Medicine for treating Duchenne muscular dystrophy and Becker muscular dystrophy

Abstract

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

Description

Technical Field

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

[0002] Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are both X-chromosome recessive single-gene genetic diseases that mainly occur in male children. The cause is a decrease in the level of dystrophin in muscle cells, which leads to muscle inflammation, fibrosis, and decreased exercise capacity. If left untreated, most patients will die before the age of 30.

[0003] Dystrophin is expressed in humans by the DMD gene, a large gene consisting of 79 exons and over 2 million total base pairs. DMD gene mutations (including exon frameshift, deletion, substitution, and duplication mutations) often lead to premature termination of mRNA or nonsense-mediated mRNA decay (NMD), thereby reducing the expression of functional dystrophin. Using oligonucleotides to skip exons of the mutated DMD gene to restore the expression of functional dystrophin is one of the means of treating DMD. Currently, four drugs have been approved by the U.S. Food and Drug Administration (FDA), and their long-term efficacy is still under evaluation.

[0004] Existing oligonucleotide therapies are limited in efficacy due to their short plasma half-life and limited muscle tissue distribution. For example, in the registrational clinical trial of eteplirsen, approved in 2016, weekly dosing of at least 30 mg / kg for 48 weeks only increased dystrophin levels in DMD patients from 0.16% before treatment to 0.44% after treatment.

[0005] Therefore, there is a need to improve the efficacy of existing drugs. Summary of the Invention

[0006] One of the objectives of the present application is 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.

[0007] Another object of the present application is to provide a conjugate comprising the above oligonucleotide and a muscle targeting agent.

[0008] Another object of the present application is to provide use of the oligonucleotide or the conjugate in preventing or treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

[0009] To solve the above problems, the present application provides conjugates that target muscle cells for delivering active ingredients to these cells.

[0010] In a first aspect, the present application provides an oligonucleotide for promoting exon skipping of dystrophin pre-mRNA and / or hybridizing with a target sequence of human DMD mRNA and mediating RNA interference against human DMD mRNA in muscle cells of a human subject, thereby promoting the increase or restoration of dystrophin expression or activity, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or a phosphorodiamidate morpholino oligomer (PMO) sequence, and the oligonucleotide binds to at least a portion of the exon and / or non-exon region of dystrophin and binds to the precursor mRNA (pre-mRNA) of the DMD gene.

[0011] Specifically, the PMO sequence refers to a six-membered morpholine ring replacing the five-membered furanosyl ring in all nucleotides.

[0012] In some embodiments, the oligonucleotide comprises or consists of a sequence that binds at least partially to any one of dystrophin pre-mRNA exons 44, 45, 50, 51, 52, and 53, and the sequence has 10 to 33 nucleotides.

[0013] In some embodiments, the sequence of the oligonucleotide is SEQ ID NO: 001-121, or a sequence having more than 90% homology thereto, preferably a sequence having more than 92% homology thereto, more than 95% homology thereto, more than 98% homology thereto, and most preferably a sequence having more than 99% homology thereto; or for sequences in RNA form, T in the above sequences SEQ ID NO: 001-121 is replaced with U.

[0014] In a second aspect, the present application provides a conjugate, which includes the above-mentioned oligonucleotide and a muscle targeting agent, wherein the muscle targeting agent is an antibody or an antigen-binding fragment.

[0015] In other embodiments, the conjugate includes a ribonucleoprotein (RNP) or an active compound for binding to the pre-mRNA of the DMD gene in the nucleus of myocytes, thereby promoting the increase or restoration of dystrophin expression or activity.

[0016] In some embodiments, the conjugate further comprises a muscle targeting agent.

[0017] In some embodiments, the muscle targeting agent is an antibody, an antigen-binding fragment, or a small molecule compound.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb) or a camelid antibody or binding fragment thereof.

[0019] In some embodiments, the muscle targeting agent is an antibody or polypeptide that can specifically bind to human transferrin receptor 1 (TfR1) (on the surface of muscle cells), thereby achieving muscle targeting and enrichment.

[0020] In some embodiments, the muscle targeting agent has 1 or 2 binding sites with human TfR1 and an affinity between 0.01 nM and 100 nM.

[0021] In some embodiments, the binding of the muscle targeting agent to human TfR1 does not affect the function of TfR1 binding to transferrin.

[0022] In some embodiments, the antibody comprises a light chain variable (VL) region and a heavy chain variable (VH) region.

[0023] In other embodiments, the muscle targeting agent can also deliver the active ingredient to the nervous system.

[0024] In some embodiments, the muscle targeting agent is covalently linked to the active ingredient via a non-cleavable or cleavable linker / linker.

[0025] In some embodiments, the linker is 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) and other chemical linkers that remain stable in plasma.

[0026] In some embodiments, the molar ratio of the active ingredient to the muscle targeting agent (or drug to antibody ratio, DAR) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or 16:1, preferably 1:1 to 8:1.

[0027] In some embodiments, the antibody peptide polypeptide comprises or consists of the sequence of SEQ ID NO: 285.

[0028] In some embodiments, the conjugate enters the endosome of the muscle cell via receptor-mediated endocytosis, and there is a certain correlation between endocytosis efficiency and affinity.

[0029] In some embodiments, the conjugate or its active ingredient is released into the cytoplasm by endosomal escape, and the release of the conjugate or its active ingredient may not be dependent on endosomal cleavage of the linker.

[0030] In some embodiments, the conjugate is used to treat Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD).

[0031] The third aspect of the present application provides a pharmaceutical composition comprising the oligonucleotide or the conjugate as described above, wherein the pharmaceutical composition is used to treat Duchenne muscular dystrophy and Becker muscular dystrophy.

[0032] In some embodiments, the pharmaceutical composition is for intravenous, subcutaneous, parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

[0033] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier, or excipient.

[0034] In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation.

[0035] In a fourth aspect, the present application provides a method for delivering the above-mentioned oligonucleotide to a cell, the method comprising coupling the above-mentioned oligonucleotide to an antibody, an antibody fragment, a polypeptide, an antigen-binding sequence or a small molecule compound.

[0036] In a fifth aspect, the present application provides a method for increasing or restoring the expression or activity of dystrophin in a cell, the method comprising contacting the cell with the oligonucleotide or conjugate described above.

[0037] In a sixth aspect, the present application provides a method for treating Duchenne muscular dystrophy and / or Becker muscular dystrophy in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of the oligonucleotide, conjugate or pharmaceutical composition as described above to treat the subject's Duchenne muscular dystrophy and / or Becker muscular dystrophy.

[0038] Preferably, wherein the subject is a human.

[0039] In a seventh aspect, the present application provides a use of the oligonucleotide or conjugate as described above for preparing a medicament for increasing or restoring the expression or activity of dystrophin, or for preventing and / or treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides a series of oligonucleotides, including but not limited to antisense oligonucleotides (ASOs) or antisense morpholino oligonucleotides (PMOs), which bind to at least a portion of the exonic and / or non-exonic regions of dystrophin pre-mRNA and promote exon skipping, thereby obtaining a protein that is nearly full-length but has partial dystrophin function. The exon skipping rate of the PMO drugs of the present invention is significantly better than that of PMO drugs published in other patents (Example 2).

[0042] The antibody-oligonucleotide conjugates provided in the embodiments of the present invention significantly alter the drug clearance pathway compared to existing oligonucleotide drugs. Existing oligonucleotide drugs, including peptide-coupled oligonucleotides, are primarily cleared through the kidneys. The molecular weight of the oligonucleotide and antibody conjugate is significantly increased, eliminating the renal clearance pathway, thereby significantly extending the plasma half-life and achieving dose-dependent muscle-targeted enrichment. At the same dose, the antibody-PMO conjugates of the present invention achieve skeletal muscle PMO concentrations two orders of magnitude higher than existing PMO drugs.

[0043] This antibody-oligonucleotide conjugate also offers efficacy advantages over existing oligonucleotide drugs. The antibody-PMO conjugate significantly increased the rate of exon skipping compared to existing PMO drugs in a dose-dependent manner. It also significantly increased full-length dystrophin levels in skeletal, cardiac, and diaphragmatic muscle in a dose-dependent manner. The antibody-PMO conjugate consistently reduced ALT, AST, and CK levels in mdx mice, demonstrating a repair effect on liver and muscle damage caused by DMD. A single dose of 10 mg / kg of the antibody-PMO conjugate was sufficient to restore 100% dystrophin levels in mdx mice and maintain this for at least 28 days. This reduced the dosing frequency and total dose required by existing PMO drugs by a factor of four and 12, respectively, while significantly increasing dystrophin levels by over 20-fold. This suggests that antibody-oligonucleotide conjugates have the potential to provide superior therapeutic outcomes for DMD patients while reducing both the dose and frequency of administration.

[0044] Secondly, the examples of the present invention also conducted mouse pharmacokinetic studies, cynomolgus monkey pharmacokinetic studies, in vitro activity, and affinity tests on the antibody-siRNA conjugates. The results showed that the antibody-siRNA conjugate extended the plasma half-life of siRNA and achieved more muscle siRNA distribution. The muscle siRNA was higher than or close to the distribution of liver siRNA, proving the muscle targeting effect of the TfR1 antibody. During the experiment, there were no significant changes in the weight and behavioral observations of mice and cynomolgus monkeys, indicating good tolerability of the conjugate. The antibody-siRNA conjugate can enter the RD cell line under in vitro co-incubation conditions without the assistance of lipofectamine and exert a dose-dependent target gene knockdown effect; the conjugation of siRNA has an adverse effect on antibody-antigen affinity, and the more conjugated, the more obvious the downward trend of antibody-antigen affinity. These works have laid the foundation for the further development of antibody-oligonucleotide conjugates.

[0045] Again, the affinity and in vitro activity study results of the polypeptide-oligonucleotide conjugates provided in the embodiments of the present invention show that polypeptide-oligonucleotide conjugates can also reduce the affinity between the polypeptide and the antigen; without the assistance of lipofectamine, they can enter the RD cell line under in vitro co-incubation conditions and exert a dose-dependent target gene knockdown effect.

[0046] The above results all indicate that conjugates comprising oligonucleotides and muscle targeting agents (including antibodies and peptides) can prolong the plasma half-life of small nucleic acid drugs, achieve more muscle distribution of small nucleic acid drugs, significantly improve the efficacy of existing small nucleic acid drugs in treating muscle-related diseases, and have more superior drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Gel electrophoresis diagram showing exon skipping of PMO in patient-derived cardiomyocytes;

[0048] Figure 2 Capillary electrophoresis diagram of PMO achieving exon skipping in patient-derived cardiomyocytes;

[0049] Figure 3 Schematic diagram of Linker-PMO and SMCC-linker-PMO;

[0050] Figure 4 is the SDS-PAGE image of TIB-219 and TIB-219-S245C;

[0051] Figure 5 is the SEC spectrum of TIB-219;

[0052] Figure 6is the SEC spectrum of TIB-219-S245C;

[0053] Figure 7 This is the purification profile of the crude product of the antibody-PMO conjugate after reaction, where fractions P2-P4 were collected separately;

[0054] Figure 8 is the IEX result of the crude product after the reaction;

[0055] Figure 9 Mass spectrometry results of the crude product collected after the reaction;

[0056] Figure 10 is the IEX spectrum of the purified antibody-PMO conjugate;

[0057] Figure 11 is the SEC profile of the purified antibody-PMO conjugate;

[0058] Figure 12 Schematic diagram of the hELISA principle and primer design;

[0059] Figure 13 The results of tissue distribution testing of CGBC-1011 in mdx mice after a single dose of CGBC-1011 are shown.

[0060] Figure 14 The results are for the muscle:liver ratio in mdx mice after a single dose of CGBC-1011.

[0061] Figure 15 Results of exon skipping efficiency (nested PCR) in muscle tissue of mdx mice after a single dose of CGBC-1011.

[0062] Figure 16 Results of exon skipping efficiency (nested PCR) in non-muscle tissues of mdx mice after a single dose of CGBC-1011.

[0063] Figure 17 The results of exon skipping efficiency (ddPCR) in muscle tissue of mdx mice after a single dose of CGBC-1011 are shown.

[0064] Figure 18 The results show the effect of a single dose of CGBC-1011 on the blood biochemistry of mdx mice.

[0065] Figure 19 Results of exon skipping efficiency (low dose, nested PCR) in muscle tissue of mdx mice after a single dose of CGBC-1011.

[0066] Figure 20To restore dystrophin levels in muscle tissue of mdx mice after a single dose of CGBC-1011 (low dose);

[0067] Figure 21 This is the molecular chromatogram of CGBC-1004-1 (A280 upper, A260 lower);

[0068] Figure 22 This is the molecular chromatogram of CGBC-1004-2 (A280 upper, A260 lower);

[0069] Figure 23 This is the molecular chromatogram of CGBC-1005-1 (A280 upper, A260 lower);

[0070] Figure 24 This is the molecular chromatogram of CGBC-1005-2 (A280 upper, A260 lower);

[0071] Figure 25 This is the molecular chromatogram of CGBC-1012-1 (A280 upper, A260 lower);

[0072] Figure 26 This is the molecular chromatogram of CGBC-1012-2 (A280 upper, A260 lower);

[0073] Figure 27 The figure shows the distribution of siRNA of CGBC-1004 (DAR1 and DAR2) in the plasma, muscle and liver of wild-type CD-1 mice;

[0074] Figure 28 The figure shows the distribution of siRNA of CGBC-1005 (DAR1 and DAR2) in the plasma, muscle and liver of wild-type CD-1 mice;

[0075] Figure 29 The muscle:liver AUC ratio test results of antibody-siRNA conjugates with different designs;

[0076] Figure 30 PK curve of CGBC-1012 (DAR1) in cynomolgus monkey plasma;

[0077] Figure 31 This is the PK curve of CGBC-1012 (DAR1) in the gastrocnemius muscle of cynomolgus monkeys;

[0078] Figure 32 The results of in vitro activity testing of CGBC-1012 (DAR1 and DAR2) in human rhabdomyosarcoma cells are shown;

[0079] Figure 33The results of antigen affinity test of CGBC-1012 (DAR1 and DAR2) and naked antibody;

[0080] Figure 34 The results of antigen affinity testing of CGBC-1004 (DAR1 and DAR2), CGBC-1005 (DAR1 and DAR2) and naked antibodies are shown;

[0081] Figure 35 This is the coupling of CGBC-1018. The coupling process was verified by HPLC and SDS-PAGE, and the molecular weight of the DAR1 coupling product was verified by mass spectrometry.

[0082] Figure 36 The results of in vitro activity test of CGBC-1018 in human rhabdomyosarcoma cells. DETAILED DESCRIPTION

[0083] The present application describes various conjugates for delivering oligonucleotide drugs to muscle cells and their uses. To further clarify the technical problems, technical solutions, and beneficial effects to be solved by the present invention, the present invention is further described below in conjunction with the examples and accompanying drawings. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0084] Definition of terms

[0085] In this application, "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by an antisense compound. The target nucleic acid can be DNA or RNA. Specifically, the target RNA can be mRNA, pre-mRNA, non-coding RNA, precursor microRNA (pre-microRNA), mature microRNA, promoter-directed RNA or natural antisense transcript. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a specific condition or disease state, or a nucleic acid molecule from a pathogenic agent.

[0086] In this application, "oligonucleotide" refers to a compound comprising a plurality of nucleosides connected to each other. Therefore, although the term "oligonucleotide" generally refers to a naturally occurring nucleotide polymer wherein nucleosides and the connection therebetween, it should be understood that the scope of the term also includes various analogs, wherein one or more of the plurality of nucleosides are modified. Oligonucleotides generally have 10-30 nucleotide residues.

[0087] In the present application, the term "antisense oligonucleotide (ASO)" refers to a single-stranded oligonucleotide molecule having a nucleobase sequence complementary to the corresponding fragment of a target nucleic acid (e.g., a target genomic sequence, an mRNA precursor, or an mRNA molecule), that is, hybridizing to the target nucleic acid and regulating the activity, processing or expression of the target nucleic acid. The length of the antisense oligonucleotide can be 12 to 30 nucleobases, and the antisense oligonucleotide can include unmodified or modified nucleic acids. The ASO first locks the target RNA and then cuts the target RNA by RNase H, thereby preventing the protein from being expressed. Since RNase H exists in the cell nucleus as well as outside the nucleus, ASO not only acts on mRNA, but can also regulate some noncoding RNA. Among them, "morpholino antisense oligonucleotides" (Phosphorodiamidate morpholino oligomers, PMO) belong to the third generation of antisense oligonucleotides, which are synthetic oligonucleotide analogs that mainly inhibit the function of the target gene by blocking the splicing process of mRNA. PMO replaces the five-membered furanosyl ring found in natural DNA and RNA with a six-membered morpholine ring. Each morpholine ring is linked by a neutral phosphorodiamidate group, rather than the negatively charged phosphate group found in natural DNA and RNA. Each phosphorodiamidate morpholine subunit contains one of the heterocyclic bases found in DNA (adenine, cytosine, guanine, or thymine).

[0088] In the examples of the present application, PMOs represented by DNA sequences (e.g., the sequences in Table 5) can be used to represent RNA sequences that can bind to the same target sequence, such as ASO sequences, after replacing T with U.

[0089] As used herein, "antisense activity" refers to any detectable and / or measurable activity based on the hybridization of an antisense compound to its target nucleic acid. Such activity can be an increase or decrease in the amount of a nucleic acid or protein, or a change in the ratio of splice variants of a nucleic acid or protein.

[0090] In the present application, " small interfering RNA " (small interfering RNA, siRNA, also referred to as short interfering RNA or silencing RNA), targets nucleic acid (for example, mRNA) for degradation by the RNA interference (RNAi) pathway in cells. The specificity of siRNA molecules can be determined by the combination of the antisense strand of the molecule with its target RNA. Generally speaking, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., constitute the specific antisense sequence of the siRNA molecule. After selecting the appropriate target RNA sequence, siRNA molecules comprising a nucleotide sequence (i.e., antisense sequence) that is complementary to all or part of the target sequence can be designed and prepared using appropriate methods. The siRNA molecule can comprise a duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure having a self-complementary sense strand and antisense strand.

[0091] In the present application, "antisense strand" refers to the strand in the region that is substantially complementary to the target sequence in a polynucleic acid molecule (e.g., dsRNA). When used in the present application, the term "region of complementarity" generally refers to the region on the antisense strand that is substantially complementary to the sequence defined in the present application (e.g., target sequence). When the region of complementarity is not completely complementary to the target sequence, mispairing can be in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[0092] In this application, "sense strand" (S) refers to a region that is substantially complementary to the region of the antisense strand defined above. "Sense strand" is sometimes referred to as "sense strand". With their sequence, the antisense strand targets the desired mRNA, while the sense strand targets different targets. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. The incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications or using 5' end caps on the sense strand.

[0093] In this application, "targeting" or "targeting to" refers to the binding of an antisense oligonucleotide to a specific target nucleic acid molecule or a specific region of nucleosides within a target nucleic acid molecule. An antisense oligonucleotide is targeted to a target nucleic acid if it is sufficiently complementary to the target nucleic acid molecule to allow hybridization under physiological conditions.

[0094] As used herein, "sequence identity" refers to the extent to which two sequences (amino acids) have identical residues at identical positions when aligned. For example, "an 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 X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y.

[0095] As used herein, "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.

[0096] As used herein, "internucleoside linkages" or "backbone" refers to the covalent bonds between adjacent nucleosides.

[0097] In this application, the term "expression" generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (eg, splicing, polyadenylation, addition of a 5'-cap), and translation.

[0098] In this application, "conjugate" refers to an atom or group of atoms that is bound to an oligonucleotide or oligomeric compound. Typically, conjugate groups change the various properties of the compound to which they are attached, for example, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are commonly used in the chemical field and are directly connected or connected to a parent compound such as an oligomeric compound via an optional linking moiety or linking group (i.e., a joint / linker).

[0099] In this application, "linker" refers to any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound. Linking groups or bifunctional linking moieties such as those known in the art are amenable to the present invention.

[0100] In this 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. The variable region of the heavy chain and light chain each contains three CDRs, designated CDR1, CDR2, and CDR3.

[0101] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0102] In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed. The intensity or affinity of the specific binding interaction can be expressed in terms of the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. The specific binding properties between two molecules can be measured using methods well known in the art, such as, using surface plasmon resonance (SPR) to measure in a BIACORE instrument.

[0103] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein. The term "variant" refers to retaining the binding specificity of its parent antibody for SARS-CoV-2, but having one or more desired properties conferred by mutations. The antibody variant comprises one or more mutations in one or more CDR sequences, one or more non-CDR sequences of the heavy chain variable region or the light chain variable region, and / or in the constant region (e.g., Fc region). For example, the antibody variant may have improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, reduced or depleted effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or compatibility with binding.

[0104] As used herein, the term "effective amount" refers to an amount of a compound of the invention sufficient to treat muscular dystrophy, slow or minimize the spread of muscular dystrophy, or provide a therapeutic benefit in the treatment or management of muscular dystrophy.

[0105] In this application, the term "subject" includes any human or non-human animal. The term "non-human animal" can include vertebrates (e.g., non-human primates, sheep, dogs) and rodents (e.g., mice, rats, and guinea pigs). The subject can preferably be a human. The term "subject" is used interchangeably with "subject" and "patient" herein.

[0106] Oligonucleotides

[0107] The oligonucleotides described in the present application can be used to bind to / target / hybridize with the pre-mRNA of the DMD gene, thereby promoting the increase or restoration of the expression or activity of dystrophin.

[0108] In some embodiments, the oligonucleotide comprises an antisense oligonucleotide (ASO) or a small interfering RNA (siRNA) sequence.

[0109] In some embodiments, the oligonucleotide is based on an antisense oligonucleotide (ASO) that is used to induce exon skipping of DMD or dystrophin pre-mRNA in a cell, organ, tissue, and / or individual. Exon skipping produces a mature DMD or dystrophin mRNA that does not contain the skipped exon, thereby resulting in a shorter protein product when the exon encodes an amino acid. Preferably, exon skipping is induced by binding the ASO to a specific exon-internal sequence containing splicing regulatory factors, splice sites, and / or intron branch point sequences.

[0110] In some embodiments, the oligonucleotide comprises a backbone of a morpholino antisense oligonucleotide (PMO) that binds to at least a portion of the sequence of an exonic and / or non-exonic region of dystrophin, wherein the bound portion has 10 to 33 nucleotides.

[0111] In some embodiments, the oligonucleotide comprises or consists of a sequence that binds to at least a portion of exons 44 to 55 of dystrophin pre-mRNA, the sequence having 10 to 33 nucleotides, and the oligonucleotide functions to promote exon skipping of dystrophin pre-mRNA.

[0112] 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, and the target sequence is SEQ ID NOs: 122-135, and the oligonucleotide is capable of promoting skipping of exon 44 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 001-014 or a sequence that differs therefrom by no more than 3 nucleotides.

[0113] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides of any one of SEQ ID NOs: 015-035, and its target sequence is SEQ ID NOs: 136-156, and the oligonucleotide is capable of promoting skipping of exon 45 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 015-035 or a sequence that differs therefrom by no more than 3 nucleotides.

[0114] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides of any one of SEQ ID NOs: 036-052, and its target sequence is SEQ ID NOs: 157-173, and the oligonucleotide is capable of promoting skipping of exon 50 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 036-052 or a sequence that differs therefrom by no more than 3 nucleotides.

[0115] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides of any one of SEQ ID NOs: 053-073, and its target sequence is SEQ ID NOs: 174-194, and the oligonucleotide is capable of promoting skipping of exon 51 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 053-073 or a sequence that differs therefrom by no more than 3 nucleotides.

[0116] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides of any one of SEQ ID NOs: 074-092, and its target sequence is SEQ ID NOs: 195-213, and the oligonucleotide is capable of promoting skipping of exon 52 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 074-092 or a sequence that differs therefrom by no more than 3 nucleotides.

[0117] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides of any one of SEQ ID NOs: 093-121, and the target sequence is SEQ ID NOs: 214-242, and the oligonucleotide is capable of promoting skipping of exon 53 of dystrophin pre-mRNA. In some embodiments, the oligonucleotide comprises a sequence selected from any one of SEQ ID NOs: 093-121 or a sequence that differs therefrom by no more than 3 nucleotides.

[0118] In some embodiments, the oligonucleotide has a small interfering RNA (siRNA) sequence, and the siRNA molecule hybridizes to a target sequence of human DMD mRNA and mediates RNA interference against human DMD mRNA in muscle cells of a human subject.

[0119] 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 that differs therefrom by no more than 3 nucleotides.

[0120] In some embodiments, the siRNA is a double-stranded structure comprising a sense strand and an antisense strand. In some embodiments, the antisense strand comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of SEQ ID NO: 268, SEQ ID NO: 270, and SEQ ID NO: 272. In some embodiments, the sequence of the antisense strand is any one of SEQ ID NO: 268, SEQ ID NO: 270, or SEQ ID NO: 272, or a sequence that differs therefrom by no more than 3 nucleotides. In some embodiments, the sense strand comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 consecutive nucleotides selected from any one of SEQ ID NO: 267, SEQ ID NO: 269, and SEQ ID NO: 271. In some embodiments, the sequence of the sense strand is any one of SEQ ID NO: 267, SEQ ID NO: 269, or SEQ ID NO: 271, or a sequence that differs therefrom by no more than 3 nucleotides.

[0121] In some embodiments, the PMO backbone and the nucleic acid monomers in the siRNA sequence are chemically modified. Specifically, the chemical modifications include modifications to the sugar moiety, modifications to the internucleoside linkages, modifications to the nucleotides themselves, and combinations thereof.

[0122] Muscle targeting agents

[0123] In some embodiments, the muscle targeting agent is an antibody, a polypeptide, an antigen-binding fragment, or a small molecule compound.

[0124] In some embodiments, the antibody or antigen-binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a divalent Fab2, a F(ab)'3 fragment, a single-chain variable fragment (scFv), a double scFv, (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.

[0125] In some embodiments, the muscle targeting agent is an antibody or antigen-binding fragment that specifically binds to human transferrin receptor 1 (TfR1) (on the cell surface of muscle cells), thereby achieving muscle targeting and enrichment.

[0126] In some embodiments, the binding of the muscle targeting agent to human TfR1 does not affect the function of TfR1 binding to transferrin.

[0127] 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.

[0128] 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 terminus of the antibody is derived from a human IgG1 subclass monoclonal antibody.

[0129] In some embodiments, the antibodies comprise one or more mutations in the framework regions, such as in the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the 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 FcγRs, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In other cases, the one or more mutations are to modulate glycosylation.

[0130] In some embodiments, one or more mutations are located in the Fc region. In some embodiments, the mutation is a LALA-LR mutation, which can reduce immunogenicity at the Fc end. In some embodiments, the mutation is an S239C, S238C, or S245C mutation, which can create a site suitable for site-directed nucleic acid conjugation.

[0131] In some embodiments, the antibody is conjugated to the oligonucleotide by random conjugation (non-specific) or site-specific conjugation (specific). In some embodiments, the conjugation process is generally applicable to different antibodies and sequences, and differences in antibody targets or sequences and differences in nucleic acid sequences do not often result in significant changes in the conjugation process.

[0132] 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 in a non-site-specific manner via a lysine residue or a cysteine ​​residue.

[0133] In some embodiments, the antibody is conjugated / coupled to the oligonucleotide in a site-specific manner. In some embodiments, the binding portion A is conjugated to the polynucleic acid molecule (B) in a site-specific manner through a lysine residue, a cysteine ​​residue, at the 5' end, the 3' end, an unnatural amino acid, or an enzymatically modified or enzymatically catalyzed residue.

[0134] In some embodiments, the muscle targeting agent has 1 or 2 binding sites with human TfR1 and an affinity between 0.01 nM and 100 nM.

[0135] 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 to the muscle targeting agent is between 1 and 8.

[0136] In some embodiments, the antibody comprises a light chain variable (VL) region and a heavy chain variable (VH) region.

[0137] In some embodiments, the 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% identical 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% identical 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.

[0138] In some embodiments, the 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.

[0139] In some embodiments, the antibody or its binding fragment is further modified using conventional techniques known in the art, for example, by using amino acid deletions, insertions, substitutions, additions, and / or by recombination and / or 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 includes modifications for modulating interaction with a receptor.

[0140] 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 comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 285. In some embodiments, the sequence of the polypeptide is SEQ ID NO: 285.

[0141] Conjugate

[0142] The conjugate described in the present application increases or restores the expression or activity of dystrophin, and the active ingredient of the conjugate is based on the above-mentioned oligonucleotide.

[0143] In other embodiments, the active ingredient of the conjugate is based on a ribonucleoprotein (RNP) or a small molecule and is configured to bind to the pre-mRNA of the DMD gene in the myocyte nucleus, thereby promoting the increase or restoration of dystrophin expression or activity.

[0144] In some embodiments, the conjugate comprises an oligonucleotide and a muscle targeting agent. In some embodiments, the muscle targeting agent also has the effect of delivering the active ingredient to the muscle. In some embodiments, the muscle is the heart, diaphragm, quadriceps, gastrocnemius, etc. In other embodiments, the muscle targeting agent also has the effect of delivering the active ingredient to the nervous system.

[0145] In some embodiments, the conjugate enters the endosomes of muscle cells via receptor-mediated endocytosis, and there is a certain correlation between endocytosis efficiency and affinity. In some embodiments, the conjugate or its active ingredient is released into the cytoplasm via endosomal escape, and the release of the conjugate or its active ingredient may not rely on endosomal cleavage of the linker. In some embodiments, the conjugate is used to treat Duchenne muscular dystrophy and Becker muscular dystrophy.

[0146] 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.

[0147] In some embodiments, a conjugate comprising an oligonucleotide and a muscle targeting agent has greater activity than an oligonucleotide that does not comprise a muscle targeting agent. In some embodiments, greater activity can enhance biologically relevant function, such as improved stability, affinity, binding, functional activity, and efficacy in the treatment or prevention of a disease state. In some embodiments, the disease state is the result of one or more mutated exons of a gene. In some embodiments, a conjugate comprising an oligonucleotide and a muscle targeting agent can increase exon skipping of one or more mutated exons compared to an oligonucleotide that does not comprise a muscle targeting agent. In some embodiments, the increase in exon skipping is at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0148] In some embodiments, the muscle targeting agent is covalently linked to the active ingredient via a linker / connector / the conjugate comprises a linker that connects the muscle targeting agent to the active ingredient. In some embodiments, the linker is a cleavable linker or a non-cleavable linker.

[0149] In some embodiments, the linker is a non-polymeric linker. A non-polymeric linker refers to a linker that does not comprise a repeating unit of a monomer produced by a polymerization process. The non-polymeric linker includes, but is not limited to, a C1-C6 alkyl (e.g., C5, C4, C3, C2, or C1 alkyl), a homobifunctional cross-linker, a heterobifunctional cross-linker, a peptide linker, a traceless linker, a self-sacrificing linker, a maleimide linker, or a combination thereof. In other cases, the non-polymeric linker does not comprise more than two linkers of the same type, such as more than two homobifunctional cross-linkers or more than two peptide linkers. In other cases, the non-polymeric linker optionally comprises one or more reactive functional groups.

[0150] In some embodiments, the linker comprises a homobifunctional linker. The homobifunctional linker includes, but is not limited to, Lomant reagent dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfosuccinimidyl tartrate). DST), ethylene glycol bis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberidate (DMS), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide containing compounds (DFDNB), for example 1 ,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrobenzene sulfone (DFDNPS), bis-[β-(4-azidosalicylamide))ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylenebis(iodoacetamide) or N,N'-hexamethylenebis(iodoacetamide), etc.

[0151] In some embodiments, the linker comprises a heterobifunctional linker. The heterobifunctional linker includes, but is not limited to, amine-reactive and sulfhydryl cross-linking agents, such as N-succinimidyl 3-(2-pyridyldithio) propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene (sMPT), 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC), sulfo-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinyl imide esters (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), N-(γ-maleimidobutyryloxy) sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-((((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), etc.

[0152] In some embodiments, the linker is 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) and other chemical linkers that remain stable in plasma.

[0153] In some embodiments, the linker is coupled to the 3' end of the PMO. In some embodiments, the 3' end of the PMO is activated with C3-NH2 and then coupled to the linker under aqueous conditions.

[0154] In some embodiments, the linker is attached to the sense strand of the siRNA. In some embodiments, the linker is attached to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the 5' end of the sense strand of the siRNA contains a C6-NH2 conjugation handle, and the linker and the conjugation handle are coupled under aqueous conditions. In some embodiments, the conjugation handle is connected to the sense strand of the siRNA via a phosphodiester at the terminal base.

[0155] In some embodiments, the muscle targeting agent is combined with the oligonucleotide to form a conjugate by random coupling or site-directed coupling.

[0156] In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent via a chemical ligation process. In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent via native ligation. In some embodiments, the oligonucleotide is conjugated to the muscle targeting agent via native ligation chemistry in a site-specific or non-specific manner.

[0157] In some embodiments, the oligonucleotide is mixed and incubated with a reduced muscle targeting agent to be conjugated in a random coupling manner. In some embodiments, the oligonucleotide is a PMO with SMCC conjugated to the 3' end or an siRNA with SMCC conjugated to the 5' end. In some embodiments, the 3' end of the PMO is activated with C3-NH2 and then coupled to SMCC under aqueous conditions. In some embodiments, the 5' end of the sense strand of the siRNA contains a C6-NH2 conjugation handle, and the linker is coupled to the conjugation handle under aqueous conditions. In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an antibody or antigen-binding fragment that specifically binds to TfR1. In some embodiments, the muscle targeting agent is reduced with TCEP (thiol reducing agent). In some embodiments, the interchain disulfide bonds of the antibody are reduced.

[0158] In some embodiments, the oligonucleotide is mixed and incubated with a muscle targeting agent that has been reduced and then oxidized to conjugate via site-specific coupling. In some embodiments, the oligonucleotide is an siRNA with SMCC conjugated to the 5' end. In some embodiments, the 5' end of the sense strand of the siRNA contains a C6-NH2 conjugation handle, and the linker is coupled to the conjugation handle under aqueous conditions. In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an antibody or antigen-binding fragment that specifically binds to TfR1. In some embodiments, the muscle targeting agent is a cysteine-mutated antibody. The muscle targeting agent is reduced with TCEP (a thiol reducing agent). In some embodiments, the antibody's cysteine ​​site-specific coupling site (depending on the specific antibody sequence, including but not limited to S239C, S238C, S245C, etc.) and interchain disulfide bonds are reduced. In some embodiments, the interchain disulfide bonds are oxidized.

[0159] In some embodiments, the oligonucleotide is coupled to the muscle targeting agent by a site-directed method using a "traceless" coupling technology (Philochem). In some embodiments, the "traceless" coupling technology utilizes a 1,2-aminothiol group at the N-terminus of the muscle targeting agent to conjugate to an oligonucleotide containing an aldehyde group.

[0160] In some embodiments, the oligonucleotide is conjugated to a muscle targeting agent by a site-directed method that utilizes an unnatural amino acid incorporated into the muscle targeting agent. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derived conjugated moiety to form an oxime bond.

[0161] Pharmaceutical composition

[0162] The present application provides a pharmaceutical composition comprising the oligonucleotide or the conjugate as described above. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier or excipient.

[0163] In some embodiments, pharmaceutical composition or medicine can comprise at least one described oligonucleotide or conjugate and one or more pharmaceutically acceptable excipients of pharmacologically effective amount.Pharmaceutically acceptable excipient is through suitable safety evaluation and is intended to be included in the material except active pharmaceutical ingredient (API, therapeutic product, such as oligonucleotide) in the drug delivery system.Excipient does not play or is not intended to play therapeutic effect under predetermined dose.Excipient can be used for a) helping the processing of drug delivery system during manufacture, b) protecting, supporting or enhancing stability, bioavailability or patient acceptability of API, c) helping product identification and / or d) enhancing any other attribute of overall safety, effectiveness of delivering API during storage or use.Pharmaceutically acceptable excipient may or may not be an inert substance.

[0164] Excipients include: absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, adhesives, buffers, carriers, coatings, pigments, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, hydrophobic agents, and wetting agents.

[0165] In some embodiments, the pharmaceutical composition or medicine 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, etc. In addition, carriers for parenteral administration include water, suitable oils, saline, aqueous glucose, and ethylene glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers may be antioxidants, for example, sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives may be benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Other pharmaceutically acceptable carriers known in the art may also be used.

[0166] In some embodiments, the oligonucleotide, the conjugate, and the pharmaceutical composition comprising the oligonucleotide or conjugate disclosed herein can be packaged or contained in a kit, container, package, or dispenser. In some embodiments, the oligonucleotide, the conjugate, or the pharmaceutical composition comprising the oligonucleotide or conjugate disclosed herein can be packaged in a prefilled syringe or vial.

[0167] In some embodiments, the pharmaceutical composition can be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form can be aerosolized or lyophilized. In some embodiments, the aerosolized or lyophilized form can be reconstituted with an aqueous or liquid solution. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation.

[0168] In some embodiments, the pharmaceutical composition is lyophilized to extend its shelf life and then prepared into a solution prior to use (e.g., administration to a subject). Thus, the excipient can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrrolidone) or a disintegration temperature regulator (e.g., dextran, ficoll, or gelatin).

[0169] In some embodiments, the pharmaceutical compositions can be formulated in buffered solutions, such as phosphate-buffered saline solutions, liposomes, micellar structures, and capsids.

[0170] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Some examples of routes of administration include parenteral administration, such as intravenous, intradermal, and subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

[0171] The pharmaceutical composition of the present application can bind to the pre-mRNA of the DMD gene, thereby promoting the increase or restoration of the expression or activity of dystrophin. In some embodiments, the pharmaceutical composition is used to treat subjects with diseases, diseases or conditions caused by premature termination of mRNA or nonsense-mediated mRNA degradation due to DMD gene mutations (including exon frameshifts, deletions, substitutions and duplication mutations). In some embodiments, the pharmaceutical composition is used to treat subjects with diseases, diseases or conditions caused by reduced or suppressed dystrophin expression. In some embodiments, the pharmaceutical composition is used to treat subjects with diseases, diseases or conditions caused by premature termination of mRNA or nonsense-mediated mRNA degradation leading to reduced or suppressed dystrophin expression. In some embodiments, diseases, diseases or conditions that will benefit from the restoration of dystrophin expression include but are not limited to: (progressive) muscular dystrophy. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD). In some embodiments, the subject is a mammal, including but not limited to humans.

[0172] Use / treatment methods

[0173] The present application provides a method for preventing and / or treating a disease or disorder, comprising administering to a target subject an effective amount of the aforementioned oligonucleotide and / or the aforementioned conjugate and / or the aforementioned pharmaceutical composition.

[0174] In some embodiments, the oligonucleotides and / or conjugates and / or pharmaceutical compositions described herein can be used to treat Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD) by increasing or restoring the expression or activity of dystrophin.

[0175] In some embodiments, a therapeutically effective amount of one or more of the oligonucleotides and / or the aforementioned conjugates and / or the aforementioned pharmaceutical compositions is administered to a subject, thereby increasing dystrophin expression in the subject (e.g., effectively increasing or restoring dystrophin expression or activity). In some embodiments, the subject can be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject has a DMD gene mutation (including exon frameshift, deletion, substitution, and duplication mutations) that may be associated with the disease.

[0176] Generally speaking, for the administration of any oligonucleotide and / or conjugate and / or pharmaceutical composition described in this application, it can be administered to the patient as a single dose (singledose), or it can also be administered in a divided treatment regimen for long-term administration in a multiple dose (multiple dose) manner. The pharmaceutical composition of the present application can change the content of the active ingredient according to the symptoms of the disease. Preferably, the preferred total dose of the composition of the present application can be about 0.01 μg to 1000 mg, most preferably 0.1 μg to 100 mg per kg of body weight per day for 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 provides maximum efficacy while minimizing the safety risks to the subject. Generally speaking, the efficacy and treatment and safety risks can be monitored throughout the treatment process. The appropriate effective dose of the pharmaceutical composition of the present application can be determined by a person with common sense in the art based on the route of administration and the number of treatments and various factors (such as the patient's age, weight, health status, sex, disease severity, diet, and excretion rate). The pharmaceutical composition according to the present application is not particularly limited in dosage form, administration route, and administration method, as long as it exhibits the effects of the present application.

[0177] 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 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.

[0178] Any suitable method can be used to assess the effectiveness of treatment. In some embodiments, the effectiveness of treatment can be assessed by observing the symptoms associated with DMD and / or BMD, by measuring the self-reported outcomes of the subject (e.g., mobility, self-care, daily activities, pain / discomfort, and anxiety / depression) or by quality of life indicators (e.g., life span).

[0179] In some embodiments, the pharmaceutical composition is administered to a subject at an effective concentration sufficient to increase the activity or expression of the target gene 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).

[0180] In some embodiments, oligonucleotide delivery technology known in the art can be used to deliver oligonucleotide described herein or compositions to cell, cell mass, tissue or experimenter.Generally, any suitable method for delivering nucleic acid molecules (in vitro or in vivo) recognized in the art can be applicable to oligonucleotide described herein.For example, delivery can be by local application (local administration) (as, direct injection, implantation or local application (topicaladministering), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal or parenteral approach, including intracranial (as intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal or local (including through cheek and sublingual) use.In some embodiments, by subcutaneous or intravenous infusion or injection administration composition.

[0181] In addition, the pharmaceutical composition of the present application can be administered as a single therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the composition of the present application and the other therapeutic agent can be administered simultaneously, separately, or sequentially. In this case, the other therapeutic agent may be a substance known to have the effect of treating or ameliorating muscular dystrophy. Other therapeutic agents also include all other anti-cancer treatments other than drug therapy, such as radiation therapy.

[0182] In other embodiments, oligonucleotides can be combined with lipids, nanoparticles, polymers, liposomes, micelles or other delivery systems available in the art. Oligonucleotides can also be chemically conjugated to targeting groups, lipids (including but not limited to cholesterol and cholesterol-based derivatives), nanoparticles, polymers, liposomes, micelles or other delivery systems available in the art. Oligonucleotides can be conjugated to delivery polymers. In some embodiments, delivery polymers are amphipathic membrane-active polyamines that are reversibly masked / modified.

[0183] use

[0184] The present application provides the aforementioned oligonucleotides and / or the aforementioned pharmaceutical compositions and / or the use of the aforementioned pharmaceutical compositions in the preparation of medicaments for preventing and / or treating diseases, disorders, or conditions 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).

[0185] In preclinical studies and clinical practice, antibody-oligo conjugate (AOC) drugs have significantly increased the plasma half-life of oligonucleotides. Antibodies or peptides targeting the transferrin receptor (TfR) enhance the muscle accumulation of oligonucleotides. Receptor-mediated endocytosis also facilitates the transmembrane and release of oligonucleotides into muscle cells, thereby promoting the expression of functional dystrophin in muscle cells. Therefore, AOC drugs have the potential to significantly enhance the efficacy of existing DMD and BMD therapies.

[0186] The present application is further described below through specific examples:

[0187] Example 1 Expression and affinity verification of anti-TfR1 antibodies

[0188] The inventors selected and expanded stable cell pools and inoculated them in shake flasks or cell bags at 37°C and 5% CO2 using Serum-free Expression medium (GenScript). On the day of transfection, the culture medium was heated to 25-37°C. 50 μM Swaisonine (MedChemExpress) was added to the culture medium 1 hour before transfection. Appropriate amounts of plasmids and reagents were added to the transfection mixture according to the instructions and mixed thoroughly. The mixture was incubated at room temperature. Appropriate feeds were added to the cell culture according to cell growth. The culture supernatant was collected on the 7th day for purification. The supernatant was loaded onto MabSelect at an appropriate flow rate. TM PrismA Crude. After washing and elution with an appropriate buffer, the eluted fraction was buffered with the final formulation buffer. The eluted protein was buffered with PBS (pH 7.2) and the purified protein was analyzed by SDS-PAGE and SEC-HPLC (column information: TSKgel G3000SWxl TOSHO) to determine molecular weight and purity. The final product was tested to have a protein purity of >98%.

[0189] The sequences of the anti-human TfR1 antibodies used in the experiments are shown in Table 1. The Fab terminus is derived from 13E4-Variant 2iii of U.S. Patent No. 10913800B2, and the Fc terminus is derived from a human IgG1 subclass monoclonal antibody. The inventors have modified the Fc terminus, using, for example, the LALA-LR mutation or the S238C mutation. The affinity of the anti-human TfR1 antibody for human TfR1 and cynomolgus monkey TfR1 was confirmed using Octet BLI, with the results shown in Table 2.

[0190] The sequence of the anti-mouse TfR1 antibody used in the experiment is shown in Table 3. Its variable region is derived from the prior art rat anti-mouse TfR1 antibody TIB-219 (light chain GenBank: ABV48920.1, heavy chain GenBank: ABV48917.1), and its Fc end is derived from a human IgG1 subclass monoclonal antibody. Among them, the inventors have modified the Fc end, such as using LALA-LR mutation or S245C mutation. Considering that the expression is a human-mouse fusion antibody, the quality of the antibody was controlled in the experiment. SDS-PAGE results showed that the purity of TIB-219 and TIB-219-S245C reached 99% regardless of reducing conditions or non-reducing conditions (see Figure 4 , where M: Marker; Lane 1: reduced TIB-219, purity 99%; Lane 2: reduced TIB-219-S245C, purity 99%; Purity: 99%; Lane 3: non-reduced TIB-219, purity 99%; Lane 4: non-reduced TIB-219-S245C, purity 99%; Lane 5: non-reduced human IgG. SEC results showed that the main peak purity of TIB-219 was 97.32%, while the main peak purity of TIB-219-S245C was 97.71% ( Figure 5-6 The affinity of the anti-mouse TfR1 antibody to mouse TfR1 was confirmed by Octet BLI, and the results are shown in Table 4.

[0191] Table 1 Anti-human TfR1 antibody sequences

[0192]

[0193]

[0194] Table 2 Anti-human TfR1 antibody affinity test results

[0195] Antibody antigen <![CDATA[Chi 2 (RU 2 )]]> 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

[0196] Table 3 Anti-mouse TfR1 antibody sequences

[0197]

[0198]

[0199] Table 4 Affinity of anti-mouse TfR1 antibodies

[0200] Antibody antigen <![CDATA[Chi 2 (RU 2 )]]> 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

[0201] Example 2 Design and Activity of PMO

[0202] The inventors designed PMO sequences for a total of 6 exons, namely 44, 45, 50, 51, 52 and 53 of the human DMD gene. The transcript sequence of the DMD gene selected in the design was derived from ENST00000357033.9. All designed PMO sequences and their target sequences are shown in Table 5, wherein SEQ ID NOs: 001-014 are used for skipping exon 44, and their target sequences are SEQ ID NOs: 122-135; SEQ ID NOs: 015-035 are used for skipping exon 45, and their target sequences are SEQ ID NOs: 136-156; SEQ ID NOs: 036-052 are used for skipping exon 50, and their target sequences are SEQ ID NOs: 157-173; SEQ ID NOs: 053-073 are used for skipping exon 51, and their target sequences are SEQ ID NOs: 174-194; SEQ ID NOs: 074-092 are used for skipping exon 52, and their target sequences are SEQ ID NOs: 195-213; and SEQ ID NOs: 093-121 are used for skipping exon 53, and their target sequences are SEQ ID NOs: 214-242.

[0203] The drug activity of PMOs is determined by four key factors: 1) sequence length between 25 and 30 nt, 2) distance from the splicing acceptor no more than 100 nt, 3) GC% no less than 40%, and 4) binding energy no greater than -40. Validation results showed that the most active PMOs were between 25 and 30 nt in length, with only 12 of 121 sequences containing less than 25 nt. Furthermore, the distance from the splicing acceptor to the PMO was generally no more than 100 nt, with only 17 of the 121 sequences containing more than 100 nt. After design, GC% and binding energy were tested using the RNAeval algorithm and RNAstructure v5.3. The calculated GC% and binding energy results are shown in Table 5.

[0204] Table 5 Sequences and activities of PMOs

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] The inventors selected some PMOs for further verification of cell activity. Nineteen sequences targeting exon 52 skipping (SEQ ID NOs: 074-092) and the Yangshen sequence H52A (-01+24) from patent US20230110479A1 were selected. Normal saline was set as a negative control. Primary cardiomyocytes from DMD patients with exon 51 deletion were transfected in vitro. The PCR fragment length after skipping was 278 bp, while the fragment length before skipping was 396 bp. The cell activity of the PMOs was assessed by two-step PCR-gel electrophoresis and capillary electrophoresis for exon 52 skipping efficiency (primers are shown in Table 6).

[0212] Table 6 Primer sequences for PMO cell activity study

[0213]

[0214] The gel electrophoresis diagram of the experimental results is as follows Figure 1 As shown, gel electrophoresis can semi-quantitatively evaluate the skipping efficiency of PMO. The 400 bp and 300 bp markers correspond to the unskip and skip PCR fragments, respectively. The skip% of each PMO can be calculated by grayscale calculation: skip / (skip+unskip)*100%. The calculation results are shown in Table 7, among which SEQ ID NO: 89 and SEQ ID NO: 90 showed better skipping efficiency than Yangshen.

[0215] Table 7 Gel electrophoresis experimental results

[0216]

[0217] Capillary electrophoresis can quantitatively evaluate the hopping efficiency of PMO. The raw data of the experimental results are as follows: Figure 2 As shown, the nucleic acid concentrations of the unskip and skip PCR fragments were calculated by integrating the peak areas of the electrophoresis patterns, and the skip% of each PMO was obtained by the following formula: skip%=skip / (skip+unskip)*100%. The calculation results are shown in Table 8, which once again proves that SEQ ID NO: 89 and SEQ ID NO: 90 exhibit better skipping efficiency than that of Yangshen.

[0218] Table 8 Capillary electrophoresis experimental results

[0219]

[0220]

[0221] Example 3 Synthesis of Antibody-PMO Conjugate

[0222] A: Synthesis of Linker-PMO and SMCC-linker-PMO

[0223] The 3'-terminal C3-NH2-activated M23D tool PMO (Linker-PMO) (EG3-GGCCAAACCTCGGCTTACCTGAAAT-C3-NH2, SEQ ID NO: 243) can be obtained by standard solid phase synthesis method and purified by HPLC. The 3'-terminal C3-NH2-activated M23D PMO is coupled with 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) linker under aqueous conditions to form a 3'-terminal SMCC-activated M23D PMO (SMCC-linker-PMO) (EG3-GGCCAAACCTCGGCTTACCTGAAAT-C3-NH-SMCC, SEQ ID NO: 244, the nucleic acid sequence of which is partially the same as SEQ ID NO: 243), which can be used for the next antibody coupling reaction after purification by HPLC. The specific structures of Linker-PMO and SMCC-linker-PMO are shown in Figure 2. Figure 3 shown.

[0224] B: Synthesis of Antibody-PMO Conjugates

[0225] TIB-219 antibody (10 mg / mL) was stored in PBS, and then 2.1 equivalents of TCEP (thiol reducing agent) aqueous solution was added and incubated at 25°C 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°C for 3 hours. 12 equivalents of L-cysteine ​​were then added to the mixture at room temperature for 30 minutes to quench unreacted maleimide.

[0226] The reaction mixture was purified using AKTAExplorer FPLC using HIC Method 1 (see Table 9). Fractions P2, P3, and P4, which were expected to contain conjugates with different drug-to-antibody ratios (DARs), were collected and analyzed using HIC Method 2 (see Table 10) and mass spectrometry for each fraction and the reaction mixture. Figure 7 The purification profile of the crude product after the reaction is shown, where P2, P3, and P4 were collected for DAR and molecular weight confirmation.

[0227] Because antibodies and nucleic acids have different absorbances at 260nm and 280nm, the absorbance ratio of the single component A260 / 280 can be used to confirm that the main components of P2, P3 and P4 are DAR2, DAR4 and DAR6 molecules, respectively (see the verification results). Figure 8 Mass spectrometry was used to confirm the molecular weight and DAR value (see Figure 9 ), where B260T01-E01-TFF-02 was component P2, B260T01-E01-TFF-03 was component P3, and B260T01-E01-TFF-04 was component P4. The difference in the main peak molecular weights essentially confirmed that P2 was the DAR2, P3 was the DAR4, and P4 was the DAR6 conjugate. This confirmed the DAR and purity of the reaction mixture and the final purified product. Figure 10 The chromatogram of the purified product analyzed by HIC method 2 is shown, which shows DAR 2 (15.274), DAR4 (16.582), and DAR6 (two peaks at 17.226 and 17.735, respectively). The numbers in brackets are retention times (minutes). This result ultimately determined that the final product was DAR4.6 (see Table 11). Figure 11 The SEC chromatogram of the purified product is shown, wherein the product purity is 96.43%.

[0228] Table 9 HIC method 1

[0229] Chromatographic columns FPLC (0.66cm) resin Capto Butyl ImpRes (Cytiva) volume 5.8 mL flow rate 150cm / h,5mL / min Buffer A 50 nM PB, 0.8 M ammonium sulfate, pH 7.0 Buffer B 50 nM PB, pH 7.0

[0230] Table 10 HIC Method 2

[0231]

[0232]

[0233] Table 11 Analysis results of antibody-PMO conjugates

[0234] Conjugate DAR % Purity (based on peak area) CGBC-1011 4.6 96.43

[0235] Example 4 In vivo efficacy of antibody-PMO conjugates in mdx mice (multiple doses)

[0236] The experimental design and dosage are shown in Table 12. The antibody-PMO conjugate CGBC-1011 was prepared according to the method of Example 3, wherein the antibody was TIB-219 in Example 2 and the PMO was the 3'-end SMCC-activated M23D PMO (SEQ ID NO: 244) in Example 1.

[0237] mdx mice were intravenously administered PBS control and antibody-PMO conjugates. Animals were euthanized 14 and 28 days after dosing. Blood was collected submandibularly and anticoagulated with EDTA-K2 to collect plasma samples. Tissue samples, including myocardium, diaphragm, gastrocnemius, and liver, were also harvested and snap-frozen in liquid nitrogen for subsequent pharmacokinetic, exon skipping efficiency, and blood biochemistry studies.

[0238] Table 12 Efficacy study of antibody-PMO conjugates in mdx mice (multiple doses)

[0239]

[0240] A: Pharmacokinetic study of antibody-PMO conjugates in mdx mice

[0241] The hybridization ELISA method was used in the experiment. The experimental method and primer design principles are as follows Figure 12 As shown, plasma and tissue PMO accumulation was quantified using a probe complementary to the M23D sequence. Figure 13 and Figure 14 Among them, the antibody-PMO conjugate extended the plasma half-life of traditional PMO drugs. The PMO blood concentration of mdx mice treated with the conjugate was still detectable within 28 days, which was much higher than the plasma half-life of traditional PMO of about 2 hours ( Figure 13 At the same time, the antibody-PMO conjugate achieved dose-dependent muscle-targeted PMO enrichment. As the dosage increased, the muscle PMO concentration increased proportionally and slowly decreased over time ( Figure 13 The antibody-PMO conjugate increased liver accumulation in a dose-dependent manner, and the PMO concentration ratio in gastrocnemius muscle:liver gradually decreased with increasing dose. This tissue distribution pattern is different from that of the antibody-siRNA conjugate, indicating that PMO may have an antibody-independent liver accumulation pathway ( Figure 13-14 ). Therefore, continuously increasing the dosage may lead to the accumulation of PMO in non-target tissues such as the liver, and a dose of 10 mg / kg is more conducive to the accumulation of PMO in muscle tissue.

[0242] B: Exon skipping efficiency of antibody-PMO conjugates in mdx mice

[0243] Two methods were used in the experiment to study the efficiency of exon skipping. Nested PCR was first used to evaluate the efficiency of exon skipping (Table 13). Animal tissues were lysed for RNA isolation. In a 20 μL reaction system, 2 μg of RNA was reverse transcribed into cDNA and diluted 4 times before a two-step nested PCR reaction (Table 13). The final amplification primers were analyzed for fragment length in a 4% TAE agarose gel. The results 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 was quantified by optical density method, and the exon skipping efficiency was calculated as follows: skip rate = skip / (skip+unskip) × 100%.

[0244] Table 13 Nested PCR primer design and experimental plan

[0245]

[0246] In addition to nested PCR, exon skipping efficiency can also be assessed using ddPCR (Table 14). This method involves RNA extraction and ddPCR using the QX200 Droplet Digital PCR System. The reaction system, primer design, and detailed steps are described below. Exon skipping efficiency is then calculated using the QX200's built-in software.

[0247] Table 14 ddPCR primer design and experimental plan

[0248]

[0249] Nested PCR results showed that the maximum jump efficiency of the antibody-PMO conjugate in the gastrocnemius, myocardium, and diaphragm of mdx mice after a single dose was 51%, 23%, and 55%, respectively. The efficacy can be maintained for at least 28 days and is clearly dose-dependent ( Figure 15 Among non-muscle tissues, only in the liver was a certain percentage of exon skipping efficiency higher than that of PBS observed, and it was generally less than 10% ( Figure 16 ). Combined with tissue distribution data ( Figure 13 ), it can be speculated that the endocytosis mediated by the antibody-receptor specific interaction plays a crucial role in the efficacy of PMO, rather than the non-specific endocytosis of the liver to the antibody plays a crucial role in the efficacy of PMO. In muscle tissue, ddPCR ( Figure 17 ) and nested PCR ( Figure 15 ) results showed no significant difference, but ddPCR significantly reduced the noise of the PBS group.

[0250] C: Effects of antibody-PMO conjugates on blood biochemistry in mdx mice

[0251] Mdx mice often show elevated levels of creatine kinase (CK) and transaminases due to long-term muscle damage and inflammation. Plasma samples were collected and assayed for changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) using a blood biochemical analyzer. The results showed that the antibody-PMO conjugate significantly and persistently reduced ALT, AST, and CK levels in mdx mice, with efficacy maintained for at least 28 days after a single dose. Figure 18 ).

[0252] Example 5 In vivo efficacy of antibody-PMO conjugates in mdx mice (low dose)

[0253] The results of Example 4 above show that the antibody-PMO conjugate dose-dependently increases the concentration of muscle PMO, but at the same time achieves PMO enrichment in the liver at a faster rate. Furthermore, as the dose increases, the gastrocnemius muscle:liver PMO concentration ratio gradually decreases, suggesting that PMO may have a non-antibody-dependent liver enrichment pathway. In clinical practice, PMO is often administered weekly and at a clinically recommended dose of 30 mg / kg or above. Based on the above, dose optimization of the antibody-PMO conjugate will help maximize the muscle enrichment of PMO and the restoration of dystrophin at a lower dosing frequency and dose. The purpose of this experiment is to evaluate the in vivo efficacy of the antibody-PMO conjugate in mdx mice at a dose lower than the clinically recommended dose, such as 10 mg / kg.

[0254] The experimental design and dosage are shown in Table 15. The antibody-PMO conjugate CGBC-1011 was prepared according to Example 3, wherein the antibody was TIB-219 in Example 2 and the PMO was the 3'-end SMCC-activated M23D PMO (SEQ ID NO: 244) in Example 1.

[0255] mdx mice were intravenously administered with PBS control and antibody-PMO conjugates. Animals were euthanized 14 and 28 days after dosing, respectively, according to the experimental design. Submandibular blood was collected and anticoagulated with EDTA-K2, and plasma samples were collected. Tissue samples, including myocardium, diaphragm, gastrocnemius, and liver, were also harvested and snap-frozen in liquid nitrogen for subsequent analysis of exon skipping efficiency and western blot analysis.

[0256] Table 15: Efficacy study of antibody-PMO conjugates in mdx mice (single dose)

[0257]

[0258] A: Exon skipping efficiency of antibody-PMO conjugates in mdx mice

[0259] The nested PCR operation steps are shown in Example 4. The results showed that 28 days after a single dose of 10 mg / kg of the antibody-PMO conjugate, the exon skipping efficiency in the gastrocnemius muscle, myocardium, and diaphragm of mdx mice was 24%, 8%, and 27%, respectively (see Figure 19 ).

[0260] B: Ability of antibody-PMO conjugates to restore muscle dystrophin content in muscle tissue of mdx mice

[0261] The dystrophin content was determined by western blot. The specific experimental protocol is as follows: After the collected muscle tissue was minced with scissors, the sample was homogenized using a steel column, the supernatant was collected, and the protein concentration was measured by BCA assay. The total protein (24 μg) was loaded onto a 3% to 8% NuPAGE. TM The electrophoresis was performed on a Tris acetate protein gel at 75 V for 1 hour and then at 150 V for another 1 hour, all on ice. TM The proteins were transferred from the gel to a polyvinylidene difluoride membrane using a 2-well transfer machine at 25 V for 10 minutes. The membrane was cut and incubated with 1:2000 anti-dystrophin antibody (Abcam catalog number ab154168) at 4°C overnight, followed by incubation with 1:5000 goat anti-rabbit IgG at room temperature for 1 hour. As a control, the remaining blot was incubated with 1:2000 anti-α-actinin antibody (Abcam catalog number ab9465) at 4°C overnight, followed by incubation with 1:10000 goat anti-mouse IgG at room temperature for 1 hour. The blot was developed using an ECLWestern detection kit (Cytiva) and quantified using an iBright FL1500 (ThermoFisher Scientific).

[0262] For each western blot experiment, a fixed ratio of mdx mouse muscle and healthy C57BL / 6J mouse muscle was weighed and homogenized to construct a standard curve. The percentage of wild-type protein in the standard samples was 64%, 32%, 16%, 8%, 4%, 2%, and 0%, respectively. The standard curve was fitted nonlinearly using the 4PL method.

[0263] The results showed that 28 days after a single dose of 10 mg / kg of the antibody-PMO conjugate, dystrophin in the gastrocnemius, myocardium, and diaphragm of mdx mice could be restored to 100%, 39%, and 88% of healthy animal tissues, respectively, and the efficacy could be maintained for at least 28 days ( Figure 20 This result suggests that antibody-conjugated PMOs have the potential to achieve better exon skipping and dystrophin recovery at a reduced dose and frequency.

[0264] Example 6 Synthesis of Antibody-siRNA Conjugates

[0265] In addition to PMOs, chemically modified antisense oligonucleotides also have the ability to achieve exon skipping. This type of nucleic acid drug is closer to siRNA in terms of physicochemical properties and metabolism. Therefore, in order to systematically study the tissue distribution of antibody-oligonucleotide drug conjugates, antibody-siRNA conjugates with higher detection sensitivity were used for in vivo pharmacokinetic studies to support subsequent drug optimization and in vivo studies.

[0266] A: Synthesis of Linker-siRNA and SMCC-linker-siRNA

[0267] For the linker-siRNA used in this experiment, the 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. The siRNA sense strand contained a C6-NH2 conjugation handle at the 5' end, which was attached to the siRNA sense strand via a phosphodiester at the terminal base.

[0268] The siRNA with C6-NH2 activation at the 5' end was coupled to the 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) linker under aqueous conditions to form a linker-siRNA with SMCC activation at the 5' end. After purification by HPLC, it can be used for the next antibody coupling reaction.

[0269] B: Conjugation of wild-type antibody and siRNA

[0270] The wild-type antibody and siRNA are conjugated using a random conjugation process. Specifically, the interchain disulfide bonds of the antibody are first reduced and then conjugated to the linker-siRNA activated by the SMCC linker at the 5' end of the siRNA sense strand, ultimately forming the antibody-siRNA conjugate.

[0271] Step 1: Reduce the antibody with TCEP

[0272] The antibody was buffer exchanged with 25mM Tris buffer (pH 8) to a concentration of 10mg / mL. 0.8 equivalents of TCEP in the same buffer were added to the solution and incubated at room temperature for 2 hours. The reaction solution was then centrifuged and replaced with 2mM EDTA in 25mM Tris buffer (pH 8), and SMCC-siRNA (3 equivalents) was added and mixed, and reacted at 22°C for 3 hours. Analysis of the reaction mixture by analytical SAX column chromatography revealed antibody-siRNA conjugates as well as unreacted antibody and siRNA.

[0273] Step 2: Purification and analysis

[0274] The crude reaction mixture was purified by AKTAPure FPLC using anion exchange chromatography (SAX) method-1 (Table 16). Fractions containing the antibody-siRNA conjugate were isolated, concentrated, and buffer exchanged with pH 7.4 PBS.

[0275] The purity of the conjugate was assessed by analytical HPLC using SAX Method-2 (Table 17).

[0276] Table 16 Anion exchange chromatography method (SAX)-1

[0277]

[0278]

[0279] Table 17 Anion Exchange Chromatography Method (SAX)-2

[0280]

[0281] C: Conjugation of cysteine-mutant antibodies and siRNA

[0282] The conjugation of cysteine-mutated antibodies and siRNAs utilizes a site-specific conjugation process. Specifically, the antibody cysteine ​​site-specific conjugation site (depending on the specific antibody sequence, including but not limited to S239C, S238C, S245C, etc.) and interchain disulfide bonds are first reduced. The interchain disulfide bonds are then oxidized while retaining the reduced state of the cysteine ​​sites. Finally, the antibody is conjugated to the 5' end of the siRNA sense strand via a linker-siRNA activated with an SMCC linker to form the antibody-siRNA conjugate.

[0283] Step 1: Reduce the antibody with TCEP

[0284] The antibody was buffer exchanged with 25mM Tris buffer (pH 8) to a concentration of 10mg / mL. 100 equivalents of DTT in the same buffer were added to the solution and incubated at room temperature for 16 hours. The reaction solution was then exchanged by ultrafiltration into 25mM Tris buffer (pH 8) with 2mM EDTA, and 20 equivalents of DHAA were added and reacted at room temperature (RT) for 2 hours. 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°C for 2 hours. Analysis of the reaction mixture by analytical SAX column chromatography showed antibody siRNA conjugates as well as unreacted antibody and siRNA.

[0285] Step 2: Purification and analysis

[0286] The crude reaction mixture was purified by AKTAPure FPLC using anion exchange chromatography method (SAX)-1 (Table 16). Fractions containing the antibody-siRNA conjugate were isolated, concentrated, and buffer exchanged with pH 7.4 PBS.

[0287] The purity of the conjugate was assessed by analytical HPLC using method SAX-2 (Table 17).

[0288] The analytical data of the conjugates synthesized in this example are shown in Table 19, with the HPLC retention time (RT) in minutes and the percentage purity determined by the chromatographic peak area. The molecular analytical data of the purified representative antibody-siRNA conjugates are shown in Table 19, and the purity is above 97%. The spectra are shown in Figure 21-26 shown.

[0289] The siRNAs used to synthesize antibody-siRNA conjugates in the experiment are shown in Table 18, where 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. The specific sequences are shown in Table 18 below. Regarding antibodies, the antibody used in CGBC-1004-1, CGBC-1004-2, CGBC-1005-1, and CGBC-1005-2 was TIB-219-S245C. The antibody used in CGBC-1012-1 and CGBC-1012-2 was Var2iii-S238C, all of which were synthesized using the above-mentioned site-directed conjugation process.

[0290] Table 18 siRNA sequences

[0291]

[0292] Modification description: m = 2'-O-methyl; i2F = 2'-fluoro; gn = glycol nucleic acid; * / = phosphorothioate linkage; / = phosphodiester linkage; VP = 5-(E)-VP vinyl phosphate modified nucleic acid

[0293] Table 19 Design and analysis results of antibody-siRNA conjugates

[0294]

[0295] Example 7 Pharmacokinetic Study of Antibody-siRNA Conjugates in Mice

[0296] The purpose of the experiment was to investigate the ability of antibody-siRNA (using the above sequence) conjugates to deliver nucleic acids to muscle in mice. The experimental design and dosage are shown in Table 20. The siRNAs used to synthesize the antibody-siRNA conjugates in the experiment are shown in Table 18.

[0297] Mice were given PBS control and antibody-siRNA conjugates by intravenous injection. The animals were euthanized 7 and 14 days later, respectively. Plasma and various tissue samples, including quadriceps and liver, were harvested and snap-frozen in liquid nitrogen. The tissues were homogenized and digested with proteinase K, and the supernatant samples were diluted and analyzed by SL-PCR. The pharmacokinetic experimental method mainly used SL-PCR to analyze the siRNA concentration in plasma and tissues. SL-PCR mainly consists of a first step of reverse transcription using a stem-loop primer sequence and a second step of quantification using qPCR. The reaction system is shown in Tables 21-22, and the primer sequences are shown in Table 23.

[0298] Table 20 Study on knockdown of target genes by antibody-siRNA conjugates in wild-type CD-1 mouse muscle

[0299]

[0300]

[0301] Table 21 SL-PCR reverse transcription reaction system

[0302] PCR reaction mixture components Volume required for 1 reaction system (μL) 100mM dNTPs (with dTTP) 0.15 MultiScribe Reverse Transcriptase,50U / μL 1 10X Reverse Transcription Buffer 1.5 RNase Inhibitor, 20 U / μL 0.19 Nuclease-free Water 7.085 5XRT primer (4 μM) 0.1875 siRNA / miRNA 5 Total 15

[0303] Table 22 SL-PCR qPCR reaction system

[0304] PCR reaction mixture components Volume required for 1 reaction system (μL) Universal PCR Master Mix 2x 5 Forward primer (10 μM) 1.5 Reverse primer (10 μM) 0.7 Probe (10 μM) 0.2 cDNA 2 H2O 0.6 Total 10

[0305] Table 23 Primer sequences for SL-PCR in mouse experiments

[0306]

[0307] A standard curve (Ct-log [concentration]) must be constructed. The pharmacokinetic standard curve is prepared by diluting the antibody-siRNA conjugate compound and adding it to a blank mouse tissue homogenate. Aside from the above steps, the actual sample processing procedures remain the same as those for the standard curve. The amplification efficiency of the standard curve must be maintained in the 90-110% range.

[0308] The antibody-siRNA conjugate showed a plasma clearance rate much lower than that of traditional siRNA drugs and a higher muscle-specific siRNA delivery efficiency. After 14 days of administration, the siRNA in the plasma could still be detected by SL-PCR ( Figures 27-28 ), which is significantly different from the plasma half-life of traditional siRNA drugs of several hours, indicating that the antibody-siRNA conjugate prolongs the plasma half-life of siRNA. In terms of tissue distribution, the antibody-siRNA conjugate achieves more muscle siRNA distribution, and the muscle siRNA is higher than or close to the distribution of liver siRNA, proving the muscle targeting effect of TfR1 antibody. In terms of molecular design, the DAR1 molecule shows higher blood concentration and more muscle enrichment than the DAR2 molecule. The design of 5-(E)-VP also significantly improves the enrichment of the antibody-siRNA conjugate in muscle ( Figure 29 ).

[0309] There were no significant changes in the body weight and behavioral observations of the mice during the experiment, indicating that the conjugate was well tolerated.

[0310] Example 8 Pharmacokinetic Study of Antibody-siRNA Conjugates in Cynomolgus Monkeys

[0311] The purpose of this experiment was to investigate the ability of antibody-siRNA conjugates to deliver nucleic acids to muscle in cynomolgus monkeys. For experimental design and dosage, see Table 24. The siRNA used to synthesize the antibody-siRNA conjugate was siRNA ID #003, as shown in Table 18. The antibody used was Var2iii-S238C, both synthesized using the site-directed conjugation process described above.

[0312] Antibody-siRNA conjugates were administered intravenously to cynomolgus monkeys. Plasma samples were collected before and 3, 6, and 12 hours after dosing, and on days 1, 3, 7, 14, 28, and 42 after dosing. Gastrocnemius muscle samples were collected by muscle puncture before and 3, 7, 14, 28, 56, and 84 after dosing for pharmacokinetic studies. The standard curve was drawn using the same method as in Example 7 and is not described here. Primer sequences are shown in Table 25.

[0313] Table 24 In vivo studies of antibody-siRNA conjugates in cynomolgus monkeys

[0314]

[0315] Table 25 Primer sequences for SL-PCR in cynomolgus monkey experiments

[0316]

[0317]

[0318] The experimental results showed that the antibody-siRNA conjugate exhibited a typical two-compartment model in cynomolgus monkey plasma and linear PK at two doses. The distribution half-life at the high dose was 27.6 hours and the elimination half-life was 279 hours. In the low-dose group, only the distribution phase curve was accurately recorded due to the detection limit of the method ( Figure 30 In terms of tissue distribution, the antibody-siRNA conjugate was enriched in the gastrocnemius muscle of cynomolgus monkeys and reached a Cmax of more than 300 pmol / g on the third day after administration. The elimination half-life of the antibody-siRNA conjugate in the gastrocnemius muscle of cynomolgus monkeys was 239 hours, and the siRNA concentration in the gastrocnemius muscle remained above 1 pmol / g for 84 days before the end of the experiment ( Figure 31 ).

[0319] There were no significant changes in the body weight and behavioral observations of cynomolgus monkeys during the experiment, indicating that the conjugate was well tolerated.

[0320] Example 9 In vitro activity of antibody-siRNA conjugates

[0321] The purpose of the experiment was to evaluate whether the antibody-siRNA conjugate could be internalized 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 specific experimental method was as follows: RD human rhabdomyosarcoma cell line (4201HUM-CCTCC00295) was cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco). The antibody-siRNA conjugate was diluted to a maximum dose of 10 mM and transfected with the antibody-siRNA conjugate at final siRNA concentrations of 200, 100, 10, 1, and 0.1 nM. 24 hours before dosing, the cells were seeded into 48-well culture plates at 10,000 cells per well. Antibody-siRNA conjugates were added to wells of a 48-well plate; PBS was added to some wells as an additional negative control; cells were placed at 37°C and 5% CO2 for 72 hours, the medium was removed from the wells, and 150 mL of Trizol (Life Technologies) was added; the plates were frozen at -80°C overnight or longer before analysis, and RNA was isolated using the Direct-zol-96 RNA kit (Zymo Research) according to the manufacturer's instructions; RNA was isolated using the ReverTra Ace TM RNA was reverse transcribed into cDNA using qPCR RT MasterMix (TOYOBO) according to the manufacturer's instructions. cDNA samples were evaluated by qPCR using Taqman FastAdvanced Master Mix (Applied Biosystems) with DMPK-specific and GAPDH-specific primers and the SYBR Green assay. % mRNA was calculated using the standard 2-ΔΔCT method, with PBS-treated cells set as 100% expression. All experiments were performed in triplicate. Primer information is shown in Table 26.

[0322] Table 26 Primer information

[0323] Primers 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)

[0324] The experimental results showed that CGBC-1011 can enter the RD cell line under in vitro co-incubation conditions without the assistance of lipofectamine and exert a dose-dependent knockdown effect on the target gene. The knockdown effect of the antibody-siRNA conjugate reached saturation at a concentration of 1nM. The maximum knockdown efficiency of the antibody-siRNA conjugate for DAR2 was approximately 40%, while the maximum knockdown efficiency of the molecule for DAR1 was approximately 50% ( Figure 32 ).

[0325] Example 10 Affinity of Antibody-siRNA Conjugates

[0326] The purpose of this experiment was to evaluate the changes in the affinity of antibody-siRNA conjugates and naked antibodies for the antigen, and to assess the impact of the coupling method and DAR value on antibody-antigen affinity. The experimental method evaluated the affinity of the conjugates and naked antibodies for the TfR1 antigen by ELISA. The specific steps included coating a 96-well plate with 1 μg / mL of antigen, incubating overnight at 4°C, washing twice in PBST, coating with 3% MPBS, incubating at 37°C for 1 hour, and washing twice in PBST. Next, serially diluted conjugates or naked antibodies were added, and detection was performed using anti-human / rat IgG-Fc-HRP. Finally, TMB was used for color development. During the experiment, the appropriate antigen and detection antibody should be selected based on the species specificity of the conjugate and naked antibody. After the experiment, the OD450 value was plotted against the antibody concentration, and the EC50 value was calculated.

[0327] The experimental results showed that the target affinity of antibody-siRNA conjugates was significantly reduced compared with naked antibodies ( Figures 33-34 In comparison of conjugates, DAR2 generally has a significantly lower affinity than DAR1 ( Figure 34 ), but the affinity of DAR2 and DAR1 in CGBC-1012 is not much different ( Figure 33 The above data show that siRNA conjugation has an adverse effect on antibody-antigen affinity, and the greater the number of conjugated siRNAs, the more obvious the downward trend in antibody-antigen affinity.

[0328] Example 11 Synthesis of polypeptide-siRNA conjugates

[0329] The nucleic acid sequence used in the experiment is the same as that in Example 8. The polypeptide CGBB2 sequence used in the experiment is as follows:

[0330] PSEEEIKKLVEELLKELSKEEAALKLVETAADVVVVTPKGIVVVKGDRETAEAVFKAAEEAFDKYPDDAEFIAEYIKKKVPKARVVLVPN (SEQ ID NO: 285).

[0331] To 30 μL of peptide (3 nmol) dissolved in pH 7.4 phosphate buffer, 6-azidomethyl-4-methoxypicolinaldehyde (30 nmol dissolved in 1 μL DMSO) and 3-maleimidopropionic acid (15 nmol dissolved in 1 μL DMSO) were added. The resulting mixture was incubated overnight at 37°C. The resulting solution was then concentrated and PBS buffer was added.

[0332] A 0.5 mL centrifugal filter was used for liquid exchange. Buffer exchange was first performed by diluting each sample to 500 μL using 50 mM pH 7.4 phosphate buffer. Each sample was then concentrated to 30 μL, and the process was repeated three times to obtain a polypeptide intermediate. DBCO-modified oligonucleotide (4.5 nmol, dissolved in 4.5 μL pH 7.4 phosphate buffer) was then added to 30 μL of protein solution (3 nmol), and the resulting mixture was incubated overnight at room temperature and purified by SEC FPLC to obtain the corresponding polypeptide-oligonucleotide conjugate, named CGBC-1018. The results of CGBC-1018 coupling, purification, and analysis are shown in Figure 35 .

[0333] Example 12 Affinity and in vitro activity studies of polypeptide-siRNA conjugates

[0334] The purpose of this experiment was to evaluate the affinity of the peptide-siRNA conjugate and whether it could be internalized by muscle cells and exert biological activity in vitro without the aid of lipofectamine. Affinity was confirmed by Octet BLI using the same experimental method as in Example 1; in vitro activity was determined using the same experimental method as in Example 9.

[0335] The results of the affinity study are shown in Table 27. They show that, like antibody-to-nucleic acid coupling, peptide-to-nucleic acid coupling can reduce the affinity between the peptide and the antigen. However, under the design of DAR1, the reduction in affinity is minimal and does not significantly affect the ability of the peptide to deliver the nucleic acid.

[0336] Table 27 Affinity of anti-human TfR1 polypeptide and coupled product

[0337]

[0338]

[0339] The results of in vitro activity studies showed that CGBC-1018 can enter the RD cell line 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 the peptide-siRNA conjugate CGBC-1018 is lower than that of the previously evaluated antibody-siRNA conjugate CGBC-1012, and requires a concentration greater than 100nM to exert a significant knockdown effect. The maximum knockdown efficiency of the peptide-siRNA conjugate is approximately 30% ( Figure 36 ).

[0340] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An oligonucleotide for promoting exon skipping of human dystrophin pre-mRNA and / or hybridizing to a target sequence of human dystrophin pre-mRNA, wherein the oligonucleotide is an antisense oligonucleotide (ASO) or 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 oligonucleotide sequence 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 includes 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.

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

7. The conjugate according to claim 6, characterized in that The muscle targeting agent has one or two binding sites with human transferrin receptor 1, and the affinity is between 0.01 nM and 100 nM.

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

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

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

11. The conjugate according to claim 6, characterized in that 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 is used for treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

13. The pharmaceutical composition according to claim 12, characterized in that The pharmaceutical compositions are 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 includes 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, characterized in that: The drug is used for promoting the expression of dystrophin, or for preventing and / or treating Duchenne muscular dystrophy and / or Becker muscular dystrophy.

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