissue in cynomolgus monkeys. The experimental design and dosing regimen are shown in Table 24. The siRNA used for synthesis of antibody-siRNA conjugates in the experiments was siRNA ID#003 from Table 18, and the antibody used was Var2iii-S238C, both synthesized by the site-specific conjugation process described above.

[0277] Antibody-siRNA conjugates were administered to cynomolgus monkeys by intravenous injection. Plasma samples were collected before dosing and at 3, 6, 12 hours and 1, 3, 7, 14, 28, and 42 days post-dosing according to the study protocol. Meanwhile, gastrocnemius muscle samples were collected by muscle biopsy before dosing and at 3, 7, 14, 28, 56, and 84 days post-dosing for pharmacokinetic studies. The standard curve preparation method was the same as described in Example 7 and is not repeated here. Primer sequences are shown in Table 25.

[0278] Table 24: In vivo study of antibody-siRNA conjugates in cynomolgus monkeys Group Test Compound N siRNA Equivalent Dose (mg / kg) Injection Volume (mL / kg) End  Point (days) 1 CGBC-1012-1 2 0.6 5 84 2 CGBC-1012-1 2 6 5 84

[0279] Table 25: Primer sequences for SL-PCR used in cynomolgus monkey experiments Primer Sequences (5’-3’) Stem-loop primer 5’- GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGA CATTCCTA-3’ (SEQ ID NO: 277) Forward 5’-GCGCATAAAATCTACAGTCA-3’ (SEQ ID NO: 278) Reverse 5’-GTGCAGGGTCCGAGGT-3’ (SEQ ID NO: 279) Probe 5’-FAM-CGCACTGGATACGACATTCC-MGB-3’ (SEQ ID NO: 280)

[0280] The experimental results showed that the antibody-siRNA conjugate exhibited a typical two-compartment model in cynomolgus monkey plasma, with linear PK observed at both doses. The distribution half-life at the high dose was 27.6 hours, and the elimination half-life was 279 hours. In the low-dose group, only the distribution phase curve was accurately recorded due to the lower limit of detection of the method (Figure 30). In terms of tissue distribution, the antibody-siRNA conjugate was enriched in cynomolgus monkey gastrocnemius muscle, reaching a Cmax of over 300 pmol / g on day 3 post-dosing. The elimination half-life of the antibody-siRNA conjugate in cynomolgus monkey gastrocnemius muscle was 239 hours, and the siRNA concentration in gastrocnemius muscle remained above 1 pmol / g throughout the 84-day period before the end of the experiment (Figure 31).

[0281] No significant changes in body weight or behavioral observations were noted in cynomolgus monkeys during the experiments, indicating good tolerability of the conjugate.

[0282] Example 9: In vitro activity of antibody-siRNA conjugates

[0283] The purpose of the experiment was to evaluate whether antibody siRNA conjugates could be internalized by muscle cells and exert biological activity in vitro without the aid of lipofectamine (a type of liposome). The antibody-siRNA conjugates used in the experiments were the same as those in Example 7. The specific experimental method was as follows: RD human rhabdomyosarcoma cells (4201HUM-CCTCC00295) were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco). Antibody-siRNA conjugates were diluted to a maximum dose of 10 mM and transfected at final siRNA concentrations of 200, 100, 10, 1, and 0.1 nM. Cells were seeded in 48-well plates at 10,000 cells per well 24 hours before dosing. Antibody-siRNA conjugates were added to the wells; PBS was added to some wells as an additional negative control. Cells were placed at 37 C and 5% CO2 for 72 hours. The medium was removed from the wells and 150 u L of Trizol (Life) was added. Plates were frozen at -80C overnight or longer before analysis. RNA was isolated using the Direct-zol-96 RNA Kit (Zymo Research) according to the manufacturer's instructions. RNA was reverse-transcribed into cDNA using ReverTra Ace™ qPCR RT Master Mix (TOYOBO) according to the manufacturer's instructions. cDNA samples were evaluated by qPCR using DMPK-specific and GAPDH-specific primers and SYBR Green method with Taqman Fast Advanced Master Mix (Applied Biosystems). % mRNA was calculated using the standard 2-AAQT method, with PBS-treated cells set as 100% expression. All experiments were performed in triplicate. Primer information is shown in Table 26. Primer Sequences (5’-3’) Human DMPK-F CACTGTCGGACATTCGGGAAGGTGC(SEQ ID NO: 281) Human DMPK-R GCTTGCACGTGTGGCTCAAGCAGCTG(SEQ ID NO: 282) Human GADPH-F ATGGGGAAGGTGAAGGTCG(SEQ ID NO: 283) Human GADPH-R GGGGTCATTGATGGCAACAATA(SEQ ID NO 284)

[0285] The experimental results showed that CGBC-1011 was able to enter RD cells under in vitro co-incubation conditions without the aid of lipofectamine, and exerted dose-dependent target gene knockdown. The knockdown effect of antibodysiRNA conjugates reached saturation at a concentration of 1 nM, wherein the maximum knockdown efficiency of the DAR2 antibody-siRNA conjugate was approximately 40%, while that of the DAR1 molecule was approximately 50% (Figure 32).

[0286] Example 10: Affinity of antibody-siRNA conjugates

[0287] The purpose of the experiment was to evaluate the changes in affinity of antibody-siRNA conjugates and unconjugated antibody for the antigen, and to assess the impact of conjugation method and DAR value selection on antibodyantigen affinity. The affinity of the conjugates and unconjugated antibody for TfR1 antigen was evaluated by ELISA. The specific steps included: coating a 96-well plate with 1 U g / mL antigen, incubating at 4 C overnight, washing twice with PBST, blocking with 3% MPBS at 37 C for 1 hour, washing twice with PBST, then adding serially diluted conjugates or unconjugated antibody, detecting with anti-human / rat IgG-Fc-HRP, and finally performing color developing with TMB. During the experiment, care was taken to select the corresponding antigen and detection antibody according to the species specificity of the conjugates and unconjugated antibody. After the experiment, curves were plotted with OD450 and antibody concentration, and EC50 was calculated.

[0288] The experimental results showed that the target affinity of antibody siRNA conjugates was significantly decreased compared to unconjugated antibody (Figures 33-34). Among the conjugates, DAR2 generally showed a more pronounced decrease in affinity compared to DAR1 (Figure 34), but the affinity difference between DAR2 and DAR1 in CGBC-1012 was relatively small (Figure 33). The above data indicate that siRNA conjugation has an adverse effect on antibody-antigen affinity, and the greater the number of conjugations, the more pronounced the decline in antibody-antigen affinity.

[0289] Example 11: Synthesis of peptide-siRNA conjugates

[0290] The nucleic acid sequences used in the experiments were the same as those in Example 8. The peptide CGBB2 sequence used in the experiments was as follows:

[0291] PSEEEIKKLVEELLKELSKEEAALKLVETAADVVVVTPKGIVVV KGDRETAEAVFKAAEEAFDKYPDDAEFIAEYIKKKVPKARVVLVPN (SEQ ID NO: 285 ) o

[0292] To a 30 pL solution of the polypeptide (3 nmol) in phosphate buffer (pH 7.4) were added 6-azidomethyl-4-methoxy-2-pyridinecarboxaldehyde (30 nmol in 1 pL DMSO) and 3-maleimidopropionic acid (15 nmol in 1 pL DMSO). The resulting mixture was incubated at 37 °C overnight. The solution was then concentrated and PBS buffer was added.

[0293] Buffer exchange was performed using 0.5 mL centrifugal filters. For buffer exchange, each sample was first diluted to 500 pL with 50 mM phosphate buffer (pH 7.4), then concentrated to 30 pL. This process was repeated three times to obtain the polypeptide intermediate. Subsequently, DBCO-modified oligonucleotide (4.5 nmol in 4.5 pL phosphate buffer, pH 7.4) was added to 30 pL of the protein solution (3 nmol), and the resulting mixture was incubated at room temperature overnight. The corresponding polypeptide-oligonucleotide conjugate, designated CGBC-1018, was purified by SEC FPLC. The conjugation, purification, and analytical results for CGBC-1018 are shown in Figure 35.

[0294] Example 12: Affinity and in vitro activity studies of polypeptide-siRNA conjugates

[0295] The purpose of the experiment was to evaluate the affinity of polypeptide-siRNA conjugates and to determine whether they could be internalized by muscle cells and exert biological activity in vitro without the aid of lipofectamine. Affinity was confirmed by Octet BLI, using the same method as in Example 1; the in vitro activity assay was performed using the same method as in Example 9.

[0296] The results of the affinity study are shown in Table 27. The results indicated that, similar to antibody-conjugated nucleic acids, conjugation of nucleic acids to polypeptides also reduced the affinity of the polypeptide for its antigen. However, with the DAR1 design, the reduction in affinity was modest and did not significantly affect the ability of the polypeptide to deliver the nucleic acid.

[0297] Table 27: Affinity of anti-human TfR1 polypeptide and conjugate products Test article Antigen Rmax ka (1 / Ms) kd (1 / s) KD (M) Dissoc Xa2 Dissoc Ra2 CGBB2 Human TfR1 0.1764 1.53E+06 5.62E-03 3.67E-9 0.0644 0.9886 CGBC-1018 Human TfR1 0.3001 7.520E+05 3.154E-04 4.195E-9 0.2994 0.9922

[0298] The results of the in vitro activity study showed that CGBC-1018 was able to enter RD cells under in vitro co-incubation conditions without the aid of lipofectamine, and exerted dose-dependent target gene knockdown. The in vitro knockdown efficiency of the polypeptide-siRNA conjugate CGBC-1018 was lower than that of the previously evaluated antibody-siRNA conjugate CGBC-1012, and required concentrations greater than 100 nM to exert significant knockdown, with a maximum knockdown efficiency of approximately 30% (Figure 36).

[0299] The foregoing description is merely illustrative of preferred embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the principles of the present invention shall fall within the scope of protection of the present invention.

Claims

1. An oligonucleotide for promoting exon skipping of human dystrophin pre-mRNA and / or hybridizing to a target sequence of human dystrophin pre-mRNA, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or a phosphorodiamidate morpholino oligomer (PMO), and the oligonucleotide binds to at least a portion of an exon and / or a non-exon region of dystrophin.

2. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises or consists of a sequence that is capable of at least partially binding to any one of dystrophin pre-mRNA exons 44, 45, 50, 51, 52, and 53, and the sequence has 10 to 33 nucleotides.

3. The oligonucleotide according to claim 1, wherein the oligonucleotide has a sequence of SEQ ID NOs: 001-121, or a sequence having at least 90% homology thereto, preferably at least 92%, 95%, 98%, or 99% homology thereto; or a sequence in RNA form in which T is replaced with U.

4. A conjugate comprising the oligonucleotide according to any one of claims 13 and a muscle-targeting agent, wherein the muscle-targeting agent is an antibody or an antigen-binding fragment.

5. The conjugate according to claim 4, wherein the antibody or antigen-binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab’, a bivalent Fab2, a single-chain variable fragment, a diabody, a minibody, a nanobody, a single-domain antibody, or a camelidantibody or binding fragment thereof.

6. The conjugate according to claim 4, wherein the muscle-targeting agent is an antibody or polypeptide capable of binding to human transferrin receptor 1.

7. The conjugate according to claim 6, wherein the muscle-targeting agent has 1 or 2 binding sites for human transferrin receptor 1, and has an affinity between 0.01 nM and 100 nM.

8. The conjugate according to claim 4, wherein the muscle-targeting agent is covalently linked to the oligonucleotide via a non-cleavable or a cleavable linker.

9. The conjugate according to claim 8, wherein the linker is N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

10. The conjugate according to claim 4, wherein a molar ratio of the oligonucleotide to the muscle-targeting agent is between 1 and 8.

11. The conjugate according to claim 6, wherein the antibody or polypeptide comprises or consists of a sequence set forth in SEQ ID NO: 285.

12. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1-3 or the conjugate according to any one of claims 4-11, wherein the pharmaceutical composition is for treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition is formulated for intravenous, subcutaneous, parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

14. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable salt,excipient, or carrier.

15. Use of the oligonucleotide according to any one of claims 1-3 or the conjugate according to any one of claims 4-11 in the manufacture of a medicament, wherein the medicament is for promoting dystrophin expression, or for preventing and / or treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

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