CN120648680A

CN120648680A - Medicament for treating duchenne muscular dystrophy and becker muscular dystrophy

Info

Publication number: CN120648680A
Application number: CN202410303445.0A
Authority: CN
Inventors: 吴昊; 盛飞; 邱祎帛; 袁梵雨; 郑竞翔; 邢亚非
Original assignee: Jiajin Biomedicine Shanghai Co ltd
Current assignee: Jiajin Biomedicine Shanghai Co ltd
Priority date: 2024-03-15
Filing date: 2024-03-15
Publication date: 2025-09-16
Also published as: WO2025190277A1

Abstract

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

Description

Medicament for treating duchenne muscular dystrophy and becker muscular dystrophy

Technical Field

The invention relates to the field of biological medicine, in particular to a medicine for treating Duchenne muscular dystrophy and Beck muscular dystrophy and application thereof.

Background

Duchenne muscular dystrophy (Duchenne muscular dystrophy, DMD) and becker muscular dystrophy (Beckermuscular dystrophy, BMD) both belong to X-chromosome recessive monogenic genetic diseases, which are mainly seen in men and children, and are caused by decreased levels of dystrophin in muscle cells, resulting in muscle inflammation, fibrosis, decreased motor skills, etc., as most patients will die before age 30 without treatment.

Dystrophin is expressed in humans by the DMD gene, a large gene that contains 79 exons and over 200 kilototal base pairs. DMD gene mutations (including exon frameshift, deletions, substitutions and repeat mutations) tend to result in premature termination of mRNA or nonsense-mediated mRNA degradation (nonsense-MEDIATED MRNA DECAY, NMD), thereby reducing expression of functional dystrophin. Exon skipping using oligonucleotides against the mutated DMD gene, thereby restoring expression of functional dystrophin, is one of the means to treat DMD. There are currently 4 batches available from the U.S. Food and drug administration (Food and DrugAdministration, FDA) whose long term efficacy is still under evaluation.

Existing oligonucleotide therapies are limited by their short plasma half-life and less muscle tissue distribution, with limited efficacy. For example, in the registered clinic of eteplirsen, available in 2016, at least 30mg/kg was administered weekly for 48 weeks, only to increase the dystrophin in DMD patients from 0.16% before treatment to 0.44% after treatment.

There is therefore a need for improvements in the efficacy of existing drugs.

Disclosure of Invention

It is an object of the present application to provide an oligonucleotide capable of increasing or restoring the expression or activity of dystrophin, thereby treating duchenne muscular dystrophy and/or becker muscular dystrophy in a subject.

It is also an object of the present application to provide a conjugate comprising the above oligonucleotide and a muscle targeting agent.

It is also an object of the present application to provide the use of said oligonucleotide or said conjugate for the prevention or treatment of duchenne muscular dystrophy and/or becker muscular dystrophy.

To address the above problems, the present application provides conjugates that target muscle cells for delivery of active ingredients to these cells.

In a first aspect, the application provides an oligonucleotide for promoting exon skipping of a dystrophin pre-mRNA and/or hybridizing to a target sequence of human DMD mRNA and mediating RNA interference with respect to human DMD mRNA in a muscle cell of a human subject, thereby promoting increased or restored expression or activity of dystrophin, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or morpholino antisense oligonucleotide (phosphorodiamidate morpholino oligomer, phosphorodiamidate morpholino oligonucleotide, abbreviated PMO) sequence that binds to a pre-mRNA (pre-mRNA) of the DMD gene in combination with at least a portion of the dystrophin exon and/or non-exon region.

Specifically, the PMO sequence refers to a six-membered morpholine ring instead of a five-membered furanosyl ring in all nucleotides.

In some embodiments, the oligonucleotide comprises or consists of a sequence capable of at least partially binding to either of the dystrophin pre-mRNA exons 44,45,50,51,52 and 53, said sequence having from 10 to 33 nucleotides.

In some embodiments, the oligonucleotide has a sequence of SEQ ID NO. 001-121, or a sequence having more than 90% homology thereto, preferably 92% homology or more, 95% homology or more, 98% homology or more, most preferably 99% homology or, for RNA sequences, the T in SEQ ID NO. 001-121 is replaced with U.

In a second aspect the application provides a conjugate comprising an oligonucleotide as described above, and a muscle targeting agent which is an antibody or antigen binding fragment.

In other embodiments, the conjugate comprises Ribonucleoprotein (RNP) or an active compound for binding pre-mRNA of DMD gene within the muscle cell nucleus, thereby promoting enhancement or restoration of dystrophin expression or activity.

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

In some embodiments, the muscle targeting agent is an antibody, antigen binding fragment, or small molecule compound.

In some embodiments, 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 (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelidae antibody or binding fragment thereof.

In some embodiments, the muscle targeting agent is an antibody or polypeptide capable of specifically binding to human transferrin receptor 1 (TfR 1) (on the surface of muscle cells) to achieve muscle targeting and enrichment.

In some embodiments, the muscle targeting agent and human TfR1 have 1 or 2 binding sites and an affinity between 0.01nM and 100nM.

In some embodiments, the binding of the muscle targeting agent to human TfR1 does not affect the function of TfR1 to bind transferrin (transferrin).

In some embodiments, wherein the antibody comprises a light chain Variable (VL) region and a heavy chain Variable (VH) region.

In other embodiments, the muscle targeting agent may also deliver an active ingredient to the nervous system.

In some embodiments, the muscle targeting agent is covalently linked to the active ingredient through a non-cleavable or cleavable linker/linker.

In some embodiments, the linker is N-hydroxysuccinimide ester of 4- (N-maleimidomethyl) cyclohexane carboxylic acid (SMCC) and other chemical linkers that remain stable in plasma.

In some embodiments, the molar ratio of active ingredient to 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.

In some embodiments, the antibody peptide polypeptide comprises or consists of the sequence of SEQ ID NO 285.

In some embodiments, the conjugate enters the endosome of the muscle cell by receptor-mediated endocytosis, and there is a correlation in endocytic efficiency and affinity.

In some embodiments, the conjugate or active ingredient thereof is released into the cytoplasm by endosomal escape, and the release of the conjugate or active ingredient thereof may be independent of endosomal cleavage of the linker.

In some embodiments, the conjugates are used for the treatment of Duchenne Muscular Dystrophy (DMD) and/or Becker Muscular Dystrophy (BMD).

In a third aspect the present application provides a pharmaceutical composition comprising an oligonucleotide as described above or a conjugate as described above for use in the treatment of duchenne muscular dystrophy and becker muscular dystrophy.

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

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

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

In a fourth aspect, the application provides a method of delivering an oligonucleotide as described above to a cell, the method comprising coupling the oligonucleotide as described above to an antibody, antibody fragment, polypeptide, antigen binding sequence or small molecule compound.

In a fifth aspect the application provides a method of increasing or restoring the expression or activity of dystrophin in a cell, said method comprising contacting said cell with an oligonucleotide or conjugate as described above.

In a sixth aspect the 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 an oligonucleotide, conjugate or pharmaceutical composition as described above to treat duchenne muscular dystrophy and/or becker muscular dystrophy in the subject.

Preferably, wherein the subject is a human.

In a seventh aspect the present application provides the use of an oligonucleotide as described above or a conjugate as described above for the preparation of a medicament for increasing or restoring the expression or activity of dystrophin or for the prevention and/or treatment of duchenne muscular dystrophy and/or becker muscular dystrophy.

Compared with the prior art, the invention has the beneficial effects that:

The present embodiments provide a range of oligonucleotides, including but not limited to antisense oligonucleotides (ASOs) or morpholino antisense oligonucleotides (PMOs), that bind to at least a portion of the exons and/or non-exonic regions of a dystrophin precursor mRNA and promote exon skipping, resulting in a protein that is near full length but has partial dystrophin function, the proportion of exon skipping of the PMO drug of the present invention being significantly better than that of PMO drugs already published in other patents (example 2).

The antibody-oligonucleotide conjugates provided by the embodiments of the present invention significantly alter the route of drug clearance relative to existing oligonucleotide drugs. Existing oligonucleotide drugs, including polypeptide-conjugated oligonucleotides, are cleared primarily by the kidneys. The molecular weight of the oligonucleotide and the antibody after conjugation is greatly improved, and the path of kidney clearance is no longer provided, so that the half-life of plasma is obviously prolonged, and the dose-dependent muscle targeting enrichment is realized. At the same dose, the skeletal muscle PMO concentration of the antibody-PMO conjugate of the invention is two orders of magnitude higher than that of the existing PMO drug.

The antibody-oligonucleotide conjugate also has the advantage of drug efficacy over existing oligonucleotide drugs. The antibody-PMO conjugates significantly increased the proportion of exon skipping compared to existing PMO drugs and exhibited dose dependence. The antibody-PMO conjugates significantly increased the levels of full length dystrophin in skeletal muscle, cardiac muscle and diaphragm muscle and exhibited dose-dependency over existing PMO drugs. antibody-PMO conjugates consistently reduced ALT, AST and CK levels in mdx mice, exhibiting repair effects on DMD-induced liver and muscle injury. The antibody-PMO conjugate is sufficient to restore 100% of the dystrophin level in mdx mice and maintain the same for at least 28 days at a dose of 10mg/kg in a single administration, the administration frequency is reduced to 1/4 of the original PMO drug administration frequency, the total administration dose is reduced to 1/12 of the original PMO drug administration frequency, and the relative dystrophin level is remarkably improved by more than 20 times of the original dystrophin level. It follows that antibody-oligonucleotide conjugates have the opportunity to deliver superior efficacy to DMD patients with reduced doses and frequency of administration.

Secondly, the embodiment of the invention also carries out mouse pharmacokinetics research, cynomolgus monkey pharmacokinetics research, in-vitro activity and affinity test on the antibody-siRNA conjugate, and the results show that the antibody-siRNA conjugate prolongs the plasma half life of siRNA, realizes more muscle siRNA distribution, has muscle siRNA higher than or close to the distribution of liver siRNA, proves the muscle targeting effect of TfR1 antibody, and has no obvious change in weight and behavioural observation of mice and cynomolgus monkeys in the experimental process, thereby showing good tolerance of the conjugate. The antibody-siRNA conjugate can enter an RD cell line under the condition of in vitro co-incubation without the assistance of lipofectamine and has the dose-dependent target gene knockdown effect, the conjugation of siRNA has adverse effect on the affinity of the antibody-antigen, and the more the conjugation quantity is, the more the tendency of the decrease of the affinity of the antibody-antigen is obvious. These efforts lay the foundation for further development of antibody-oligonucleotide conjugates.

Furthermore, the affinity and in vitro activity research results of the polypeptide-oligonucleotide conjugate provided by the embodiment of the invention show that the affinity of the polypeptide and the antigen can be reduced as the polypeptide is coupled with the oligonucleotide, and the polypeptide can enter an RD cell line under the in vitro co-incubation condition without the assistance of lipofectamine and perform the dose-dependent target gene knockdown effect.

The results show that the conjugate containing the oligonucleotide and the muscle targeting agent (comprising the antibody and the polypeptide) can prolong the plasma half-life of the small nucleic acid medicine, realize more muscle distribution of the small nucleic acid medicine, remarkably improve the drug effect of the existing small nucleic acid medicine for treating muscle related diseases and have more excellent patent medicine performance.

Drawings

FIG. 1 is a gel electrophoresis diagram of PMO effecting exon skipping in patient-derived cardiomyocytes;

FIG. 2 is a capillary electrophoresis diagram of PMO effecting exon skipping in patient-derived cardiomyocytes;

FIG. 3 is a schematic diagram of Linker-PMO and SMCC-Linker-PMO;

FIG. 4 is a SDS-PAGE of TIB-219 and TIB-219-S245C;

FIG. 5 is a SEC spectrum of TIB-219;

FIG. 6 is a SEC spectrum of TIB-219-S245C;

FIG. 7 is a crude purification profile of an antibody-PMO conjugate after reaction, collecting the P2-P4 fractions, respectively;

FIG. 8 shows IEX results for crude products after reaction;

FIG. 9 is a mass spectrum of the crude collected components after the reaction;

FIG. 10 is an IEX profile of the antibody-PMO conjugate after purification;

FIG. 11 is a SEC profile of the antibody-PMO conjugate after purification;

FIG. 12 is a schematic diagram of the principle and primer design of hELISA;

FIG. 13 is a graph showing the results of tissue distribution assays in mdx mice after a single dose of CGBC-1011;

FIG. 14 shows the results of the myoliver ratio assay in mdx mice after a single dose of CGBC-1011;

FIG. 15 shows the results of exon skipping efficiency (nested PCR) detection of muscle tissue in mdx mice after single dose CGBC-1011;

FIG. 16 shows the results of exon skipping efficiency (nested PCR) detection of non-muscle tissue in mdx mice after single dose CGBC-1011;

FIG. 17 shows the results of exon skipping efficiency (ddPCR) detection of muscle tissue in mdx mice after single dose CGBC-1011;

FIG. 18 is a graph showing the results of the detection of the effect of CGBC-1011 on the biochemical blood production of mdx mice after a single administration;

FIG. 19 shows the results of exon skipping efficiency (low dose, nested PCR) detection of muscle tissue in mdx mice after single dose CGBC-1011;

FIG. 20 is a graph showing muscle tissue recovery of dystrophin levels (low dose) in mdx mice after a single dose of CGBC-1011;

FIG. 21 is a CGBC-1004-1 molecular chromatogram (A280 upper, A260 lower);

FIG. 22 is a CGBC-1004-2 molecular chromatogram (A280 upper, A260 lower);

FIG. 23 is a CGBC-1005-1 molecular chromatogram (A280 upper, A260 lower);

FIG. 24 is a CGBC-1005-2 molecular chromatogram (A280 upper, A260 lower);

FIG. 25 is a CGBC-1012-1 molecular chromatogram (A280 upper, A260 lower);

FIG. 26 is a CGBC-1012-2 molecular chromatogram (A280 upper, A260 lower);

FIG. 27 is a graph of siRNA profile assays for CGBC-1004 (DAR 1 and DAR 2) in wild-type CD-1 mouse plasma, muscle and liver;

FIG. 28 is a graph of siRNA profile assays for CGBC-1005 (DAR 1 and DAR 2) in wild-type CD-1 mouse plasma, muscle and liver;

FIG. 29 shows the results of muscle to liver AUC ratio measurements for different designs of antibody-siRNA conjugates;

FIG. 30 is a PK profile of CGBC-1012 (DAR 1) in cynomolgus monkey plasma;

FIG. 31 is a PK profile of CGBC-1012 (DAR 1) in cynomolgus gastrocnemius muscle;

FIG. 32 is a graph showing the in vitro activity assay of CGBC-1012 (DAR 1 and DAR 2) in human rhabdomyosarcoma cells;

FIG. 33 is a plot of antigen affinity detection results for CGBC-1012 (DAR 1 and DAR 2) and naked antibodies;

FIG. 34 is a plot of antigen affinity detection results for CGBC-1004 (DAR 1 and DAR 2), CGBC-1005 (DAR 1 and DAR 2) and naked antibodies;

FIG. 35 is a coupling of CGBC-1018. The coupling process is verified by HPLC and SDS-PAGE, and the DAR1 coupled product is verified by the molecular weight of the mass spectrum;

FIG. 36 shows the results of in vitro activity assay of CGBC-1018 in human rhabdomyosarcoma cells.

Detailed Description

The examples describe various conjugates for delivery of oligonucleotide drugs to muscle cells and uses thereof. In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application 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 for purposes of illustration only and are not intended to limit the scope of the application.

Definition of terms

In the present application, "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be regulated by an antisense compound. The target nucleic acid may be DNA or RNA, in particular, the target RNA may be mRNA, pre-mRNA, non-coding RNA, pre-microRNA (pre-microRNA), mature microRNA, promoter-directed RNA or natural antisense transcripts. For example, a target nucleic acid may be a nucleic acid molecule that expresses a cellular gene (or mRNA transcribed from a gene) associated with a particular disorder or disease state, or from a pathogenic agent.

In the present application, an "oligonucleotide" refers to a compound that contains a plurality of nucleosides linked to each other. Thus, while the term "oligonucleotide" generally refers to a polymer of nucleotides in which the nucleosides and linkages between them are naturally occurring, it is understood that the scope of the term also includes various analogs in which one or more of the plurality of nucleosides is modified. Oligonucleotides generally have about 10-30 nucleotide residues.

In the present application, the term "antisense oligonucleotide (ASO)" refers to a single stranded oligonucleotide molecule having a nucleobase sequence complementary to a corresponding fragment of a target nucleic acid (e.g., a genomic sequence of interest, a pre-mRNA, or an mRNA molecule), i.e., hybridizing to the target nucleic acid and modulating the activity, processing, or expression of the target nucleic acid. Antisense oligonucleotides can be 12 to 30 nucleobases in length, and can include unmodified or modified nucleic acids. ASO locks target RNA first, then cuts target RNA through RNase H, and makes protein unable to express. Since RNase H is present both in the nucleus and outside the nucleus, ASO acts not only on mRNA but also on some noncoding (non-coding) RNAs. Wherein the "morpholino antisense oligonucleotide" (Phosphorodiamidate morpholino oligomers, PMO) is a third generation antisense oligonucleotide, a synthetic oligonucleotide analog that inhibits the function of the gene of interest, primarily by blocking the mRNA splicing process. PMO replaces five-membered furanosyl rings in natural DNA and RNA with six-membered morpholino rings, each of which is linked by an electrically neutral phosphorodiamidate group, rather than by a negatively charged phosphate in natural DNA and RNA. Each phosphorodiamidate morpholino subunit comprises a heterocyclic base (adenine, cytosine, guanine or thymine) in DNA.

In the present embodiment, PMO expressed as a DNA sequence (e.g., the sequence in table 5), after replacing T with U, can be used to represent an RNA sequence, such as an ASO sequence, that binds to the same target sequence.

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

In the present application, a "small interfering RNA" (SMALL INTERFERING RNA, SIRNA, also referred to as a short interfering RNA or silencing RNA) targets a nucleic acid (e.g., mRNA) for degradation via an RNA interference (RNAi) pathway in a cell. The specificity of an siRNA molecule can be determined by the binding of the antisense strand of the molecule to its target RNA. Typically, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., constitute a specific antisense sequence of the siRNA molecule. After selecting the appropriate target RNA sequence, an siRNA molecule comprising a nucleotide sequence (i.e., an antisense sequence) that is complementary to all or a portion of the target sequence can be designed and prepared using an appropriate method. The siRNA molecule may comprise a duplex (duplex), asymmetric duplex, hairpin, or asymmetric hairpin secondary structure having a self-complementary sense strand and antisense strand.

In the present application, an "antisense strand" refers to a strand of a region of a polynucleic acid molecule (e.g., dsRNA) that is substantially complementary to a target sequence. As used herein, the term "complementarity region" generally refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, the mismatch may be in the interior or terminal region of the molecule. Typically, the most tolerated mismatches are within the terminal region, e.g., 5, 4, 3 or 2 nucleotides at the 5 'end and/or 3' end.

In the present application, the "sense strand" (S) refers to a region substantially complementary to a region of the term antisense strand defined above. The "sense strand" is sometimes referred to as the "sense 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 the use of modifications on the sense strand or the use of 5' end caps.

In the present application, "targeting" or "targeting" refers to the binding of an antisense oligonucleotide to a specific target nucleic acid molecule or a specific region of a nucleoside 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 permit hybridization under physiological conditions.

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

In the present application, a "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.

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

In the present application, the term "expression" generally means 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.

In the present application, "conjugate" refers to an atom or group of atoms that is bound to an oligonucleotide or oligomeric compound. In general, the conjugation groups alter various properties of the compounds to which they are attached, e.g., pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cell uptake, charge and clearance. Conjugation groups are commonly used in the chemical arts and are linked to parent compounds (parent compounds) such as oligomeric compounds either directly or via an optional linking moiety or linking group (i.e., linker/linker).

In the present application, "linker/linker" refers to any atom or group of atoms used to attach the conjugate to the oligonucleotide or oligomeric compound. Linking groups or difunctional linking moieties such as those known in the art are amenable to the present application.

In the present application, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains.

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

In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present application, 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 antigen. Specific binding properties between two molecules can be determined using methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument.

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

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

In the present application, the term "subject" includes any human or non-human animal. The term "non-human animal" can be vertebrates (e.g., non-human primates, sheep, dogs) and rodents (e.g., mice, rats, and guinea pigs). The subject may preferably be a human. The term "subject" is used interchangeably herein with "subject" and "patient".

Oligonucleotides

The oligonucleotides of the application can be used to bind/target/hybridize to pre-mRNA of the DMD gene, thereby promoting increased or restored dystrophin expression or activity.

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

In some embodiments, the oligonucleotide is based on an antisense oligonucleotide (ASO) for inducing exon skipping of DMD or dystrophin pre-mRNA in a cell, organ, tissue, and/or individual. Exon skipping produces mature DMD or dystrophin mRNA that does not contain the exon being skipped, and thus, when encoding the exon for an amino acid, produces a protein product that expresses less. Preferably, the skipping of exons is induced by binding the ASO to specific exon-internal sequences comprising splice regulator, splice site and/or intron branch point sequences.

In some embodiments, the oligonucleotide comprises a backbone of morpholino antisense oligonucleotides (PMOs) that bind to at least a portion of the sequence of a dystrophin exon and/or non-exon region, said bound portion having from 10 to 33 nucleotides.

In some embodiments, the oligonucleotide comprises or consists of a sequence that binds to at least a portion of the dystrophin pre-mRNA exons 44 to 55, said sequence having 10 to 33 nucleotides, the function of the oligonucleotide being to promote exon skipping of the dystrophin pre-mRNA.

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 SEQ ID NOs 001-014, with a target sequence of SEQ ID NOs 122-135, which oligonucleotide is capable of promoting skipping of exon 44 of a dystrophin precursor mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOS.001-014 or not differing therefrom by more than 3 nucleotides.

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 SEQ ID NOs 015-035, with a target sequence of SEQ ID NOs 136-156, which oligonucleotide is capable of promoting skipping of exon 45 of a dystrophin precursor mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOS 015-035 or NO more than 3 nucleotides different therefrom.

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 SEQ ID NOs 036-052, the target sequence of which is SEQ ID NOs 157-173, the oligonucleotide being capable of promoting skipping of exon 50 of a dystrophin precursor mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOS 036-052 or differing therefrom by NO more than 3 nucleotides.

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 SEQ ID NOs 053-073, the target sequence of which is SEQ ID NOs 174-194, the oligonucleotide being capable of promoting skipping of exon 51 of a dystrophin precursor mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs 053-073 or differing therefrom by NO more than 3 nucleotides.

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 SEQ ID NOs 074-092, the target sequence of which is SEQ ID NOs 195-213, the oligonucleotide being capable of promoting skipping of exon 52 of a dystrophin precursor mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOS 074-092 or differing therefrom by NO more than 3 nucleotides.

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 SEQ ID NOs 093-121, with a target sequence of SEQ ID NOs 214-242, said oligonucleotide being capable of promoting skipping of exon 53 of a dystrophin precursor mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOS 093-121 or differing therefrom by NO more than 3 nucleotides.

In some embodiments, the oligonucleotides have small interfering RNA (siRNA) sequences that hybridize to a target sequence of human DMD mRNA and mediate RNA interference against human DMD mRNA in muscle cells of a human subject.

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 SEQ ID NOS 267-272. In some embodiments, the siRNA has a sequence of any one of SEQ ID NOS 267-272 or a sequence differing therefrom by NO more than 3 nucleotides.

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 contiguous nucleotides selected from any one of SEQ ID No. 268, SEQ ID No. 270, and SEQ ID No. 272. In some embodiments, the antisense strand has a sequence of any one of SEQ ID NO. 268, SEQ ID NO. 270, or SEQ ID NO. 272 or a sequence that differs from it 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 contiguous nucleotides selected from any of SEQ ID NO:267, SEQ ID NO:269, and SEQ ID NO: 271. In some embodiments, the sense strand has a sequence of any one of SEQ ID NO:267, SEQ ID NO:269, or SEQ ID NO:271 or a sequence that differs from it by NO more than 3 nucleotides.

In some embodiments, the nucleic acid monomers in the PMO backbone and siRNA sequence are chemically modified, in particular, the chemical modification comprises modification of a sugar moiety, modification of internucleoside linkages, modification of the nucleotide itself, and combinations thereof.

Muscle targeting agents

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

In some embodiments, 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 F (ab)' ₃ fragment, a single chain variable fragment (scFv), a diav, (scFv) ₂, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, or a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

In some embodiments, the muscle targeting agent is an antibody or antigen binding fragment that specifically binds to human transferrin receptor 1 (TfR 1) (on the surface of muscle cells) to achieve muscle targeting and enrichment.

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.

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 end of the antibody is from a human IgG1 subclass monoclonal antibody.

In some embodiments, the antibody comprises one or more mutations in the framework region, e.g., in the CHl domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, one or more mutations are to stabilize the antibody and/or increase half-life. In some embodiments, the one or more mutations are to modulate Fc receptor interactions to reduce or eliminate Fc effector functions, such as fcγr, antibody dependent cell-mediated cytotoxicity (ADCC), or Complement Dependent Cytotoxicity (CDC). In other cases, one or more mutations are to modulate glycosylation.

In some embodiments, one or more mutations are located in the Fc region. In some embodiments, the mutation is a LALA-LR mutation, capable of reducing immunogenicity of the Fc-terminus. In some embodiments, the mutation is a S239C, S C or S245C mutation, capable of creating a site suitable for site-directed coupling of nucleic acids.

In some embodiments, the antibody binds to the oligonucleotide by random (non-specific) or site-directed (specific) coupling. In some embodiments, the coupling process is universally applicable to different antibodies and sequences, and differences in antibody targets or sequences, as well as differences in nucleic acid sequences, often do not result in significant changes in the coupling process.

In some embodiments, the antibody is non-specifically conjugated/coupled to the oligonucleotide. In some embodiments, the antibody is non-specifically conjugated/coupled to the siRNA. In some embodiments, the antibody is conjugated to the oligonucleotide via a lysine residue or a cysteine residue in a non-site specific manner.

In some embodiments, the antibody is conjugated/coupled to the oligonucleotide in a site-specific manner, in some embodiments, binding moiety a is conjugated to polynucleic acid molecule (B) via a site-specific manner via a lysine residue, a cysteine residue, a residue at the 5 'end, the 3' end, an unnatural amino acid, or an enzyme modified or catalyzed residue.

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

In some embodiments, an anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the VH region comprises a sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 245/247/249/251, and the VL region comprises a sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 246/248/250/252, 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.

In some embodiments, an anti-transferrin receptor antibody comprises a VH region and a VL region, wherein the sequence of the VH region is SEQ ID No. 245/247/249/251 and the sequence of the VL region is SEQ ID No. 246/248/250/252, 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.

In some embodiments, the antibody or binding fragment thereof is further modified using conventional techniques known in the art, e.g., by using amino acid deletions, insertions, substitutions, additions, and/or by recombination, or by any other modification known in the art (e.g., post-translational and chemical modifications, such as glycosylation and phosphorylation), alone or in combination. In some embodiments, the modification further comprises a modification for modulating interaction with a receptor.

In some embodiments, the muscle targeting agent is a polypeptide. In some embodiments, the polypeptide comprises a polypeptide sequence comprising at least one CDR. The sequence of the polypeptide includes a sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 285. In some embodiments, the sequence of the polypeptide is SEQ ID NO 285.

Conjugate(s)

The conjugates of the application increase or restore the expression or activity of dystrophin, the active ingredients of which are based on the oligonucleotides described above.

In other embodiments, the active ingredient of the conjugate is based on Ribonucleoprotein (RNP) or a small molecule configured to bind pre-mRNA of DMD gene within the muscle cell nucleus, thereby facilitating the enhancement or restoration of dystrophin expression or activity.

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 an active ingredient to the muscle. In some embodiments, the muscle is the myocardium (heart), the diaphragm (diaphragm), the quadriceps (quadriceps), the gastrocnemius (gastro), and the like. In other embodiments, the muscle targeting agent also has the effect of delivering an active ingredient to the nervous system.

In some embodiments, the conjugate enters the endosome of the muscle cell by receptor-mediated endocytosis, and there is a correlation in endocytic efficiency and affinity. In some embodiments, the conjugate or active ingredient thereof is released into the cytoplasm by endosomal escape, and the release of the conjugate or active ingredient thereof may be independent of endosomal cleavage of the linker. In some embodiments, the conjugates are used in the treatment of duchenne muscular dystrophy and becker muscular dystrophy.

In some embodiments, the muscle targeting agent has an effect of improving 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.

In some embodiments, the conjugate comprising the oligonucleotide and the muscle targeting agent has greater activity than an oligonucleotide that does not comprise the muscle targeting agent. In some embodiments, the higher activity is capable of enhancing biologically relevant functions such as 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 mutant exons of a gene. In some embodiments, a conjugate comprising an oligonucleotide and a muscle targeting agent with an oligonucleotide that does not comprise a muscle targeting agent is capable of increasing exon skipping of one or more mutant exons. In some embodiments, the increase in exon skipping is at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%.

In some embodiments, the muscle targeting agent is covalently linked to the active ingredient via a linker/the conjugate comprises a linker linking the muscle targeting agent to the active ingredient. In some embodiments, the linker is a cleavable linker or a non-cleavable linker.

In some embodiments, the linker is a non-polymeric linker. Non-polymeric linkers refer to linkers that do not contain repeating units of monomers resulting from the polymerization process. The non-polymeric linkers include, but are not limited to, C1-C6 alkyl (e.g., C5, C4, C3, C2, or C1 alkyl), homobifunctional crosslinking agents, heterobifunctional crosslinking agents, peptide linkers, traceless linkers, self-sacrificing linkers, maleimide-based linkers, or combinations thereof. In other cases, the non-polymeric linker does not comprise more than two linkers of the same type, e.g., more than two homobifunctional crosslinkers or more than two peptide linkers. In other cases, the non-polymeric linker optionally comprises one or more reactive functional groups.

In some embodiments, the linker comprises a homobifunctional linker. Such homobifunctional linkers include, but are not limited to, lomant reagent dithiobis (succinimidyl propionate) DSP, 3' -dithiobis (sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfoDST), ethyleneglycol bis (succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N, N ' -disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelate (DMP), dimethyl suberoyl imide (DMS), dimethyl 3,3' -Dithiodipropionate (DTBP), 1, 4-di-3 ' - (2 ' -pyridyldithio) propionamido) butane (DPDPB), dimaleimide hexane (BMH), aryl-containing halides (DFDNB)), such as 1, 5-difluoro-2, 4-dinitrobenzene or 1, 3-difluoro-4, 6-dinitrobenzene, 4' -difluoro-3, 3' -dinitrobenzenesulfone (DFDNPS), bis- [ beta- (4-azidosalicylamide)) ethyl ] disulfide (BASED), formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3 '-dimethylbenzidine, benzidine, α' -p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N '-ethylenebis (iodoacetamide) or N, N' -hexamethylenebis (iodoacetamide), and the like.

In some embodiments, the linker comprises a heterobifunctional linker. The heterobifunctional linkers include, but are not limited to, amine reactive and thiol cross-linkers such as N-succinimidyl 3- (2-pyridyldithio) propionate (sPDP), long chain N-succinimidyl 3- (2-pyridyldithio) propionate (LC-sPDP), water soluble long chain N-succinimidyl 3- (2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-alpha-methyl-alpha- (2-pyridyldithio) toluene (sMPT), N-hydroxysuccinimidyl 4- (N-maleimidomethyl) cyclohexanecarboxylate (SMCC), sulfo-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide (MBs), m-maleimidobenzoyl-N-hydroxysuccinimide (sulfo-MBs), N-succinimidyl (4-iodoacetyl) amino-alpha- (2-pyridyldithio) toluene (sMPT), N- (N-maleimidomethyl) cyclohexanecarboxylate (GMB) amino-4- ((6-succinimidyl) butaneimide (GMB) amino) benzoate (GMB), 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.

In some embodiments, the linker binds to the 3' end of the PMO. In some embodiments, the 3' end of the PMO is coupled to the linker under aqueous conditions after activation with C ₃-NH₂.

In some embodiments, the linker is bound to the sense strand of the siRNA. In some embodiments, the linker binds 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 C ₆-NH₂ conjugation handle to which the linker is coupled under aqueous conditions. In some embodiments, the conjugation handle is linked to the sense strand of the siRNA through a phosphodiester on the terminal base.

In some embodiments, the muscle targeting agent binds to the oligonucleotide by random or site-directed coupling to form a conjugate.

In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent by a chemical ligation process. In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent through a natural linkage. In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent in a site-specific or non-specific manner by natural ligation chemistry.

In some embodiments, the oligonucleotides are conjugated in a random coupling manner by mixed incubation with the reduced muscle targeting agent. In some embodiments, the oligonucleotide is a PMO with SMCC bound at the 3 'end or an siRNA with SMCC bound at the 5' end. In some embodiments, the 3' end of the PMO is coupled to the SMCC under aqueous conditions after activation with C ₃-NH₂. In some embodiments, the 5' end of the sense strand of the siRNA contains a C ₆-NH₂ conjugation handle to which the linker is coupled 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 TfR 1. In some embodiments, the muscle targeting agent is reduced by TCEP (thiol-based reducing agent). In some embodiments, the interchain disulfide bonds of the antibody are reduced.

In some embodiments, the oligonucleotide is conjugated in a site-directed coupling manner by mixed incubation with a muscle targeting agent that is oxidized after reduction. In some embodiments, the oligonucleotide is an siRNA with SMCC bound to the 5' end. In some embodiments, the 5' end of the sense strand of the siRNA contains a C ₆-NH₂ conjugation handle to which the linker is coupled 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 TfR 1. In some embodiments, the muscle targeting agent is a cysteine mutant antibody. The muscle targeting agent is reduced by TCEP (thiol-based reducing agent). In some embodiments, the cysteine-site-directed coupling sites (including but not limited to S239C, S238C, S245C, etc., depending on the particular antibody sequence) and interchain disulfide bonds of the antibody are reduced. In some embodiments, the interchain disulfide bonds are oxidized.

In some embodiments, the oligonucleotide is coupled to the muscle targeting agent by a site-directed method using a "traceless" coupling technique (Philochem). In some embodiments, a "traceless" coupling technique utilizes the conjugation of a1, 2-aminothiol group at the N-terminus of a muscle targeting agent to an oligonucleotide containing an aldehyde group.

In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent by a site-directed method that utilizes unnatural amino acids that are 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 is selectively coupled to an alkoxy-amine derived conjugated moiety to form an oxime bond.

Pharmaceutical composition

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

In some embodiments, a pharmaceutical composition or medicament may comprise a pharmacologically effective amount of at least one of the oligonucleotides or conjugates and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are substances other than the active pharmaceutical ingredient (API, therapeutic product, such as oligonucleotide) that are suitably safely evaluated and intentionally included in a drug delivery system. The excipient does not exert or is not intended to exert a therapeutic effect at the predetermined dose. Excipients may be used a) to aid in handling of the drug delivery system during manufacture, b) to protect, support or enhance stability, bioavailability or patient acceptability of the API, c) to aid in product identification and/or d) to enhance any other attribute of overall safety, effectiveness of delivering the API during storage or use. The pharmaceutically acceptable excipient may or may not be an inert substance.

Excipients include absorption enhancers, anti-adherents, defoamers, antioxidants, binders, adhesives, buffers, 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, water-repellent agents, and wetting agents.

In some embodiments, a pharmaceutical composition or medicament may comprise a pharmacologically effective amount of at least one of the oligonucleotides or conjugates and one or more pharmaceutically acceptable carriers. As a pharmaceutically acceptable carrier, for example, a carrier for oral administration or a carrier 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 dextrose, and glycols and the like, and may further include stabilizers and preservatives. Suitable stabilizers may be antioxidants, for example sodium bisulphite, sodium sulphite or ascorbic acid. Suitable preservatives may be benzalkonium chloride methyl parahydroxybenzoate or parahydroxybenzoate propyl hydroxybenzoate and chlorobutanol. Other pharmaceutically acceptable carriers known in the art may also be used.

In some embodiments, the oligonucleotides, the conjugates, and pharmaceutical compositions comprising the oligonucleotides or conjugates disclosed herein may be packaged or contained in a kit, container, package, or dispenser. In some embodiments, the oligonucleotide, the conjugate, or a pharmaceutical composition comprising an oligonucleotide or conjugate disclosed herein may be packaged in a prefilled syringe or vial.

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 atomized or lyophilized. In some embodiments, the atomized or lyophilized form can be reconstituted with an aqueous or liquid solution. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation.

In some embodiments, the pharmaceutical composition is lyophilized for extended shelf life and then made into a solution prior to use (e.g., administration to a subject). Thus, the excipient may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a disintegration temperature regulator (e.g., dextran, ficoll, or gelatin).

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

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, e.g., intravenous, intradermal, subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

The pharmaceutical composition of the present application can bind to pre-mRNA of DMD gene, thereby promoting the enhancement or restoration of the expression or activity of dystrophin. In some embodiments, the pharmaceutical compositions are used to treat a subject suffering from a disease, disorder, or condition in which DMD gene mutations (including exon frameshifting, deletions, substitutions, and repeat mutations) result in premature termination of mRNA or nonsense-mediated mRNA degradation. In some embodiments, the pharmaceutical composition is used to treat a subject suffering from a disease, disorder, or condition caused by reduced or inhibited dystrophin expression. In some embodiments, the pharmaceutical composition is used to treat a subject for a disease, disorder, or condition caused by premature termination of mRNA or nonsense-mediated mRNA degradation resulting in reduced or inhibited dystrophin expression. In some embodiments, the disease, disorder or condition that would benefit from restoration of dystrophin expression includes, but is 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.

Methods of use/treatment

The present application provides a method of preventing and/or treating a disease or disorder, the method comprising administering to a subject of interest an effective amount of a pharmaceutical composition comprising the foregoing oligonucleotide and/or the foregoing conjugate and/or the foregoing.

In some embodiments, the oligonucleotides and/or conjugates and/or pharmaceutical compositions described herein are useful for treating Duchenne Muscular Dystrophy (DMD) and/or Becker Muscular Dystrophy (BMD) by increasing or restoring the expression or activity of dystrophin.

In some embodiments, a therapeutically effective amount of one or more of the oligonucleotides and/or conjugates of the foregoing and/or pharmaceutical compositions of the foregoing is administered to a subject, thereby increasing the expression of dystrophin in the subject (e.g., effective to increase or restore the expression or activity of dystrophin). 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 has mutations (including exon frameshift, deletions, substitutions, and repeat mutations) that optionally comprise a disease-associated DMD gene.

Generally, for administration of any of the oligonucleotides and/or conjugates and/or pharmaceutical compositions described herein, the administration may be to the patient as a single dose (singledose) or may be by a split-up treatment regimen with prolonged administration by multiple doses (multipledose). The pharmaceutical composition of the present application can vary the content of the active ingredient according to the symptoms of the disease. Preferably, the preferred total dose of the composition of the application may be from about 0.01 μg to 1000mg, most preferably from 0.1 μg to 100mg per day per 1 kg body weight of the patient. In some embodiments, the treatment will be administered once. In some embodiments, the treatment will be administered daily, every two weeks, weekly, every two months, monthly, or at any time interval that minimizes the safety risk to the subject while providing maximum efficacy. Generally, efficacy and treatment as well as safety risks can be monitored throughout the course of treatment. The appropriate effective dosage of the pharmaceutical composition of the present application can be determined by those having ordinary skill in the art based on the administration route and the number of treatments and various factors such as the age, weight, health condition, sex, disease severity, diet and excretion rate of the patient. The pharmaceutical composition according to the present application is not particularly limited in terms of dosage form, administration route and administration method as long as it exhibits the effects of the present application.

In some embodiments, a single administration or administration of the pharmaceutical composition to a subject is sufficient to increase or restore expression or activity of dystrophin 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. In some embodiments, a single administration or administration of the pharmaceutical composition to a 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 administration of the pharmaceutical composition to a subject is sufficient to increase or restore dystrophin expression or activity for at least 1, 2,3, 4, 5, or 6 months.

The efficacy of the treatment may be assessed using any suitable method. In some embodiments, the efficacy of a treatment may be assessed by evaluating observations of symptoms associated with DMD and/or BMD, by measuring the subject's self-reported outcome (e.g., mobility, self-care, daily activity, pain/discomfort, and anxiety/depression), or by quality of life indicators (e.g., longevity).

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).

In some embodiments, the oligonucleotides or compositions of the application may be delivered to a cell, cell population, tissue or subject using oligonucleotide delivery techniques known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) may be suitable for use with the oligonucleotides described herein. For example, delivery may be by local administration (local administration) (e.g., direct injection, implantation, or local administration (topical administering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, or topical (including buccal and sublingual), in some embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

Furthermore, the pharmaceutical compositions 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 compositions of the present application and the other therapeutic agents may be administered simultaneously, separately or sequentially. In this case, the other therapeutic agent may be a substance known to have an effect of treating or improving muscular dystrophy. Other therapeutic agents also include all other anti-cancer treatments besides drug treatments, such as radiation therapy.

In other embodiments, the oligonucleotides may be combined with lipids, nanoparticles, polymers, liposomes, micelles, or other delivery systems available in the art. Oligonucleotides 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 amphiphilic membrane active polyamine.

Use of the same

The present application provides the use of the aforementioned oligonucleotides and/or the aforementioned pharmaceutical compositions in the manufacture of a medicament for the prevention and/or treatment of 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).

In preclinical research and clinical practice, an antibody-coupled oligonucleotide (AOC) drug significantly improves the plasma half-life of the oligonucleotide, an antibody or polypeptide targeting the transferrin receptor (TfR) improves the muscle enrichment of the oligonucleotide, and simultaneously the receptor-mediated endocytosis (receptor-mediated endocytosis) also helps the oligonucleotide to realize membrane penetration and release in muscle cells, thereby promoting the expression of functional dystrophin in muscle cells. Thus, the antibody-conjugated oligonucleotide drug has the opportunity to significantly improve the efficacy of existing DMD and BMD therapies.

The application is further described by the following specific examples:

Example 1 expression and affinity validation of anti-TfR 1 antibodies

The inventors selected, expanded a stable cell pool and inoculated it in shake flasks or cell bags using Serum-free Expression medium (GenScript) at 37℃and 5% CO ₂. On the day of transfection, the medium was heated to 25-37 ℃. 50 μ M Swaisonine (MedChemExpress) was added to the medium 1 hour before transfection. And adding a proper amount of plasmids and reagents into the transfection mixture according to the instruction, and fully and uniformly mixing. The mixture was incubated at room temperature. Appropriate supplements are added to the cell culture according to the cell growth. Culture supernatants were collected on day 7 for purification. The supernatant was loaded onto a MabSelect ^TM PrismA Crude at the appropriate flow rate. After washing and elution with the appropriate buffer, the buffer of the eluted fraction is replaced with the final formulated buffer. The purified protein was analyzed by SDS-PAGE and SEC-HPLC (column information: TSKgel G3000SWxl TOSHO) by changing the buffer for eluting the protein to PBS (pH 7.2) to determine the molecular weight and purity. The protein purity of the final product was >98% as detected.

The anti-human TfR1 antibody sequences used in the experiments are shown in table 1. The Fab end is from 13E4-Variant 2iii of U.S. Pat. No. 3, 10913800B2 and the Fc end is from a monoclonal antibody of the human IgG1 subclass. Among them, the inventors have engineered the Fc-terminus, such as with LALA-LR mutation or S238C mutation. The affinity of the anti-human TfR1 antibodies for human TfR 1/cynomolgus TfR1 was confirmed by Octet BLI and the results are shown in table 2.

The sequences of the anti-mouse TfR1 antibodies used in the experiments are shown in Table 3, the variable regions of which are derived from the prior art rat anti-mouse TfR1 antibody TIB-219 (light chain GenBank: ABV48920.1, heavy chain GenBank: ABV 48917.1), and the Fc terminal of which is derived from a human IgG1 subclass monoclonal antibody. Among them, the inventors have engineered the Fc-terminus, such as using LALA-LR mutation or S245C mutation. The quality of the antibodies was controlled in experiments considering that human murine fusion antibodies were expressed. SDS-PAGE showed 99% Purity of TIB-219 and TIB-219-S245C (see FIG. 4, wherein M: marker; lane 1: reduced TIB-219, 99% Purity; lane 2: reduced TIB-219-S245C, 99% Purity; purity:99%, 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 a main peak Purity 97.32% of TIB-219, and a main peak Purity 97.71% of TIB-219-S245C (FIGS. 5-6). The affinity of anti-mouse TfR1 antibody to mouse TfR1 was confirmed by Octet BLI, results are shown in Table 4.

TABLE 1 anti-human TfR1 antibody sequences

TABLE 2 affinity assay results for anti-human TfR1 antibodies

Antibodies to

Antigens

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

TABLE 3 anti-mouse TfR1 antibody sequences

TABLE 4 affinity of anti-mouse TfR1 antibodies

Antibodies to

Antigens

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

Design and Activity of example 2 PMO

The inventors designed PMO sequences for the total of 6 exons of 44, 45, 50, 51, 52 and 53 of the human DMD gene, the transcript sequences of the DMD gene selected for the design being derived from ENST00000357033.9. All PMO sequences designed and their target sequences are shown in Table 5, wherein SEQ ID NO. 001-014 is used for the jump of exon 44, SEQ ID NO. 122-135;SEQ ID NO:015-035 is used for the jump of exon 45, SEQ ID NO. 136-156;SEQ ID NO:036-052 is used for the jump of exon 50, SEQ ID NO. 157-173;SEQ ID NO:053-073 is used for the jump of exon 51, SEQ ID NO. 174-194;SEQ ID NO:074-092 is used for the jump of exon 52, SEQ ID NO. 195-213;SEQ ID NO:093-121 is used for the jump of exon 53 and SEQ ID NO. 214-242.

The pharmaceutical activity of PMO is focused on four factors, 1) the sequence length is 25-30nt, 2) the position at splicing acceptor (splice acceptor) is no more than 100nt, 3) GC% is no less than 40%, 4) the binding energy to the target sequence is no more than-40. The verification result shows that the length of the PMO sequence with better activity is mostly 25-30nt, only 12 PMOs in 121 sequences are lower than 25nt, the position of the PMO with better activity at the distance splicing acceptor is generally not more than 100nt, and only 17 PMOs in 121 sequences are more than 100nt. GC% and binding energy testing was performed using RNAeval algorithm and RNAstructure v 5.3.3 after the design was completed and the GC% and binding energy calculations are shown in table 5.

The sequences and Activity of Table 5 PMO

The inventors selected a portion of PMO for further validation of cellular activity. 19 sequences (SEQ ID NO: 074-092) for exon skipping of 52 and the positive reference sequence H52A (-01+24) from patent US20230110479A1 were selected, physiological saline was set as negative control, primary cardiomyocytes from DMD patients with exon deletion of 51 were transfected in vitro, the length of the PCR fragment after skipping was 278bp, and the fragment length before skipping was 396bp. The cellular activity of PMO was assessed by two-step PCR-gel electrophoresis and capillary electrophoresis for jumping efficiency of exon 52 (primers see Table 6).

TABLE 6 PMO primer sequences for cell Activity studies

Gel electrophoresis of experimental results is shown in fig. 1, gel electrophoresis can semi-quantitatively evaluate jumping efficiency of PMOs, PCR fragments of unskip and skip are respectively corresponding to the vicinity of markers of 400bp and 300bp, skip% = skip/(skip + unskip) ×100% of each PMO can be obtained through gray scale calculation, and calculation results are shown in table 7, wherein SEQ ID NO:89 and SEQ ID NO:90 show better jumping efficiency than yang ginseng.

TABLE 7 gel electrophoresis test results

Capillary electrophoresis quantitatively evaluates the jumping efficiency of PMOs, the initial data of the experimental results are shown in fig. 2, the nucleic acid concentrations of the unskip and skip PCR fragments are calculated respectively through the peak area integral of an electropherogram, the skip% = skip/(skip + unskip) ×100% of each PMO can be obtained through the following formula, the calculated results are shown in table 8, and again, the SEQ ID NO:89 and SEQ ID NO:90 are proved to show better jumping efficiency than that of the yang ginseng.

TABLE 8 capillary electrophoresis test results

EXAMPLE 3 Synthesis of antibody-PMO conjugates

Synthesis of Linker-PMO and SMCC-Linker-PMO

The 3' -end C ₃-NH₂ -activated M23D tool PMO (Linker-PMO) (EG 3-GGCCAAACCTCGGCTTACCTGAAAT-C ₃-NH₂, SEQ ID NO: 243) was obtained using standard solid phase synthesis methods and purified by HPLC. 3 '-terminal C ₃-NH₂ activated M23D PMO was coupled with a 4- (N-maleimidomethyl) cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) linker under aqueous conditions to form 3' -terminal SMCC activated M23D PMO (SMCC-linker-PMO) (EG 3-GGCCAAACCTCGGCTTACCTGAAAT-C ₃ -NH-SMCC, SEQ ID NO:244, the nucleic acid sequence portion of which was identical to SEQ ID NO: 243) which was used in the next step of antibody coupling reaction after purification by HPLC. Specific structures of Linker-PMO and SMCC-Linker-PMO are shown in FIG. 3.

Synthesis of antibody-PMO conjugates

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

Using HIC method 1 (see Table 9), the reaction mixture was purified with AKTAExplorer FPLC. Fractions P2, P3 and P4 of the conjugates predicted to contain different drug to antibody ratios (DAR) were collected and analyzed for each fraction and reaction mixture using HIC method 2 (see table 10) and mass spectrometry. FIG. 7 shows a purification profile of the crude product after the reaction, where P2, P3 and P4 were collected for DAR and molecular weight confirmation.

Since the antibody and the nucleic acid have different absorbance at 260nm and 280nm, it can be confirmed that the main components of P2, P3 and P4 are molecules of DAR2, DAR4 and DAR6 in this order by the absorbance ratio of the single component A260/280 (see FIG. 8 for verification results). Mass spectrometry was used to perform molecular weight and DAR value determinations (see figure 9), where B260T01-E01-TFF-02 was the P2 component, B260T01-E01-TFF-03 was the P3 component, and B260T01-E01-TFF-04 was the P4 component. P2 was essentially determined to be DAR2, P3 was DAR4, and P4 was DAR6 conjugate by the difference in the molecular weights of the main peaks. Thereby determining the DAR and purity of the reaction mixture and final purified product. FIG. 10 shows a chromatogram of the purified product analyzed by HIC method 2, showing DAR2 (15.274), DAR4 (16.582), DAR6 (two peaks, 17.226, 17.735 respectively), the numbers in brackets being retention times (minutes), which ultimately determines the final product as DAR4.6 (see Table 11). Fig. 11 shows SEC chromatograms of the purified product, wherein the product purity was 96.43%.

Table 9 HIC method 1

Chromatographic column	Sedan FPLC (0.66 cm)
		Resin composition	Capto Butyl ImpRes (Cytiva) volume 5.8mL
Flow rate	150cm/h,5mL/min
		Buffer A	50NM PB,0.8M ammonium sulphate, pH7.0
Buffer B	50nM PB,pH7.0

Table 10 HIC method 2

TABLE 11 analysis of antibody-PMO conjugates

Conjugate(s)	DAR	% Purity (in terms of peak area)
			CGBC-1011	4.6	96.43

Example 4 in vivo efficacy of antibody-PMO conjugate in mdx mice (multiple doses)

Experimental design and dosages are shown in table 12. antibody-PMO conjugate CGBC-1011 was prepared as in example 3, the antibody was TIB-219 in example 2, and PMO was M23D PMO (SEQ ID NO: 244) activated by 3' SMCC in example 1.

PBS control and antibody-PMO conjugate were given to mdx mice by intravenous injection, and animals were euthanized 14 days and 28 days after dosing, respectively. Blood is collected under the jaw, EDTA-K2 is anticoagulated and a blood plasma sample is collected, and simultaneously tissue samples including cardiac muscle (heart), diaphragm (diaphragm), gastrocnemius (gastro) and liver (liver) are harvested and stored in a liquid nitrogen flash. Subsequent studies on pharmacokinetics, exon skipping efficiency and hematochemistry were used.

TABLE 12 study of the efficacy of antibody-PMO conjugates in mdx mice (multiple doses)

Pharmacokinetic study of antibody-PMO conjugates in mdx mice

In the experiment, the hybridization ELISA method, the experimental method and the primer design principle are shown in FIG. 12, and the probe complementary to the M23D sequence is used for quantifying the accumulation of PMO in plasma and tissues. The results are shown in figures 13 and 14, where the antibody-PMO conjugate prolonged the plasma half-life of the conventional PMO drug, and PMO plasma concentrations in mdx mice treated with the conjugate were still detectable within 28 days, much higher than the plasma half-life of conventional PMO for about 2 hours (figure 13). At the same time, the antibody-PMO conjugate achieved a dose-dependent muscle-targeted PMO enrichment, proportionally increasing the PMO concentration of the muscle with increasing dosing, and slowly decreasing over time (fig. 13). The antibody-PMO conjugate dose-dependent increased the enrichment of the liver and as the dose increased the gastrocnemius to liver PMO concentration ratio gradually decreased, a tissue distribution pattern that was different from the antibody-siRNA conjugate, indicating that PMO may be present in a non-antibody dependent liver enrichment pathway (fig. 13-14). Thus, increasing the dose of the drug may lead to enrichment of PMO in non-target tissues such as liver, and a dose of 10mg/kg is more conducive to enrichment of PMO in muscle tissue.

Exon skipping efficiency of antibody-PMO conjugate in mdx mice

In experiments, two methods were used to study the efficiency of exon skipping, nested PCR was first used to evaluate the efficiency of exon skipping (table 13). Animal tissue is lysed for isolation of RNA. In a 20. Mu.L reaction system, 2. Mu.g of RNA was reverse transcribed into cDNA, diluted 4-fold, and subjected to a nested PCR reaction (Table 13). The final amplification primer was analyzed for fragment length in a 4% TAE agarose gel, which showed that the wild-type DMD product had 788 base pairs, while the exon-skipping product DMDΔ23 had 575 base pairs. Finally, the product content is quantified by optical density, and the calculation formula of the exon skipping efficiency is skip=skip/(skip+ unskip) ×100%.

TABLE 13 nested PCR primer design and Experimental protocol

In addition to nested PCR, the efficiency of exon skipping can also be assessed by ddPCR (Table 14). The method comprises the steps of extracting RNA and performing ddPCR reaction by using a QX200 Droplet DIGITAL PCR system, wherein the reaction system, the primer design and the detailed steps are as follows, and finally, the exon skipping efficiency is calculated by the QX200 self-contained software.

Table 14 ddPCR primer design and Experimental protocol

The results of the nested PCR experiments showed that the maximum jump efficiency of the antibody-PMO conjugate in the gastrocnemius, myocardium, diaphragm after a single administration in mdx mice was 51%, 23%, 55%, respectively, the efficacy was maintained for at least 28 days, and there was a clear dose dependency (fig. 15). In non-muscle tissue, a proportion of exon skipping efficiencies above PBS were observed in the liver alone, and were typically below 10% (fig. 16). In combination with tissue distribution data (fig. 13), it is speculated that antibody-receptor specific interactions mediated endocytosis plays a critical role in the efficacy of PMO, rather than liver non-specific endocytosis. In muscle tissue, the results of ddPCR (fig. 17) and nested PCR (fig. 15) were not significantly different, but ddPCR significantly reduced noise in PBS group.

Influence of antibody-PMO conjugate on blood biochemistry of mdx mice

Mdx mice often exhibit elevated Creatine Kinase (CK) and transaminases due to long-term muscle injury and inflammatory response. Plasma samples were collected and assayed for changes in alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and Creatine Kinase (CK) in plasma using a hemocytometer. The results demonstrate that antibody-PMO conjugates significantly and consistently reduced ALT, AST and CK levels in mdx mice, with efficacy maintained for at least 28 days after a single administration (fig. 18).

Example 5 in vivo efficacy of antibody-PMO conjugate in mdx mice (low dose)

The results of example 4 above show that the antibody-PMO conjugate increases the concentration of muscle PMO dose-dependently, but at the same time achieves enrichment of PMO in the liver at a faster rate. And as the dose increases, the gastrocnemius to liver PMO concentration ratio gradually decreases, which suggests that PMO may present an antibody-independent liver enrichment pathway. In clinical practice, PMO is often administered at weekly frequency and at clinically recommended doses of 30mg/kg or more. Taken together, it was determined that optimization of antibody-PMO conjugate dose would help maximize PMO muscle enrichment and dystrophin recovery at lower dosing frequency and dose. The aim of this experiment was to evaluate the in vivo efficacy of the antibody-PMO conjugate in mdx mice at doses below the clinically recommended dose, e.g. at 10 mg/kg.

See table 15 for experimental design and dosages. antibody-PMO conjugate CGBC-1011 was prepared as in example 3, the antibody was TIB-219 in example 2, and PMO was 3' SMCC-activated M23D PMO in example 1 (SEQ ID NO: 244).

PBS control and antibody-PMO conjugate were given to mdx mice by intravenous injection, and animals were euthanized according to the experimental design at 14 and 28 days after dosing, respectively. Blood is collected under the jaw, EDTA-K2 is anticoagulated and a blood plasma sample is collected, and simultaneously tissue samples including cardiac muscle (heart), diaphragm (diaphragm), gastrocnemius (gastro) and liver (liver) are harvested and stored in a liquid nitrogen flash. Subsequent studies on exon skipping efficiency, and western blot.

TABLE 15 study of the efficacy of antibody-PMO conjugates in mdx mice (single dose)

Exon skipping efficiency of antibody-PMO conjugates in mdx mice

The procedure of nested PCR is described in example 4. The results showed that the exon skipping efficiencies in gastrocnemius, cardiac muscle, diaphragm in mdx mice were 24%, 8%, 27% respectively 28 days after single administration of the antibody-PMO conjugate (see fig. 19).

B ability of antibody-PMO conjugate to restore muscle dystrophin content in muscle tissue in mdx mice

The dystrophin content was determined by western blot. Specific protocols are described below, after shearing the collected muscle tissue with scissors, the supernatant was collected using steel column homogenate samples and protein concentration was measured by BCA assay. Total protein (24 μg) was loaded onto 3% to 8% NuPAGE ^TM Tris acetate protein gel, run for 1 hour at 75V followed by running for 1 hour at 150V and run all the way on ice, then proteins were transferred from the gel onto polyvinylidene fluoride membrane using an iBlot ^TM 2 transfer membrane apparatus, run for 10 minutes at 25V, split, and incubated overnight at 4℃in 1:2000 anti-dystrophin (dystrophin) antibody (Abcam catalog No. ab 154168), then 1 hour at room temperature in 1:5000 goat anti-rabbit IgG, as a control, the remaining blotting membrane was incubated overnight at 4℃with 1:2000 anti-alpha-actin (ALPHA ACTININ) antibody (Abcam catalog No. ab 9465), then 1 hour at room temperature with 1:10000 goat anti-mouse IgG, developed using ECLWESTERN detection kit (Cytiva), and quantified using iBright FL (Thermo FISHER SCIENTIFIC).

In each western blot experiment, a fixed proportion of mdx mouse muscle and healthy C57BL/6J mouse muscle of the same class were weighed and homogenized to establish a standard curve for western blot. The percentage of wild-type protein in the standard of the standard curve was 64%,32%,16%,8%,4%,2% and 0% from high to low. The standard curve was fitted non-linearly using the 4PL approach.

The results showed that the antibody-PMO conjugate could recover 100%, 39%, 88% of the muscle dystrophin in gastrocnemius, myocardium, diaphragm in mdx mice, respectively, to healthy animal tissues 28 days after single administration of 10mg/kg, and the efficacy could be maintained for at least 28 days (fig. 20). This result suggests that antibody-conjugated PMOs have the opportunity to achieve better exon skipping and recovery of dystrophin with reduced dosing and frequency.

EXAMPLE 6 Synthesis of antibody-siRNA conjugates

In addition to PMO, chemically modified antisense oligonucleotides also have the ability to effect exon skipping. The nucleic acid drugs are closer to siRNA in terms of physical and chemical properties and metabolism, so that in order to systematically study the tissue distribution of the antibody-oligonucleotide coupled drugs, in vivo pharmacokinetic studies are performed by using antibody-siRNA conjugates with higher detection sensitivity so as to support subsequent drug optimization and in vivo studies.

Synthesis of Linker-siRNA and SMCC-Linker-siRNA

For linker-siRNA used in this experiment, single strands were fully assembled on solid phase using standard phosphoramidite chemistry and purified using HPLC, base, sugar and phosphate modifications well described in the RNAi field were used to optimize duplex stability. Wherein the 5' end of the sense strand of the siRNA contains a C ₆-NH₂ conjugate handle, and the conjugate handle is connected to the sense strand of the siRNA through a phosphodiester on a terminal base.

The siRNA activated at the 5 'end C ₆-NH₂ is coupled with N-hydroxysuccinimide ester (SMCC) linker of 4- (N-maleimidomethyl) cyclohexane carboxylic acid under aqueous condition to form linker-siRNA activated at the 5' end by SMCC, and can be used for the next antibody coupling reaction after purification by HPLC.

Coupling of wild-type antibody and siRNA

The coupling of wild-type antibody and siRNA employed a random coupling process. Specifically, the interchain disulfide bond of the antibody is reduced first, and conjugated with linker-siRNA activated by SMCC linker at the 5' end of the siRNA sense strand, to finally form an antibody-siRNA conjugate.

Step 1 reduction of antibodies with TCEP

The antibody was buffer exchanged with 25mM Tris buffer (pH 8) and brought to a concentration of 10mg/mL. To this solution was added 0.8 equivalent of TCEP in the same buffer and incubated for 2 hours at room temperature. The reaction was then exchanged by ultrafiltration to 2mM EDTA in 25mM Tris buffer (pH 8) and mixed with SMCC-siRNA (3 eq.) and reacted at 22℃for 3 hours. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugates, unreacted antibodies and siRNAs.

Step 2 purification and analysis

The crude reaction mixture was purified by AKTAPure FPLC using anion exchange chromatography (SAX) method-1 (Table 16). The fractions containing the antibody-siRNA conjugate were isolated, concentrated, and buffer exchanged with PBS pH 7.4.

The purity of the conjugates was assessed by analytical HPLC using SAX method-2 (table 17).

TABLE 16 anion exchange chromatography (SAX) -1

TABLE 17 anion exchange chromatography (SAX) -2

Coupling of cysteine mutant antibodies and siRNA

The coupling of the cysteine mutant antibody and the siRNA adopts a site-directed coupling process. Specifically, the antibody cysteine site-directed coupling site (including but not limited to S239C, S238C, S245C, etc., depending on the specific antibody sequence) and the interchain disulfide bond are first reduced, then the interchain disulfide bond is oxidized and the reduced state of the cysteine site is preserved, and finally the antibody-siRNA conjugate is formed by conjugation with linker-siRNA activated by SMCC linker at the 5' end of the siRNA sense strand.

Step 1 reduction of antibodies with TCEP

The antibody was buffer exchanged with 25mM Tris buffer (pH 8) and brought to a concentration of 10mg/mL. To this solution 100 equivalents of DTT in the same buffer were added and incubated for 16 hours at room temperature. The reaction was then changed to 2mM EDTA in 25mM Tris buffer (pH 8) by ultrafiltration centrifugation, and 20 equivalents of DHAA were added and reacted at Room Temperature (RT) for 2 hours, and the resulting reaction mixture was passed through a desalting column to remove excess DHAA and mixed with SMCC-siRNA (0.8 equivalents) and reacted at 22℃for 2 hours. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugates, unreacted antibodies and siRNAs.

Step 2 purification and analysis

The crude reaction mixture was purified by AKTAPure FPLC using anion exchange chromatography (SAX) -1 (Table 16). The fractions containing the antibody-siRNA conjugate were isolated, concentrated, and buffer exchanged with PBS pH 7.4.

The purity of the conjugates was assessed by analytical HPLC using method SAX-2 (table 17).

Analytical data for the conjugates synthesized in this example are shown in table 19 for representative antibody-siRNA conjugate molecule analysis data after purification, both in terms of HPLC Retention Time (RT) in minutes and percent purity as determined by chromatographic peak area, at a purity above 97% and the spectra are shown in fig. 21-26.

The siRNAs used in the experiments to synthesize the antibody-siRNA conjugates are shown in Table 18, in which siRNA ID#001 was used to synthesize CGBC-1004-1 and CGBC-1004-2, siRNA ID#002 was used to synthesize CGBC-1005-1 and CGBC-1005-2, and siRNA ID#003 was used to synthesize CGBC-1012-1 and CGBC-1012-2, with the specific sequences shown in Table 18 below. In terms of antibodies, CGBC-1004-1, CGBC-1004-2, CGBC-1005-1 and CGBC-1005-2 were used with TIB-219-S245C. The antibodies used for CGBC-1012-1 and CGBC-1012-2 were Var2iii-S238C, both synthesized by the site-directed coupling procedure described above.

Table 18 siRNA sequence

Modification description m=2 '-O-methyl, i2f=2' -fluoro, gn=ethylene glycol nucleic acid, x/=phosphorothioate linkage, v/=phosphodiester linkage, vp=5- (E) -VP vinyl phosphate modified nucleic acid

TABLE 19 design and analysis results of antibody-siRNA conjugates

EXAMPLE 7 mouse pharmacokinetic Studies of antibody-siRNA conjugates

The aim of the experiment was to investigate the ability of antibody-siRNA (using the sequences described above) conjugates to deliver nucleic acids in muscle targeting in mice. See table 20 for experimental design and dosages. The sirnas used in the experiments to synthesize the antibody-siRNA conjugates are shown in table 18.

PBS control and antibody-siRNA conjugate were given to mice by intravenous injection, animals were euthanized after 7 and 14 days, respectively, and plasma and different tissue samples including quadriceps (quadriceps), liver (liver) were harvested and cryopreserved in liquid nitrogen. Tissue homogenates were taken and after digestion with proteinase K supernatant samples were diluted for SL-PCR analysis. Pharmacokinetic experiments mainly used SL-PCR to analyze siRNA concentration in plasma and tissues, which consisted mainly of a reverse transcription using stem-loop primer sequences in the first step and a quantitative reaction using qPCR in the second step. The reaction system is shown in tables 21 to 22, and the primer sequences are shown in Table 23.

TABLE 20 study of antibody-siRNA conjugates for knockdown of target genes in wild-type CD-1 mouse muscle

TABLE 21 SL-PCR reverse transcription reaction System

PCR reaction solution composition	1 Reaction System required volume (μL)
		100mM dNTPs(withdTTP)	0.15
MultiScribe Reverse Transcriptase,50U/μL	1
		10X Reverse Transcription Buffer	1.5
RNase Inhibitor,20U/μL	0.19
		Nuclease-free Water	7.085
5XRT primer(4μM)	0.1875
		siRNA/miRNA	5
Total	15

Table 22 SL-qPCR reaction System

PCR reaction solution composition	1 Reaction System required volume (μL)
		Universal PCR Master Mix 2x	5
Forward primer(10μM)	1.5
		Reverse primer(10μM)	0.7
Probe(10μM)	0.2
		cDNA	2
H2O	0.6
		Total	10

TABLE 23 primer sequences for SL-PCR in mouse experiments

A standard curve of Ct-log [ concentration ] is drawn in the experiment. Standard pharmacokinetic curves were prepared by diluting the antibody-siRNA conjugate compound and adding it to blank mouse tissue homogenates. In addition to the above steps, the actual sample processing step and the sample processing step of the standard curve remain identical. The amplification efficiency of the standard curve needs to be ensured to be in the range of 90-110%.

The antibody-siRNA conjugates exhibit much lower plasma clearance rates and higher muscle-specific siRNA delivery efficiencies than traditional siRNA drugs. The siRNA in plasma could still be detected by SL-PCR after 14 days of dosing (fig. 27-28), significantly different from the plasma half-life of conventional siRNA drugs for several hours, indicating that antibody-siRNA conjugates prolonged the plasma half-life of siRNA. In terms of tissue distribution, the antibody-siRNA conjugate achieved more muscle siRNA distribution, with muscle siRNA higher or closer than liver siRNA distribution, demonstrating muscle targeting of TfR1 antibodies. In terms of molecular design, the molecules of DAR1 showed higher blood concentration and more muscle enrichment than the DAR2 molecules, and the design of 5- (E) -VP also significantly improved the enrichment of antibody-siRNA conjugates in the muscle (fig. 29).

Neither the body weight of the mice nor the behavioral observations changed significantly during the experiment, showing good tolerability of the conjugates.

Example 8 cynomolgus monkey pharmacokinetic study of antibody-siRNA conjugates

The aim of the experiment was to investigate the ability of antibody-siRNA conjugates to deliver nucleic acids in muscle targeting in cynomolgus monkeys. Experimental design and dosages are shown in table 24. The siRNA used in the experiments to synthesize the antibody-siRNA conjugates was shown in Table 18 as siRNA ID #003, and the antibody Var2iii-S238C was used and synthesized by the site-directed coupling procedure described above.

The pharmacokinetic studies were performed by intravenous injection of antibody-siRNA conjugates into cynomolgus monkeys, collecting plasma samples 3, 6, 12 hours and 1,3, 7, 14, 28, 42 days before and after administration according to the experimental protocol, while gastrocnemius (gastro) samples were collected by muscle puncture 3, 7, 14, 28, 56, 84 days before and after administration. The method of drawing the standard curve is consistent with example 7 and will not be described again. Primer sequences are shown in Table 25.

Table 24 in vivo studies of antibody-siRNA conjugates in cynomolgus monkeys

Table 25 primer sequences for SL-PCR in cynomolgus monkey experiments

Experimental results showed that the antibody-siRNA conjugate exhibited a typical two-compartment model in cynomolgus plasma, with linear PK at two doses, where the distribution half-life at high dose was 27.6 hours, clearance half-life was 279 hours, and the profile of the distribution phase was accurately recorded for the low dose group because of the lower limit of detection of the method (fig. 30). In terms of tissue distribution, the antibody-siRNA conjugate was enriched in cynomolgus gastrocnemius muscle and reached Cmax exceeding 300pmol/g on day 3 post-dose. The clearance half-life of the antibody-siRNA conjugate in the calf muscle of cynomolgus monkeys was 239 hours, and the calf muscle siRNA concentration was always higher than 1pmol/g for 84 days before the end of the experiment (fig. 31).

Neither the cynomolgus monkey body weight nor the behavioural observations changed significantly during the experiment, showing good tolerability of the conjugates.

Example 9 in vitro Activity of antibody-siRNA conjugates

The aim of the experiment was to assess whether the antibody-siRNA conjugate could be engulfed by muscle cells and exert biological activity in vitro without the aid of lipofectamine (liposomes). The antibody-siRNA conjugate used in the experiment was the same as in example 7. The experimental details were as follows, the RD human rhabdomyosarcoma cell line (4201 HUM-CCTCC 00295) was cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco), the antibody-siRNA conjugate was diluted to a maximum dose of 10mM, the antibody-siRNA conjugate was transfected with final siRNA concentrations of 200, 100, 10, 1 and 0.1nM, and cells were seeded onto 48 well plates at 10,000 cells per well 24h prior to dosing. antibody-siRNA conjugates were added to wells of 48-well plates, PBS was added to some wells as an additional negative control, cells were placed at 37℃and 5% CO ₂ for 72 hours, medium was removed from the wells and 150mL Trizol (Life) was added, plates were frozen overnight or longer at-80℃before analysis, RNA was isolated using Direct-zol-96RNA kit (Zymo Research) according to the instructions, RNA was reverse transcribed to cDNA according to the instructions using REVERTRA ACE ^TM QPCR RT MASTER Mix (TOYOBO), cDNA samples were evaluated by qPCR using TAQMAN FASTADVANCED MASTER Mix (Applied Biosystems) using DMPK-specific and GAPDH-specific primers and SYBR Green methods. . % mRNA was calculated using standard 2- ΔΔCT method, PBS-treated cells were set to 100% expression. All experiments were performed in triplicate. Primer information is shown in Table 26.

TABLE 26 primer information

Primer(s)	Sequence (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 NO284)

Experimental results show that CGBC-1011 can enter an RD cell line under the condition of in vitro co-incubation without the assistance of lipofectamine, and the dose-dependent target gene knockdown effect is achieved. The knockdown effect of the antibody-siRNA conjugate was saturated at a concentration of 1nM, with a maximum knockdown efficiency of the antibody-siRNA conjugate of DAR2 of about 40% and a maximum knockdown efficiency of the molecule of DAR1 of about 50% (fig. 32).

Example 10 affinity of antibody-siRNA conjugates

The aim of the experiment was to evaluate the change in affinity of antibody-siRNA conjugates and bare antibodies to antigen, and to evaluate the effect of the coupling mode and the selection of DAR values on the affinity of antibody-antigen. Experimental methods the affinity of conjugates and naked antibodies to TfR1 antigen was assessed by ELISA, specific steps comprising coating 96-well plates with 1. Mu.g/mL antigen, washing 2 times in PBST after overnight at 4 ℃, coating with 3% MPBS, washing twice in PBST after 1 hour at 37 ℃, followed by addition of gradient diluted conjugates or naked antibodies and detection using anti-human/rat IgG-Fc-HRP, and finally development using TMB. Care should be taken in the experiments to select the corresponding antigen and detection antibody based on the species specificity of the conjugate and naked antibody. After the end of the experiment, EC50 was calculated by plotting OD450 and antibody concentration.

The experimental results showed a significant decrease in target affinity for the antibody-siRNA conjugates compared to the naked antibody (fig. 33-34). In the comparison of conjugates, there was generally a significant decrease in affinity for DAR2 over DAR1 (FIG. 34), but the affinity for DAR2 in CGBC-1012 was not much different than that for DAR1 (FIG. 33). The above data demonstrate that conjugation of siRNA has a detrimental effect on antibody-antigen affinity, and the greater the number of conjugates, the more pronounced the trend toward decrease in antibody-antigen affinity.

EXAMPLE 11 Synthesis of polypeptide-siRNA conjugates

The nucleic acid sequences used in the experiments were identical to those of example 8, and the polypeptide CGBB2 sequences used in the experiments were as follows:

PSEEEIKKLVEELLKELSKEEAALKLVETAADVVVVTPKGIVVVKGDRETAEAVFKAAEEAFDKYPDDAEFIAEYIKKKVPKARVVLVPN(SEQ ID NO:285).

To 30. Mu.L of the polypeptide (3 nmol) in phosphate buffer pH 7.4 were added 6-azidomethyl 4-methoxypyridine formal (30 nmol in 1. Mu.L of DMSO) and 3-maleimidopropionic acid (15 nmol in 1. Mu.L of DMSO). The resulting mixture was incubated overnight at 37 ℃. The resulting solution was then concentrated and PBS buffer was added.

The liquid was exchanged using a 0.5mL centrifugal filter. Buffer exchange each sample was first diluted to 500 μl using 50mm pH 7.4 phosphate buffer. Each sample was concentrated to 30. Mu.L and the procedure repeated 3 times to give a polypeptide intermediate. DBCO modified oligonucleotide (4.5 nmol in 4.5. Mu.L pH 7.4 phosphate buffer) was then added to 30. Mu.L protein solution (3 nmol) and the resulting mixture incubated overnight at room temperature and purified via SEC FPLC to give the corresponding polypeptide-oligonucleotide conjugate, designated CGBC-1018.CGBC-1018 coupling, purification and analysis results are shown in FIG. 35.

EXAMPLE 12 affinity and in vitro Activity Studies of polypeptide-siRNA conjugates

The aim of the experiment was to assess the affinity of the polypeptide-siRNA conjugate and whether it could be engulfed by muscle cells and exert biological activity in vitro without the aid of lipofectamine. Affinity was confirmed by Octet BLI, experimental procedure was the same as in example 1, and in vitro activity was the same as in example 9.

The results of the affinity studies are shown in Table 27, and indicate that the same polypeptide-conjugated nucleic acid as the antibody-conjugated nucleic acid reduces the affinity of the polypeptide to the antigen. However, under the design of DAR1, the magnitude of the affinity decrease was not large, and the ability of the polypeptide to deliver nucleic acid was not significantly affected.

Table 27 affinity of anti-human TfR1 Polypeptides and conjugation products

The results of in vitro activity studies show that CGBC-1018 can enter RD cell lines under in vitro co-incubation conditions without the assistance of lipofectamine and exert a dose-dependent target gene knockdown effect. The in vitro knockdown efficiency of polypeptide-siRNA conjugates CGBC-1018 was lower than previously evaluated antibody-siRNA conjugates CGBC-1012 and required to exert significant knockdown at concentrations greater than 100nM, with a maximum knockdown efficiency of the polypeptide-siRNA conjugates of about 30% (fig. 36).

The above description is only of the preferred embodiments of the present invention and is not intended to limit the invention, but any modifications, equivalents, improvements, etc. within the principles of the present invention should be included in the scope 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 an antisense morpholino oligonucleotide (PMO), and the oligonucleotide binds to at least a portion of an exon and/or non-exon region of dystrophin.

2. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises or consists of a sequence that binds to at least a portion of 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 sequence of the oligonucleotide is SEQ ID NO: 001-121, or a sequence having 90% or more homology thereto, preferably a sequence having 92% or more, 95% or more, 98% or more, or 99% or more homology thereto;

Or a sequence expressed in the form of RNA in which T is replaced by U.

4. A conjugate comprising the oligonucleotide according to any one of claims 1 to 3, 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, characterized in that 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 divalent Fab2, a single-chain variable fragment, a diabody, a minibody, a nanobody, a single domain antibody or a camelid antibody or binding fragment thereof.

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 one or two binding sites with human transferrin receptor 1 and an affinity between 0.01 nM and 100 nM.

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

9 . The conjugate according to claim 8 , wherein the linker is 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester.

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

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

12. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the oligonucleotide according to any one of claims 1 to 3 or the conjugate according to any one of claims 4 to 11, and the pharmaceutical composition is used to treat Duchenne muscular dystrophy and/or Becker muscular dystrophy.

13. The pharmaceutical composition of 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, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable salt, excipient or carrier.

15. Use of the oligonucleotide according to any one of claims 1 to 3 or the conjugate according to any one of claims 4 to 11 for preparing a medicament, wherein the medicament is used to promote the expression of dystrophin, or to prevent and/or treat Duchenne muscular dystrophy and/or Becker muscular dystrophy.