Compositions targeting muscle cells and uses thereof

Abstract

Disclosed herein are compositions useful for targeting signal transduction pathway modulators to muscle cells. In some cases, such compositions are useful for treating muscle disorders, such as muscular dystrophy.

Description

[0001] Cross-referencing This application claims the benefits of U.S. Provisional Application No. 63 / 598,767, filed November 14, 2023, and U.S. Provisional Application No. 63 / 671,556, filed July 15, 2024, which are incorporated herein by reference in their entirety. Summary of the Invention

[0002] This disclosure provides a pharmaceutical agent for selectively targeting muscle cells, the agent comprising (a) an antigen-binding domain of mammalian ITGA7 selectively binding to the antigen-binding domain of said mammalian ITGA7 and (b) cargo coupled to said antigen-binding domain selectively binding to said mammalian ITGA7. In some embodiments, the cargo comprises a vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is adeno-associated virus (AAV). In some embodiments, the cargo comprises lipid nanoparticles. In some embodiments, the cargo comprises nucleic acid. In some embodiments, the nucleic acid comprises ribonucleic acid (RNA). In some embodiments, the RNA is small interfering RNA (siRNA). In some embodiments, the RNA is short hairpin RNA (shRNA). In some embodiments, the cargo comprises antisense oligonucleotides (ASO). In some embodiments, the ASO is morpholino oligonucleotide. In some embodiments, the cargo comprises a drug. In some embodiments, the cargo comprises a toxin. In some embodiments, the cargo comprises an immunomodulator. In some embodiments, the immunomodulator is an interleukin. In some embodiments, the immunomodulator reduces the activity of the immune system. In some embodiments, the interleukin is IL-10, IL-35, IL-4, IL-13, IL-27, or IL-37. In some embodiments, the antigen-binding domain binds to an epitope expressed in mammalian cells selected from muscle satellite cells, skeletal muscle cells, cardiomyocytes, and myofibrils. In some embodiments, the antigen-binding domain includes an antigen-binding fragment (Fab) domain or a single-stranded variable fragment (scFv). In some embodiments, the antigen-binding domain comprises any one of SEQ ID NO: 31-40. In some embodiments, the antigen-binding domain comprises: VH CDR1, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 31; VH CDR2, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 32; and VHCDR3, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 33.In some embodiments, the antigen-binding domain comprises: VL CDR1, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 34; VL CDR2, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 35; and VL CDR3, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 36. In some embodiments, the antigen-binding domain comprises: a variable light (VL) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 38; and a variable heavy (VH) chain pestle containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 37. In some embodiments, the linker conjugates the cargo to the antigen-binding domain that selectively binds to the mammalian ITGA7. In some embodiments, the agent further comprises a crystallizable fragment domain (Fc domain). In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain comprises a pestle-and-mortar structure. In some embodiments, the antigen-binding domain comprises (i) a human IgG1 heavy chain having a pestle mutation and an N297G mutation and (ii) a light chain. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39. In some embodiments, the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 40.

[0003] In another aspect, this disclosure provides a pharmaceutical composition comprising (i) the pharmaceutical agents described herein and (ii) one or more pharmaceutically acceptable excipients.

[0004] In another aspect, this disclosure provides a method for regulating muscular dystrophy, the method comprising administering an agent described herein to a subject. In another aspect, this disclosure provides a method for selectively targeting muscle cells, the method comprising administering an agent described herein to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the agent is delivered to muscle cells or a region adjacent to muscle cells. In some embodiments, the administration comprises injecting the subject at a location near skeletal muscle, muscle satellite cells (e.g., skeletal muscle satellite cells), skeletal muscle, cardiac muscle, smooth muscle, or muscle fibers. In some embodiments, the muscular dystrophy includes Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), and / or congenital muscular dystrophy.

[0005] In another aspect, this disclosure provides a pharmaceutical agent configured to selectively target mammalian cells expressing integrin α7 (ITGA7) with a modulator, the pharmaceutical agent comprising: (a) a modulator; and (b) an antigen-binding domain of mammalian ITGA7 selectively bound; wherein the modulator is coupled to the antigen-binding domain of mammalian ITGA7 selectively bound. In another aspect, this disclosure provides a pharmaceutical agent configured to selectively target mammalian cells expressing integrin α7 (ITGA7) with a signal transduction pathway modulator, the pharmaceutical agent comprising: (a) a signal transduction pathway modulator; and (b) an antigen-binding domain of mammalian ITGA7 selectively bound; wherein the signal transduction pathway modulator is coupled to the antigen-binding domain of mammalian ITGA7 selectively bound. In some embodiments, the signal transduction pathway modulator regulates the insulin-like growth factor 1 (IGF-1) signal transduction pathway. In some embodiments, the signal transduction pathway modulator comprises an IGF-1 peptide. In some embodiments, the IGF-1 peptide is a wild-type form of IGF-1. In some embodiments, the IGF-1 peptide comprises a peptide having a reduced ability to activate the IGF-1 receptor (IGF-1R) relative to endogenous IGF-1. In some embodiments, the peptide's ability to activate the IGF-1R is reduced by at least about 5-fold. In some embodiments, the IGF-1 peptide comprises an IGF-1 variant. In some embodiments, the IGF-1 variant comprises an amino acid sequence of any one of SEQ ID NO: 1-11, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in any one of SEQ ID NO: 1-11. In some embodiments, the signal transduction pathway regulator modulates the growth hormone (GH) signal transduction pathway. In some embodiments, the signal transduction pathway regulator modulates the growth hormone receptor (GHR). In some embodiments, the signal transduction pathway regulator comprises a GHR activator. In some embodiments, the signal transduction pathway regulator comprises a GH peptide. In some embodiments, the GH polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or 13, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 12 or 13. In some embodiments, the signal transduction pathway modulator regulates the basic fibroblast growth factor (bFGF) signal transduction pathway. In some embodiments, the signal transduction pathway modulator regulates the fibroblast growth factor receptor (FGFR). In some embodiments, the signal transduction pathway modulator includes an FGFR activator.In some embodiments, the signal transduction pathway regulator comprises a bFGF polypeptide. In some embodiments, the bFGF polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 14-17, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with any of the sequences shown in SEQ ID NO: 14-17. In some embodiments, the signal transduction pathway regulator regulates the interleukin 4 (IL4) signal transduction pathway. In some embodiments, the signal transduction pathway regulator regulates the interleukin 4 receptor (IL4R). In some embodiments, the signal transduction pathway regulator comprises an IL4R activator. In some embodiments, the signal transduction pathway regulator comprises an IL4 polypeptide. In some embodiments, the IL4 polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 18-21, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with any of the sequences shown in SEQ ID NO: 18-21. In some embodiments, the signal transduction pathway modulator regulates activin type II receptor (ActRII). In some embodiments, the signal transduction pathway modulator includes an ActRII inhibitor. In some embodiments, the signal transduction pathway modulator includes bimagrumab. In some embodiments, the signal transduction pathway modulator is an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises: a VH complementarity-determining region 1 (VH CDR1) containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 22; a VH CDR2 containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 23; and a VH CDR3 containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 24.In some embodiments, the antibody or antibody fragment comprises: VL CDR1 (VL CDR1), which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 25; VL CDR2, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 26; and VL CDR3, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 27. In some embodiments, the antibody or antibody fragment comprises a variable weight (VH) chain, the VH chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 28. In some embodiments, the antibody or antibody fragment comprises a variable light (VL) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 29. In some embodiments, the antibody or antibody fragment comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 30 or 44. In some embodiments, the antigen-binding domain binds to an epitope expressed in mammalian cells selected from muscle satellite cells, skeletal muscle cells, cardiomyocytes, and myofibrils. In some embodiments, the antigen-binding domain comprises an antigen-binding fragment (Fab) domain or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises: a VH complementarity-determining region 1 (VH CDR1) containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 31; a VH CDR2 containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 32; and a VH CDR3 containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 33.In some embodiments, the antigen-binding domain comprises: VL CDR1 (VL CDR1), which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 34; VL CDR2, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 35; and VL CDR3, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 36. In some embodiments, the antigen-binding domain comprises: a variable light (VL) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 38; and a variable heavy (VH) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 37. In some embodiments, the signal transduction pathway modulator is coupled to the N-terminus of the antigen-binding domain. In some embodiments, the signal transduction pathway modulator is coupled to the C-terminus of the antigen-binding domain. In some embodiments, the linker couples the signal transduction pathway modulator to the antigen-binding domain that selectively binds to the mammalian ITGA7. In some embodiments, the agent further comprises a crystallizable fragment domain (Fc domain). In some embodiments, the Fc domain is the human IgG1 Fc domain. In some embodiments, the Fc domain comprises a mortar and pestle structure. In some embodiments, (a) the signal transduction pathway modulator comprises an amino acid sequence of any one of SEQ ID NO: 1-30, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with any one of the sequences shown in SEQ ID NO: 1-30, wherein the C-terminus or N-terminus of the signal transduction pathway modulator is coupled to a linker of GGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGS (SEQ ID NO: 42) and a human IgG1 Fc domain having a mortar and N297G mutation; and (b) the antigen-binding domain comprises a human IgG1 heavy chain and a light chain having a mortar and N297G mutation. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39.In some embodiments, the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 40. In some embodiments, the pharmaceutical agent comprises: a human IgG1 heavy chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39; and a light chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 40. In some embodiments, the pharmaceutical agent comprises a human IgG1 heavy chain containing SEQ ID NO: 39 and a light chain containing SEQ ID NO: 40. In some embodiments, the pharmaceutical agent comprises: a heavy chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 2; a human IgG1 heavy chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39; and a light chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 40. In some embodiments, the pharmaceutical agent comprises the heavy chain containing SEQ ID NO: 2, the human IgG1 heavy chain containing SEQ ID NO: 39, and the light chain containing SEQ ID NO: 40.

[0006] In another aspect, this disclosure provides a pharmaceutical composition comprising (i) the pharmaceutical agents described herein and (ii) one or more pharmaceutically acceptable excipients.

[0007] In another aspect, this disclosure provides a method for regulating muscular dystrophy, the method comprising administering an agent described herein to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the agent is delivered to a muscle cell or a region adjacent to a muscle cell. In some embodiments, the administration comprises injecting the subject at a location near skeletal muscle, muscle satellite cells (e.g., skeletal muscle satellite cells), skeletal muscle, cardiac muscle, smooth muscle, or muscle fibers. In some embodiments, the agent induces a reduction or inhibition of ActRII signaling in the IGF-1 signaling pathway, the GH signaling pathway, the bFGF signaling pathway, and the IL4 signaling pathway. In some embodiments, the muscular dystrophy includes Duchenne muscular dystrophy (DMD), Benedictine muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), and / or congenital muscular dystrophy.

[0008] Other aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the disclosure are shown and described. As will be appreciated, other and different embodiments of the disclosure may be adopted, and several details thereof may be modified in various obvious respects without departing from the disclosure. Therefore, the drawings and descriptions should be regarded as illustrative rather than restrictive in nature.

[0009] Incorporation All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is explicitly and individually indicated to be incorporated by reference. In the event of any conflict between a publication, patent, or patent application incorporated by reference and the disclosure contained herein, this specification is intended to supersede and / or give precedence to any such conflicting material. Attached Figure Description

[0010] The various features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description and accompanying drawings, which illustrate embodiments utilizing the principles of this disclosure, in which: Figure 1A The in vitro potency of IGF-1 targeting ITGA7 in non-muscle cells (DU145) was described compared with that of IGF-1. Figure 1B The in vitro potency of IGF-1 targeting ITGA7 in differentiated myocytes (C2C12) was described compared with that of IGF-1.

[0011] Figure 2The plasma half-lives of IGF-1 targeting ITGA7 and antibody scaffolds with ITGA7 targeting arms were depicted after the antibodies were injected into mice.

[0012] Figure 3A The relative concentrations (normalized relative to the control group) of human IgG antibody (control), IGF-1 targeting ITGA7, and antibody scaffold with ITGA7 targeting arm in the lung, liver, heart, diaphragm, forearm, and tibia were depicted 72 hours after injection of the corresponding antibody. Figure 3B The relative concentrations (normalized relative to the control group) of human IgG antibody (control), IGF-1 targeting ITGA7, and antibody scaffolds with ITGA7 targeting arms (corresponding to each column from left to right) in plasma were depicted 72 hours after injection of the corresponding antibodies; statistical analysis: one-way ANOVA, Dunnett's, n=5 animals / group. p < 0.05, p < 0.01; Figure 3C The study described the localization of human IgG antibody (control) and IGF-1 targeting ITGA7 in myofibrils in vivo after systemic administration, as determined by immunohistochemistry (IHC). Figure 3D The expression of phosphorylated Akt (S473) in the gastrocnemius and diaphragm, as detected by Western blot, was depicted at 4 h and 24 h after injection of IGF-1 targeting Fab or targeting ITGA7 only, and its normalized quantification relative to baseline levels. Figure 3E The expression of phosphorylated Akt (S473) in the kidneys and lungs, detected by Western blot assay, and its quantification relative to baseline levels, were depicted at 4 h and 24 h after injection of human IgG antibody or IGF-1 targeting ITGA7.

[0013] Figure 4 The relative concentrations of IGF-1 targeting ITGA7 in plasma, lung, liver, kidney, heart, diaphragm, forearm, and gastrocnemius muscle were depicted at 4 and 24 hours after injection of the corresponding antibody (normalized relative to the control group); statistical analysis: two-way ANOVA, Sidak multiple comparisons, 4 hours vs. 24 hours, n = 7 animals / group. p < 0.05, p < 0.01.

[0014] Figures 5A-5C The study showed that treatment with an IGF-1 fusion molecule targeting ITGA7 increased body weight and muscle mass in young mice. Figure 5A The results showed that mice administered 10 mg / kg of IGF-1 fusion molecules targeting ITGA7 intravenously every 4 days for 14 days showed an increase in body weight percentage from baseline compared with untreated mice. Figure 5BAnatomical images at the end of the 14-day treatment period are shown, depicting a qualitative increase in forearm muscle mass in mice treated with IGF-1 fusion molecules targeting ITGA7 compared to untreated mice. Figure 5C Mice treated with IGF-1 targeting ITGA7 showed increased isolated tibialis anterior (TA) muscle mass compared to untreated mice. Figure 5A The statistical analysis used was a two-way ANOVA, n=4-5 animals / group, in the time × column factor (weight of the untreated group relative to the CAV-003 group over time). p < 0.0001. Figure 5C The statistical analysis used unpaired t-tests, with n=4-5 animals / group. p < 0.05 p < 0.01.

[0015] Figures 6A-6B The results show the body weight and muscle mass of aged mice after 14 days of intravenous treatment with an IGF-1 fusion molecule targeting ITGA7 every 4 days. Figure 6A The study showed that mice treated with IGF-1 targeting ITGA7 for 14 days had a percentage increase in body weight from baseline compared to untreated mice. Figure 6B The study showed that mice treated with IGF-1 targeting ITGA7 had increased isolated tibialis anterior (TA) muscle mass compared to untreated mice. Figure 6A The statistical analysis used was a two-way ANOVA, n=4-5 animals / group, in the time × column factor (weight of the untreated group relative to the CAV-003 group over time). p < 0.05. Figure 6B The statistical analysis used unpaired t-tests, with n = 8-10 animals per group. p < 0.05 p < 0.01.

[0016] Figure 7 Results were presented in mice with a hindlimb plaster-fixed atrophy model treated with an IGF1 fusion molecule targeting ITGA7. Specifically, the right leg of mice was fixed in plaster for 2 weeks, during which time the IGF1 fusion molecule targeting ITGA7 was administered intraperitoneally every 4 days at a dose of 10 mg / kg. Subsequent gastrocnemius muscle dissection and weight measurements were performed. Results showed that, compared with the mediator-treated group, mice treated with the IGF-1 fusion molecule targeting ITGA7 exhibited increased muscle mass in both the unfixed (left leg) and plaster-fixed (right leg) muscles. Figure 7 The statistical analysis used an unpaired t-test, with n=10 animals / group. p < 0.05 p < 0.01.

[0017] Figures 8A-8C The study showed that, compared with wild-type mice and untreated FSHD mice, treatment with an IGF-1 fusion molecule targeting ITGA7 resulted in increased body muscle mass and strength production in transgenic facioscapulohumeral muscular dystrophy (FSHD). Figure 8A The results showed that, compared with untreated FSHD mice and wild-type (WT) mice treated with the drug, FSHD mice treated with an IGF-1 fusion molecule targeting ITGA7 at a dose of 10 mg / kg every 4 days via the intraperitoneal route had a percentage increase in body weight from baseline. Figure 8B The study showed that FSHD mice treated with IGF-1 targeting ITGA7 had a significant increase in isolated gastrocnemius (Gastroc) muscle mass compared to untreated FSHD mice and wild-type (WT) mice treated with the vector. Figure 8C The study showed that FSHD mice treated with IGF-1 targeting ITGA7 had a significant increase in maximal muscle strength compared to untreated FSHD mice and wild-type (WT) mice treated with the vector. Figures 8A-8C The statistical analysis used was one-way ANOVA and Dunnett's multiple comparison test, with n=8-10 animals / group. p < 0.05 p < 0.01, p < 0.001, p < 0.0001.

[0018] Figures 9A-9C The study showed increases in body weight and muscle mass, as well as changes in body composition, in adult mice after one month of IGF-1 treatment targeting ITGA7. Figure 9A Adult mice (6 months old) treated with an IGF1 fusion molecule targeting ITGA7 via the intraperitoneal route for 28 days showed a significant increase in body weight compared to animals treated with a medium (saline). Figure 9BThe weights of isolated organs (liver, kidney, and heart) and isolated skeletal muscles (including extensor digitorum longus (EDL), tibialis anterior (TA), and gastrocnemius) were shown in treated and untreated mice. Results showed no difference in the weight of isolated organs (liver, kidney, and heart) between the treatment and vector groups, but a significant increase in the mass of isolated skeletal muscle was observed between the treatment and vector groups. Figure 9C The results of time-domain MRI scans used to measure lean body mass percentage and fat mass percentage in treated and untreated mice are shown. Treated mice showed a significant increase in lean body mass percentage from baseline to two and four weeks, and a decrease in fat mass percentage. Figures 9A-9B The statistical analysis used was an unpaired t-test. Figure 9C The statistical analysis used one-way ANOVA and multiple comparisons were performed between baseline and 4-week time points, n=8-10 animals / group. p < 0.05.

[0019] Figure 10 Results of an in vitro power assay are shown, which measures the in vitro power of IGF-1 targeting ITGA7 compared to variants of IGF-1 with power-reducing mutations. EC50 data for IGF-1, IGF-1 targeting ITGA7, and its variants (1790-3, 1790-4, 1790-5) are provided. 50 value.

[0020] Figures 11A-11C The results of in vitro power assays of IGF-1 targeting ITGA7 in wild-type C2C12 cells and variants with reduced-potency mutations in IGF-1 are shown compared to ITGA7 knockout (KO) C2C12 cells. Figure 11A Immunofluorescence assay of ITGA7 using 1707-4 (ITGA7 antibody) in WT or ITGA7 KO C2C12 cells is shown. Figure 11B The levels of phosphorylated AKT (P-AKT) were shown by ELISA 15 minutes after stimulating WT and ITGA7 KO C2C12 cells with IGF-1. Figure 11C The levels of phosphorylated AKT (P-AKT) were shown by ELISA 15 minutes after stimulating WT and ITGA7 KOC2C12 cells with IGF-1 or an IGF-1 variant targeting ITGA7. Detailed Implementation

[0021] In one aspect, this document provides a drug that selectively targets mammalian cells expressing integrin α7 (ITGA7) with a modulator. In one aspect, the drug comprises (a) a signal transduction pathway modulator; and (b) an antigen-binding domain of mammalian ITGA7 selectively bound; wherein the modulator is coupled to the antigen-binding domain of mammalian ITGA7. In some embodiments, the modulator is a transcriptional modulator. Transcriptional modulators can regulate gene expression or gene accessibility. In some embodiments, transcriptional modulators include NF-κB, STAT proteins, p53, DNA methyltransferases, or histone deacetylases. In some embodiments, the modulator is a cell signal transduction modulator. In some embodiments, cell signal transduction modulators include cytokines, growth factors, or hormones. In some embodiments, the modulator is a metabolic modulator. Metabolic modulators can regulate metabolic pathways. In some embodiments, metabolic modulators are enzymes, metabolites, or nutrients. In some embodiments, the modulator is an immune system modulator. Immune system modulators can regulate or suppress immune cell activation or regulate immune responses. In some embodiments, the immune system modulator includes immune checkpoint inhibitors, antibodies, or antigens. In some embodiments, the modulator is an ion channel modulator. In some embodiments, the ion channel modulator includes a calcium channel modulator, a potassium channel modulator, or a sodium channel modulator. In some embodiments, the modulator is a pharmacological modulator. In some embodiments, the pharmacological modulator includes a receptor agonist or a receptor antagonist.

[0022] On the other hand, this article provides a drug that selectively targets mammalian cells expressing integrin α7 (ITGA7) with a signal transduction pathway regulator. In one aspect, the drug comprises (a) a signal transduction pathway regulator; and (b) an antigen-binding domain of mammalian ITGA7 that selectively binds to it; wherein the signal transduction pathway regulator is coupled to the antigen-binding domain of mammalian ITGA7.

[0023] In one aspect, signal transduction pathway modulators regulate the insulin-like growth factor 1 (IGF-1) signal transduction pathway. IGF-1 signal transduction pathway modulators may regulate the insulin-like growth factor 1 receptor (IGF-1R), hybrid insulin-like growth factor receptor / insulin receptor, insulin-like growth factor binding protein, or combinations thereof. IGF-1 signal transduction pathway modulators may include IGF-1 receptor (IGF-1R) or hybrid IGF-1R / IR activators. In some cases, IGF-1 signal transduction pathway modulators comprise IGF-1 peptides. For example, an IGF-1 peptide may comprise an IGF-1 peptide having a reduced ability to activate the IGF-1 receptor relative to endogenous IGF-1. The reduced activity can be assessed in functional assays, such as those described herein. In some cases, the activity of the IGF-1 peptide is reduced by at least about 5, 6, 7, 8, 9, 10, 15, 20, or more. In some cases, the IGF-1 peptide comprises a variant of IGF-1. IGF-1 variants may have, for example, the amino acid sequences of any one of SEQ ID NO: 1-11 and 46-50, or amino acid sequences that are at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NO: 1-11 and 46-50 (Table 1). In some cases, IGF-1 variants contain about one, about two, about three, about four, or about five amino acid modifications of the amino acid sequences of any one of SEQ ID NO: 1-11. In some cases, IGF-1 variants contain amino acid substitutions in the Fc region of the antibody to reduce its effector function. In some embodiments, the amino acid substitutions include a leucine-to-alanine substitution (LALA) at positions 234 and 235 in the Fc region of the antibody. In some embodiments, the amino acid substitutions include LALA and an additional mutation (LALA-PG) containing a proline-to-glycine substitution at another site.

[0024] On the other hand, the IGF-1 peptide is the wild-type form of IGF-1. In some embodiments, the wild-type form of IGF-1 comprises at least 90%, 95%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 1. In some embodiments, the wild-type form of IGF-1 comprises SEQ ID NO: 1. On the other hand, signal transduction pathway regulators regulate the growth hormone (GH) signal transduction pathway. GH signal transduction pathway regulators can modulate the growth hormone receptor (GHR). GH signal transduction pathway regulators may include GHR activators. In some cases, GH signal transduction pathway regulators include GH peptides. For example, GH peptides may include GH peptides that have a reduced ability to activate GHR relative to endogenous GH. The reduced activity can be assessed in functional assays, such as those described herein. In some cases, GH peptides include variants of GH. GH variants may have, for example, the amino acid sequence of SEQ ID NO: 12 or 13, or an amino acid sequence that is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO: 12 or 13 (Table 2). In some cases, GH variants contain about one, about two, about three, about four, or about five amino acid modifications of the amino acid sequence of SEQ ID NO: 12 or 13.

[0025] On the other hand, signal transduction pathway modulators regulate the basic fibroblast growth factor (bFGF) signal transduction pathway. bFGF signal transduction pathway modulators can regulate the fibroblast growth factor receptor (FGFR). bFGF signal transduction pathway modulators may include FGFR activators. In some cases, bFGF signal transduction pathway modulators include bFGF peptides. For example, a bFGF peptide may include a bFGF peptide with a reduced ability to activate FGFR relative to endogenous bFGF. The reduced activity can be assessed in functional assays, such as those described herein. In some cases, the bFGF peptide includes a variant of bFGF. The bFGF variant may have, for example, the amino acid sequence of any one of SEQ ID NO: 14-17, or an amino acid sequence that is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NO: 14-17 (Table 2). In some cases, the bFGF variant contains about one, about two, about three, about four, or about five amino acid modifications of the amino acid sequence of any one of SEQ ID NO: 14-17.

[0026] On the other hand, signal transduction pathway modulators regulate the interleukin-4 (IL4) signal transduction pathway. IL4 signal transduction pathway modulators can regulate the interleukin-4 receptor (IL4R). IL4 signal transduction pathway modulators may include IL4R activators. In some cases, IL4 signal transduction pathway modulators include IL4 peptides. For example, an IL4 peptide may include an IL4 peptide with a reduced ability to activate IL4R relative to endogenous IL4. The reduced activity can be assessed in functional assays, such as those described herein. In some cases, the IL4 peptide includes a variant of IL4. The IL4 variant may have, for example, the amino acid sequence of any one of SEQ ID NO: 18-21, or an amino acid sequence that is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NO: 18-21 (Table 2). In some cases, the IL4 variant contains about one, about two, about three, about four, or about five amino acid modifications of the amino acid sequence of any one of SEQ ID NO: 18-21.

[0027] On the other hand, signal transduction pathway modulators regulate activin type II receptor (ActRII). Signal transduction pathway modulators may include ActRII inhibitors. In some cases, signal transduction pathway modulators include bimagglutinumab. In some cases, the antibody or antibody fragment comprises: a variable heavy chain complementarity-determining region 1 (VH CDR1) containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 22; VH CDR2 containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 23; and VH CDR3 containing an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 24. In some cases, the antibody or antibody fragment comprises: VH CDR1, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 22; VH CDR2, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 23; and VH CDR3, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 24. In some cases, the antibody or antibody fragment comprises: a variable light chain complementarity-determining region 1 (VL CDR1), which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 25; VL CDR2, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 26; and VLCDR3, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 27. In some cases, the antibody or antibody fragment comprises: VL CDR1, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 25; VL CDR2, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 26; and VL CDR3, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 27.In some cases, the antibody or antibody fragment comprises a variable weight (VH) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 28. In some cases, the antibody or antibody fragment comprises a VH chain containing an amino acid sequence modified with about one, about two, about three, about four, or about five amino acids of the sequence shown in SEQ ID NO: 28. In some cases, the antibody or antibody fragment comprises a variable light (VL) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 29. In some cases, the antibody or antibody fragment comprises a VL chain containing an amino acid sequence modified with about one, about two, about three, about four, or about five amino acids of the sequence shown in SEQ ID NO: 29. In some cases, the antibody or antibody fragment contains the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 30 (Table 2). In some cases, the antibody or antibody fragment contains about one, about two, about three, about four, or about five amino acid modifications of the amino acid sequence shown in SEQ ID NO: 30. In one respect, the antigen-binding domain binds to epitopes expressed in muscle tissue. In some cases, the antigen-binding domain binds to epitopes expressed in myocytes. In some cases, the antigen-binding domain binds to epitopes expressed in mammalian muscle satellite cells. In some cases, the antigen-binding domain binds to epitopes expressed in mammalian skeletal muscle cells. In some cases, the antigen-binding domain binds to epitopes expressed in mammalian cardiomyocytes. In some cases, the antigen-binding domain binds to epitopes expressed in mammalian muscle fibers.

[0028] The antigen-binding domain can be, for example, an antibody or an antigen-binding fragment. When the target portion is an antigen-binding fragment, the antigen-binding fragment can be, for example, an antigen-binding fragment (Fab) domain or a single-chain variable fragment (scFv). In some cases, the antigen-binding domain comprises: VH complementarity-determining region 1 (VH CDR1), which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 31; VHCDR2, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 32; and VH CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 33. In some cases, the antigen-binding domain comprises: VHCDR1, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 31; VHCDR2, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 32; and VHCDR3, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 33. In some cases, the antigen-binding domain comprises: VL CDR1, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 34; VL CDR2, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 35; and VL CDR3, which comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 36. In some cases, the antigen-binding domain comprises: VL CDR1, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 34; VL CDR2, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 35; and VL CDR3, which comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in SEQ ID NO: 36.In some cases, the antigen-binding domain comprises: a variable light (VL) chain containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 38; and a variable heavy (VH) chain pestle containing an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 37. In some cases, the antigen-binding domain comprises: a variable light (VL) chain containing an amino acid sequence modified with about one, about two, about three, about four, or about five amino acids of the sequence shown in SEQ ID NO: 38; and a variable heavy (VH) chain pestle containing an amino acid sequence modified with about one, about two, about three, about four, or about five amino acids of the sequence shown in SEQ ID NO: 37 (Table 3). The signal transduction pathway modulator and the targeting moiety can be oriented in any way. In one non-limiting embodiment, the signal transduction pathway modulator is attached to the amino terminus of the targeting moiety. In another non-limiting embodiment, the signal transduction pathway modulator is attached to the carboxyl terminus of the targeting moiety.

[0029] In some cases, the drug may also contain a linker. In some cases, the drug may also contain an Fc domain. The Fc domain may be, for example, the human IgG1 Fc domain. The Fc domain may contain a club-and-mortar structure. In some cases, the drug may also contain both a linker and an Fc domain.

[0030] In one non-limiting embodiment, (a) the signal transduction modulator comprises an amino acid sequence of any one of SEQ ID NO: 1-30, or an amino acid sequence that is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NO: 1-30, wherein the C-terminus or N-terminus of the signal transduction pathway modulator is coupled to a GGGGSGGGGSGGGGS linker (SEQ ID NO: 41) and an hIgG1 FC domain having β- and N297G mutations; and (b) the antigen-binding domain comprises the heavy chain and light chain of human IgG1 having β- and N297G mutations. In another non-limiting embodiment, (a) the signal transduction modulator comprises an amino acid sequence of any one of SEQ ID NO: 1-30, or an amino acid sequence having at least 90%, 95%, 98%, or 99% identity with any one of SEQ ID NO: 1-30, wherein the C-terminus or N-terminus of the signal transduction pathway modulator is coupled to GGGGSGGGGS (SEQ ID NO: 42) and an hIgG1 FC domain having the β- and N297G mutations; and (b) the antigen-binding domain comprises a human IgG1 heavy chain and a light chain having the β- and N297G mutations. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence shown in SEQ ID NO: 39. In some embodiments, the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 40. In some embodiments, the light chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence shown in SEQ ID NO: 40 (Table 3). In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in any one of SEQ ID NO: 51, 53, and 55. In some embodiments, the human IgG1 heavy chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications of the sequence shown in any one of SEQ ID NO: 51, 53, and 55. In some embodiments, the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in any one of SEQ ID NO: 52, 54, and 56.In some embodiments, the light chain comprises an amino acid sequence having about one, about two, about three, about four, or about five amino acid modifications having the sequence shown in any one of SEQ ID NO: 52, 54, and 56.

[0031] In one aspect, the cargo includes a vector. The vector can be any suitable macromolecule or molecular complex containing a polynucleotide to be delivered to target cells. In some embodiments, the vector is a gene-editing vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a recombinant vector. In some embodiments, the vector includes more than one vector described herein.

[0032] In one aspect, the vector is a non-viral vector. In some embodiments, the non-viral vector is uncapped. In some embodiments, the non-viral vector is a deoxyribonucleic acid (DNA) vector. In some embodiments, the non-viral vector is a ribonucleic acid (RNA) vector. In some embodiments, the non-viral vector is derived from a plasmid. In some embodiments, the non-viral vector is produced in a host cell.

[0033] On the other hand, the vector is a viral vector. In some embodiments, the viral vector is a replicating viral vector. In some embodiments, the viral vector is a non-replicating viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. The AAV vector may include a polynucleotide of interest (e.g., a transgene) flanked by an AAV terminal repeat (ITR) sequence and may be packaged into an infectious viral particle. The AAV vector can be replicated when it is present in a host cell (such as a host cell that has been transfected with a vector encoding and expressing the rap and cap gene products). In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the vector includes a recombinant viral vector containing one or more nucleic acids described herein. In some embodiments, the viral vector is used to provide long-term gene expression. In some embodiments, the viral vector is used to provide short-term gene expression. In some embodiments, the viral vector is used to express a therapeutic molecule. In some embodiments, the therapeutic molecule is a nucleic acid, a protein, or an antibody.

[0034] On the other hand, the cargo includes nucleic acids. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is double-stranded. In some embodiments, the RNA is a molecule that induces RNA interference (RNAi), including but not limited to siRNA, double-stranded RNA (dsRNA), hourly RNA (stRNA), short hairpin RNA (shRNA), microRNA (miRNA), and their gene-silencing variants. In some embodiments, the nucleic acid is double-stranded RNA (dsRNA). In some embodiments, dsRNA reduces the expression of target genes in cells. In some embodiments, the cells are muscle cells. In some embodiments, reducing the expression of target genes treats diseases caused by the expression of target genes.

[0035] In some embodiments, the dsRNA has a first oligonucleotide sequence and a second oligonucleotide sequence. In some embodiments, the second oligonucleotide sequence is annealed with the first oligonucleotide sequence under biological conditions. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 60 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 50 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 40 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 30 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 10 nucleotides to about 20 nucleotides in length. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 100 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 80 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 70 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 60 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 50 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 40 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 20 nucleotides to about 30 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 100 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 90 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 80 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 70 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 60 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 50 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 30 nucleotides to about 40 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 100 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 100 nucleotides.In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 90 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 80 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 70 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 60 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 40 nucleotides to about 50 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 50 nucleotides to about 100 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 50 nucleotides to about 90 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 50 nucleotides to about 80 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 50 nucleotides to about 70 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 50 nucleotides to about 60 nucleotides. In some embodiments, the length of the first oligonucleotide sequence is from about 60 nucleotides to about 100 nucleotides. In some embodiments, the first oligonucleotide sequence is about 60 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 60 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 60 nucleotides to about 70 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 70 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 70 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 70 nucleotides to about 80 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 80 nucleotides to about 100 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 80 nucleotides to about 90 nucleotides in length. In some embodiments, the first oligonucleotide sequence is about 90 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 50 nucleotides in length.In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 40 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 30 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 10 nucleotides to about 20 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 40 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 20 nucleotides to about 30 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 30 nucleotides to about 40 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 40 nucleotides to about 50 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 50 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 50 nucleotides to about 90 nucleotides in length.In some embodiments, the second oligonucleotide sequence is about 50 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 50 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 50 nucleotides to about 60 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 60 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 60 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 60 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 60 nucleotides to about 70 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 70 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 70 nucleotides to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 70 nucleotides to about 80 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 80 nucleotides to about 100 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 80 to about 90 nucleotides in length. In some embodiments, the second oligonucleotide sequence is about 90 to about 100 nucleotides in length.

[0036] In some embodiments, the dsRNA contains a sequence fully complementary to the target sequence of the target gene. In some embodiments, the dsRNA has an asymmetric structure. In some embodiments, the dsRNA has two separate oligonucleotides linked by a third structure. In some embodiments, the dsRNA is a small interfering RNA (siRNA). In some embodiments, the dsRNA is a precursor of siRNA. In some embodiments, the dsRNA is processed by Dicer to produce siRNA. The dsRNA may be derived from a single RNA oligonucleotide that has undergone intramolecular annealing. In some embodiments, the dsRNA contains at least one modification. In some embodiments, at least one modification facilitates Dicer binding to the double-stranded RNA structure in an orientation that maximizes the effectiveness of the double-stranded RNA structure in repressing gene expression. In some embodiments, the first and second oligonucleotides do not need to be fully complementary. In some embodiments, the siRNA is 19 nucleotides long. In some embodiments, the siRNA is 20 nucleotides long. In some embodiments, the siRNA is 21 nucleotides long. In some embodiments, the siRNA is 22 nucleotides long. In some embodiments, the siRNA is 23 nucleotides long. In some embodiments, the siRNA is 24 nucleotides long. In some embodiments, the siRNA is 25 nucleotides long. In some embodiments, the siRNA is 26 nucleotides long. In some embodiments, the siRNA is 27 nucleotides long. In some embodiments, the siRNA is 28 nucleotides long. In some embodiments, the siRNA is 29 nucleotides long. In some embodiments, the siRNA is 30 nucleotides long.

[0037] siRNA may comprise an antisense strand and a sense strand. In some embodiments, the sense strand is about 20 to about 30 nucleotides long, or about 22 to about 28 nucleotides long. In some embodiments, the antisense strand is at least about 19 nucleotides long. In some embodiments, the antisense strand is modified at the 3' end and / or the 5' end. In some embodiments, the antisense strand of dsRNA has a 3' overhang. In some embodiments, the antisense strand of siRNA is modified to include about 1 to about 9 ribonucleotides at the 5' end to obtain a length of about 20 to about 30 nucleotides.

[0038] In some embodiments, siRNA is chemically modified. In some embodiments, siRNA exhibits reduced immunostimulatory activity compared to its corresponding unmodified RNA molecule. In some embodiments, siRNA exhibits increased serum stability compared to its corresponding unmodified RNA molecule.

[0039] In some embodiments, siRNA is shRNA. shRNA can function as siRNA. shRNA may have a double-stranded hairpin-like structure to increase stability. In some embodiments, the hairpin-like structure is about 1 to about 30, about 1 to about 24, or about 1 to about 10 nucleotides in length. The sequence of the hairpin-like structure may include nucleotide residues unrelated to the target gene. In some embodiments, the hairpin-like structure is 5'-UU-3'. In some embodiments, the hairpin-like structure includes a non-nucleotide portion. In some embodiments, the hairpin-like structure does not include any non-nucleotide portion. shRNA may also include RNA with stem-loop structures containing mismatches and / or protrusions. In some embodiments, the hairpin-like structure comprises nucleotides, non-nucleotides, or a combination of nucleotides and non-nucleotides.

[0040] On the other hand, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is an antisense oligonucleotide (ASO). In some embodiments, the ASO contains a sequence fully complementary to the messenger RNA (mRNA) of the target gene of the target cell. In some embodiments, the target cell is a myocyte. In some embodiments, the ASO targets precursor mRNA. In some embodiments, the ASO contains a backbone modification including phosphate thioester bonding or phosphodiamid bonding. In some embodiments, the ASO contains a phosphodiamid morpholino oligonucleotide, locked nucleic acid, peptide nucleic acid, 2'-O-methyl, 2'-fluoro, or 2'-O-methoxyethyl moiety. In some embodiments, the ASO is a morpholino oligonucleotide. In some embodiments, the ASO contains at least one modified sugar moiety. In some embodiments, the ASO is 8 to 50 nucleotides in length. In some embodiments, the ASO is 8 to 40 nucleotides in length. In some embodiments, the ASO is 8 to 35 nucleotides in length. In some embodiments, the ASO is 8 to 30 nucleotides in length. In some embodiments, the ASO is 8 to 25 nucleotides in length. In some embodiments, the ASO is 8 to 20 nucleotides in length. In some embodiments, the ASO is 8 to 15 nucleotides in length. In some embodiments, the ASO is 9 to 50 nucleotides in length. In some embodiments, the ASO is 9 to 40 nucleotides in length. In some embodiments, the ASO is 9 to 35 nucleotides in length. In some embodiments, the ASO is 9 to 30 nucleotides in length. In some embodiments, the ASO is 9 to 25 nucleotides in length. In some embodiments, the ASO is 9 to 20 nucleotides in length. In some embodiments, the ASO is 9 to 15 nucleotides in length. In some embodiments, the ASO is 10 to 50 nucleotides in length. In some embodiments, the ASO is 10 to 40 nucleotides in length. In some embodiments, the ASO is 10 to 35 nucleotides in length. In some embodiments, the ASO is 10 to 30 nucleotides in length. In some embodiments, the ASO is 10 to 25 nucleotides in length. In some embodiments, the ASO is 10 to 20 nucleotides in length. In some embodiments, the ASO is 10 to 15 nucleotides in length. In some embodiments, the ASO is 11 to 50 nucleotides in length. In some embodiments, the ASO is 11 to 40 nucleotides in length. In some embodiments, the ASO is 11 to 35 nucleotides in length. In some embodiments, the ASO is 11 to 30 nucleotides in length. In some embodiments, the ASO is 11 to 25 nucleotides in length. In some embodiments, the ASO is 11 to 20 nucleotides in length. In some embodiments, the ASO is 11 to 15 nucleotides in length.In some embodiments, the ASO is 12 to 50 nucleotides in length. In some embodiments, the ASO is 12 to 40 nucleotides in length. In some embodiments, the ASO is 12 to 35 nucleotides in length. In some embodiments, the ASO is 12 to 30 nucleotides in length. In some embodiments, the ASO is 12 to 25 nucleotides in length. In some embodiments, the ASO is 12 to 20 nucleotides in length. In some embodiments, the ASO is 12 to 15 nucleotides in length.

[0041] On the other hand, the cargo includes lipid nanoparticles (LNPs). LNPs contain lipid formulations that can be used to deliver the carrier to target sites of interest (e.g., cells, tissues, organs, etc.).

[0042] In some embodiments, nucleic acids are encapsulated in LNPs. In some embodiments, the LNP contains lipids selected from: ionizable or cationic lipids, cofactor phospholipids, conjugated lipids, PEG-conjugated lipids, cholesterol-based lipids, and combinations thereof. In some embodiments, the ionizable lipid is ALC-0315, SM-102, or MC-3. In some embodiments, the cholesterol-based lipid is cholesterol or a derivative thereof. In some embodiments, the cofactor phospholipid is DSPC. In some embodiments, the conjugated lipid is a PEG-lipid conjugate, a polyoxazoline (POZ)-lipid conjugate, a polyamide-lipid conjugate (such as an ATTA-lipid conjugate), a cationic polymer lipid (CPL) conjugate, or a mixture thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as a (methoxy polyethylene glycol) conjugated lipid. In some embodiments, the PEG-conjugated lipid is ALC-0159. In some embodiments, the PEG-lipid conjugate is PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG-dialkoxypropylcarbamate, N-(carbonyl-methoxy-polyethylene glycol 2000)-1,2-distearate-sn-glycerol-3-phosphate ethanolamine sodium salt, or mixtures thereof.

[0043] In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the LNP comprises a noncationic lipid. In some embodiments, the noncationic lipid is a neutral, uncharged lipid, a zwitterionic lipid, or anionic lipid. In some embodiments, the nonionic lipid is selected from distearyl-sn-glycerol-phosphoethanolamine, distearyl-phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexyl Alkyl-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine (such as 16-O-monomethylPE), dimethylphosphatidylethanolamine (such as 16-O-dimethylPE), 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), methionine phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOP) 5) Sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearate phosphatidylglycerol (DSPG), disqualoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), ditransoleoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysophosphatidyl lecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, esphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecosyl phosphate, lysophosphatidylcholine, and dilinoleoyl phosphatidylcholine.

[0044] In some embodiments, ionizable lipids are present at a molar ratio of 35% to 55%. In some embodiments, PEG-conjugated lipids are present at a molar ratio of 0.25% to 3%. In some embodiments, accessory phospholipids are present at a molar ratio of 5% to 20%. In some embodiments, cholesterol-based lipids are present at a molar ratio of 20% to 45%. In some embodiments, cationic lipids may be present in an amount of about 10% by weight of LNP to about 85% by weight of lipid nanoparticles, or about 50% by weight of LNP to about 75% by weight of LNP. All lipid molar ratios described herein are relative to the total lipid content of the LNP. The molar ratio of lipids (e.g., ionizable lipids, non-cationic lipids, sterols, and PEG / conjugated lipids) in the LNP can be varied as needed.

[0045] In some embodiments, the LNP does not contain any phospholipids. In some embodiments, the LNP contains one or more additional compounds. In some embodiments, the additional compounds are administered separately. In some embodiments, the additional compounds may be included in the LNP provided herein. In some embodiments, the additional compounds may be selected from small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and their derivatives, peptide mimics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0046] In another aspect, the goods include pharmaceuticals. In yet another aspect, the goods include two or more types of pharmaceuticals. In some embodiments, the pharmaceuticals are therapeutic agents for muscle diseases. In some embodiments, the pharmaceuticals are conjugated to an antigen-binding domain via a linker. In some embodiments, the linker is cleavable. In some embodiments, the linker can be cleaved by a lysing agent present in the intracellular environment (e.g., lysosomes, endosomes, or pits). In some embodiments, the linker contains a peptide (e.g., a linker containing an oligopeptide or a dipeptide, tripeptide, tetrapeptide, or pentapeptide). In some embodiments, the linker contains a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide. In some embodiments, the pharmaceuticals conjugated to the antigen-binding domain function as a targeted drug delivery system. In some embodiments, the conjugation of the pharmaceuticals to the antigen-binding domain is site-specific. In some embodiments, the conjugation of the pharmaceuticals to the antigen-binding domain is not site-specific. In some embodiments, the pharmaceuticals are prodrugs activated upon administration to a subject. In some embodiments, the pharmaceuticals are small molecule pharmaceuticals. In some embodiments, the pharmaceuticals are peptide pharmaceuticals. In some embodiments, the pharmaceuticals are protein pharmaceuticals.

[0047] In some embodiments, the drug is a toxin. In some embodiments, the toxin is a cytotoxic molecule. In some embodiments, the toxin is a radionuclide. In some embodiments, the toxin is a chemotherapeutic drug. In some embodiments, the toxin is an anticancer agent. In some embodiments, the anticancer agent is adriamycin, daunomycin, mitomycin, cisplatin, vincristine, epirubicin, methotrexate, 5-fluorouracil, aclacinomycin, nitrogen mustard, cyclophosphamide, bleomycin, daunomycin, doxorubicin, vincristine, vinblastine, or combinations thereof. In some embodiments, the toxin is cell membrane permeable. In some embodiments, the toxin is dianthin or saporin. In some embodiments, the toxin is a Pseudomonas aeruginosa exotoxin (PE) or a cytotoxic fragment thereof (e.g., PE38). In some embodiments, the toxin is monomethyl auristatin E (MMAE) or MMAF.

[0048] On the other hand, the goods include immunomodulators. In some embodiments, the immunomodulator is an immunosuppressant. In some embodiments, the immunomodulator reduces the activity of the immune system. In some embodiments, the immunomodulator is immunostimulatory. In some embodiments, the immunomodulator increases the activity of the immune system. In some embodiments, the immunomodulator is an interleukin (IL). In some embodiments, the interleukin is IL-10. In some embodiments, the interleukin is IL-35. In some embodiments, the interleukin is IL-4. In some embodiments, the interleukin is IL-13. In some embodiments, the interleukin is IL-27. In some embodiments, the interleukin is IL-37. In some embodiments, the interleukin is a combination of the interleukins provided herein. In some embodiments, the immunomodulator is a chemokine. In some implementations, the chemokine is CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, or a combination thereof.

[0049] On the other hand, this article provides a pharmaceutical composition comprising (i) any pharmaceutical agent and (ii) one or more pharmaceutically acceptable excipients.

[0050] Functional testing The agents described herein can be tested in any in vitro and / or in vitro functional assays (such as those described below).

[0051] In vitro functional testing The binding affinity and kinetics of amino acid-substituted variants of human insulin-like growth factor I (IGF-I) with recombinant IGF-binding protein 1 (IGFBP-1) and the soluble form of IGF type I receptor (sIGF-IR) were analyzed. Ligand binding kinetics were determined using BIAcore biosensor interaction analysis. Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable-selective secondary structure calculations.

[0052] In vivo function testing Obtain male mdx Mice (3-6 months old; 4-5 mice per group). Animal care and experimental procedures were conducted in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes.

[0053] Mice were administered a negative control, an IGF-1 variant, a GH variant, a bFGF variant, an IL4 variant, or bimagglutinumab alone, an antigen-binding domain alone, or the agent described herein. Treatment was administered to each group once weekly via injection (e.g., intravenous injection) for three weeks. Mice were monitored and weighed every other day after treatment began until the end of the study. Body weight and muscle mass were assessed before treatment, during treatment, and at the end of the study.

[0054] Adjustment method This document provides a method for modulating muscular dystrophy (MD) comprising delivering any of the pharmaceutical agents described herein to a region adjacent to muscle. In some cases, the administration is directed to a subject, and the subject is a mammal. A mammal may be, for example, a human. Administration may include injection into the subject at a location near skeletal muscle, muscle satellite cells (e.g., skeletal muscle satellite cells), skeletal muscle, cardiac muscle, smooth muscle, or muscle fibers. Injection may be via any suitable route of administration, including but not limited to intravenous (IV), subcutaneous (SQ), intramuscular (IM), intrathecal (IT), and intraperitoneal. Administration of the pharmaceutical agent to the subject induces signal transduction of the IGF-1 signaling pathway, GH signaling pathway, bFGF signaling pathway, IL4 signaling pathway, or inhibits ActRII. In some cases, a pharmaceutical composition is administered to the subject, wherein the pharmaceutical composition comprises a pharmaceutical agent and one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or excipient used for administering an active substance. A pharmaceutically acceptable carrier may include any substance or medium suitable for delivering the pharmaceutical agent to the subject. This term refers to any pharmaceutical carrier that can be administered without excessive toxicity. Suitable carriers may be, for example, one or more optional stabilizers, diluents and / or excipients.

[0055] Muscular dystrophy to be treated with the methods described herein includes, for example, Duchenne muscular dystrophy (DMD), Benedictine muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), and / or congenital muscular dystrophy.

[0056] Subjects to be treated with the methods described herein have been diagnosed with muscular dystrophy. The diagnosis may be based on one or more of the following: family history; genetic testing; cardiomyopathy; calf muscle hypertrophy; cognitive impairment; delayed speech and language development; elevated serum creatine kinase; muscle loss; weakness of the facial, shoulder girdle, and upper arm muscles, while the deltoid muscle is relatively unaffected; or any combination thereof. Assessment may also include nerve conduction velocity testing (NCV) and electromyography (EMG).

[0057] In one instance, a subject treated with the medication described herein experienced at least a 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, or greater improvement in one or more symptoms. The improvement may be compared to placebo treatment or to the same subject before treatment.

[0058] Some definitions All terms are intended to be understood in the same way as would be understood by one of ordinary skill in the art to which this disclosure pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as would be commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0059] The following definitions supplement those in the art and are specific to this application and should not be attributed to any related or unrelated cases, such as any jointly owned patents or applications. While similar or equivalent methods and materials to those described and used herein may be used to test the practice of this disclosure, preferred materials and methods are described herein.

[0060] The terminology used herein is for the purpose of describing specific cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless otherwise expressly stated. As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to include the plural forms.

[0061] In this application, unless otherwise stated, the use of “or” means “and / or”. The terms “and / or” and “any combination thereof” and their grammatical equivalents are used interchangeably as used herein. These terms can express any combination specifically conceived. For illustrative purposes only, the phrases “A, B and / or C” or “A, B, C or any combination thereof” can refer to “A alone; B alone; C alone; A and B; B and C; A and C; and A, B and C”. The term “or” can be used either conjoined or separately unless the context clearly indicates separate use.

[0062] The term “about” refers to a range of about 10% of a given value.

[0063] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unlisted elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of this disclosure, and vice versa. Furthermore, the compositions of this disclosure can be used to implement the methods of this disclosure.

[0064] The terms "some embodiments," "an embodiment," "one embodiment," or "other embodiments" used in this specification refer to specific features, structures, or characteristics described in connection with these embodiments that are included in at least some, but not necessarily all, embodiments of this disclosure. For ease of understanding of this disclosure, the following terms and phrases are defined.

[0065] The ranges provided in this document should be understood as abbreviations of all values ​​within that range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or subrange of any group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intermediate decimal values ​​between the aforementioned integers, such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Regarding subranges, "nested subranges" extending from either endpoint of the range are particularly considered. For example, nested subranges of the example range 1 to 50 can contain 1 to 10, 1 to 20, 1 to 30 and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20 and 50 to 10 in another direction.

[0066] The term "pharmaceutical acceptable" means that something is approved or permitted by federal or state regulatory agencies, or listed in the United States Pharmacopeia (USP) or other generally accepted pharmacopoeias, for use in animals (including humans).

[0067] The term "object" refers to an animal that is the subject of treatment, observation, or experimentation. By way of example only, objects include, but are not limited to, mammals, including but not limited to, humans or non-human mammals such as non-human primates, cattle, horses, dogs, sheep, or cats.

[0068] The terms “optional” or “optionally” indicate that the event or situation described below may but does not need to occur, and the description includes both scenarios in which the event or situation occurs and scenarios in which the event or situation does not occur.

[0069] Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first value in a series of two or more values, the terms "at least," "greater than," or "greater than or equal to" apply to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0070] Whenever the terms "not exceeding," "less than," or "less than or equal to" precede the first value in a series of two or more values, the terms "not exceeding," "less than," or "less than or equal to" apply to each value in that series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0071] The "sequence identity percentage" between the reference amino acid sequence and the query amino acid sequence (i.e., the amino acid sequence being analyzed to determine whether it falls within a specific sequence identity percentage of the reference amino acid sequence) is determined by performing optimal alignment of the sequences using the Needleman-Wunsch alignment algorithm (where the gap presence penalty is 11 and the gap extension penalty is 1) and comparing the sequences. The number of exact matches is determined by dividing by the total number of positions in the alignment (which corresponds to the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) and expressed as a percentage. This is the sequence identity percentage between the query amino acid sequence and the reference amino acid sequence (i.e., sequence identity percentage = (number of exact matches) / (total number of positions in the alignment)). 100). The “Global Align” BLAST program, available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, can be used to generate alignments using the Needleman-Wunsch alignment algorithm (where the penalty for a gap is 11 and the penalty for a gap extension is 1).

[0072] The "sequence identity percentage" between the reference nucleic acid sequence and the query nucleic acid sequence (i.e., the nucleic acid sequence being analyzed to determine whether it falls within a specific sequence identity percentage of the reference nucleic acid sequence) is determined by performing optimal alignment of the sequences using the Needleman-Wunsch alignment algorithm (where match / mismatch scores are 2 and -3, a gap presence penalty is 5, and a gap extension penalty is 2) and comparing the aligned nucleic acids. The number of exact matches is determined by dividing by the total number of nucleotides in the alignment (which corresponds to the number of nucleotides in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) and expressed as a percentage. This is the sequence identity percentage between the query nucleic acid sequence and the reference nucleic acid sequence (i.e., sequence identity percentage = (number of exact matches) / (total number of nucleotides in the alignment)). 100). The “Global Align” BLAST program, available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, can be used to generate alignments using the Needleman-Wunsch alignment algorithm (where the match / mismatch score is 2, -3, the gap presence penalty is 5, and the gap extension penalty is 2).

[0073] Example Example 1: Methods and Procedures The methods and procedures described herein are applied in all embodiments.

[0074] In vitro potency assay (pAKT assay) 15 x 10 cm will be frozen in generation 5 (P5) 6 DU145 cells (obtained from ATCC) were reseeded in T175 culture flasks containing DMEM / F12 (1:1) medium (Gibco 11320-033) supplemented with 10% FBS (Gibco 10437-028) and 10 μg / mL gentamicin (Gibco 15710-064) (hereinafter referred to as culture medium). At 80%–90% confluence, cells were digested with trypsin and manually counted using a hemocytometer at 3 x 10⁻⁶ cells / mL. 5Cells / mL were resuspended in culture medium and then reseeded in 24-well plates (Corning 3524) at 0.5 mL aliquots per well (150,000 cells / well). 14–16 hours after reseeding, the culture medium was removed, followed by washing with 1 mL PBS and incubation for 6 h in 225 µL of serum-free medium supplemented with 0.2% BSA (Millipore A3059). Two 25 µL aliquots of the following growth factor dilutions were added to the cells: 4570, 1370, 410, 125, 37, 11.1, and 3.3 nMrhIGF1 (PeproTech#100-11) (SEQ ID NO: 1) and fusion antibodies (containing three polypeptide chains: SEQ ID NO: 2, 39, and 40). After incubation at 37°C for 15 min, the culture medium was aspirated, and cells were lysed in 150 µL of 1xPTR buffer (5x PTR extraction buffer, Abcam ab193970), which contained 1x extraction enhancer buffer (50x extraction enhancer buffer, Abcam ab193971) and a protease / phosphatase inhibitor (one Pierce microtablet [A32959] / 10 mL 1xPTR). After incubation on ice for 15–30 min, the plate was vortexed, and the lysate was transferred to a microcentrifuge tube (Eppendorf tube) and stored at -80°C to assess IGF1R-AKT signal transduction intensity using a human / mouse / rat phosphorylated Akt (S473) panspecific DuoSet IC ELISA (RnD # DYC887B).

[0075] C2C12 mouse myoblasts (ATCC#CRL-1772) were cultured in DMEM supplemented with 10% FBS (Gibco 10437-028) and 10 µg / mL gentamicin (15710-064) containing 4.5 g / L glucose (Corning 10-013-CV). At 70%–80% confluence, cells were digested with trypsin and manually counted using a hemocytometer at 2 x 10⁻⁶ cells / mL. 5C2C12 cells / mL were resuspended in differentiation medium (DMEM (Corning 10-013-CV)) supplemented with 2% horse serum (Sigma, #H1138) and 10 μg / mL gentamicin (Gibco 15710-064) and seeded in 24-well plates (Corning 3524) at 0.5 mL aliquots / well (100,000 cells / well). After 5 days on differentiation medium, C2C12 cells were washed with 1 mL PBS and then serum starved for 6 h in 225 μL of serum-free medium supplemented with 0.2% BSA (Millipore A3059). The remainder of the protocol is the same as described above for DU145 cells.

[0076] Following the manufacturer's recommendations with slight modifications, perform ELISA in 384-well plates. Briefly, coat the wells of the 384-well plate with 25 μL of phosphorylated Akt1 (S473) capture antibody (RnD #841692) in PBS (6 μg / mL) (25 μL / 384 wells), seal, and incubate overnight at room temperature. Wash the wells four times with 100 μL of PBS containing 0.05% Tween 20, and block with 50 μL of PBS containing 2% BSA for 1–2 h at room temperature (RT). Wash the wells four times with 100 μL of PBS containing 0.05% Tween 20, and incubate for 2 h at room temperature with 25 μL of lysate or P-AKT standard (RnD #841694). Wash wells four times with 100 μL of PBS containing 0.05% Tween 20, then incubate for 1 h at room temperature with 25 μL of phosphorylated Akt1 (S473) detection antibody (RnD #843081) diluted 100 ng / mL in PBS containing 1% BSA. Wash wells four times with 100 μL of PBS containing 0.05% Tween 20, and incubate for 20 min at room temperature with 25 μL of streptavidin-HRP A (RnD #890803) diluted 1:200 in PBS containing 1% BSA. Wash wells four times with 100 μL of PBS containing 0.05% Tween 20, and incubate for 2–20 min at room temperature with 25 μL of TMB substrate solution (Abcam TMB ELISA high-sensitivity substrate, #ab171523) (time depends on the intensity of the colorimetric reaction). Add 12.5 μL of stop solution (RnD #DY994) and measure the OD450 of each well using a BMG Labtech CLARIOstar® Plus microplate reader. Calculate the EC50 value using Prism 10 software.

[0077] Efficacy deviation measurement Undifferentiated wild-type (WT) and ITGA7 knockout (KO) C2C12 cells were used at 0.5 x 10⁻⁶ cells per cell line. 5 Cells / mL were resuspended in culture medium and seeded in 24-well plates (Corning, #3524) at 0.5 mL aliquots per well (25,000 cells / well). The rest of the protocol was identical to that described above for DU145 cells, except for the volume of the lysis buffer (200 μL for C2C12 cells).

[0078] Animal research Wild-type (WT) males C57BL / 6J (Jackson Labs, strain #000664) or C57BL / 10ScSn-Dmdmdx / J(mdx) (Jackson Labs, strain #001801) were purchased from Jackson Laboratories at 8 weeks of age. All animal studies were conducted in accordance with protocols approved by the Institutional AnimalCare and Use Committee of Explora Biolabs.

[0079] In vivo PK analysis (human IgG assay) Wild-type (WT) males C57BL / 6J (Jackson Labs, strain #000664) were purchased at 8 weeks of age and transported to the CavalryBiosciences animal housing facility managed by Explora Biolabs in San Francisco, CA. After at least one week of acclimatization, the mice were weighed and administered a custom-made therapeutic protein from Fusion Antibodies, Inc. via lateral tail vein injection using an insulin injector (BD, 329424) at a dose volume of 5–10 ml / kg in a dose range of 0.3–15 mg / kg. Individual animals were administered at time points collected at 1, 4, 24, 48, 96, 144, and 240 hours post-administration, with 3–5 animals at each time point. At appropriate time points, animals were anesthetized with 5% isoflurane using an RC2 rodent loop controller (922100, VetEquip), and plasma was harvested from blood collected from the inferior vena cava by centrifugation in a microvolume containing dipotassium EDTA (BD, 365974). Samples were frozen on dry ice and stored at -80°C until future analysis. For analysis, plasma samples were diluted as empirically determined for each dose level, and the concentration of therapeutic proteins was assessed by a sandwich ELISA targeting the humanized Fc region of the therapeutic protein (Human IgG ELISA Kit, ab195215, Abcam) according to the manufacturer's instructions. Standard curves for each therapeutic protein analyzed were generated in sample diluents provided with the assay at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 ng / ml. The standard curve and OD450 data for each sample were collected using a Promega GloMAX Discover microplate reader. The sample data were interpolated against the standard curve, the dilution factor was corrected, plotted, and the half-life was calculated using two-phase decay in Prism 9 (Graphpad).

[0080] In vivo distribution (human IgG assay) Custom-made therapeutic proteins from Fusion Antibodies, Inc. were administered via lateral tail vein injection using an insulin injector (BD, 329424) at doses ranging from 0.3 to 15 mg / kg at a dose volume of 5–10 ml / kg. Individual animals were weighed for either C57BL / 10ScSn-Dmdmdx / J(mdx) (Jackson Labs, strain #001801) or an age-matched control wild-type (WT) male C57BL / 6J (Jackson Labs, strain #000664). Dosing was administered to individual animals at time points collected at 4, 24, or 72 hours post-dosing, with 5–8 animals per therapeutic protein per time point. At appropriate time points, animals were anesthetized with 5% isoflurane using an RC2 rodent circuit controller (922100, VetEquip), and plasma was harvested from blood collected from the inferior vena cava by centrifugation in a microcapsule containing dipotassium EDTA (BD, 365974). Animals were then perfused via the apex of the heart with 20–30 ml of Dulbecco phosphate-buffered saline (DPBS, Corning, 21030CV). Multiple tissues were collected, including some or all of the following: heart, kidneys, liver, lungs, gastrocnemius, diaphragm, tibialis anterior, and forearm muscles. Tissues were weighed and rapidly frozen on dry ice and stored at -80°C until later analysis. Using NextAdvance Bullet Blender Gold (BB24AU) with a green, dark blue, or red Eppendorf lysis kit (NextAdvance), homogenize samples at 1 ml / 250 mg tissue in 5X PTR (Abcam#ab193970) containing extraction buffer with protease and phosphatase inhibitors (Thermo Scientific, A32959). Dilute samples as empirically determined for each tissue and / or each dose level, and assess the concentration of therapeutic proteins using a sandwich ELISA targeting the humanized Fc region of the therapeutic protein (Human IgG ELISA Kit, ab195215, Abcam) according to the manufacturer's instructions. Standard curves for each therapeutic protein analyzed are generated using sample diluents provided with the assay at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 ng / ml. Standard curves and OD450 data for each sample were collected using a Promega GloMAX Discover microplate reader. Sample data were interpolated against the standard curve, dilution factors were corrected, plotted, and IgG levels were plotted in ng / ml / sample using Prism 9 (Graphpad).

[0081] In vivo PD analysis To assess the therapeutic potential (PD) of the therapeutic protein, IGFR1 activation was evaluated by determining the relative tissue level of phosphorylated AKT using a pan-specific DuoSet ICELISA (RnD #DYC887B-2) of phosphorylated Akt (S473) and treating the tissue as described above. The ELISA was performed in 384-well plates according to the manufacturer's recommendations with slight modifications. Briefly, the wells of the 384-well plate were coated with 25 µl of phosphorylated Akt1 (S473) capture antibody (#841692) in PBS (25 µl / 384 wells), sealed, and incubated overnight at room temperature. The wells were washed four times with 100 µl of PBS containing 0.05% Tween 20 and blocked for 1–2 h at room temperature (RT) with 50 µl of PBS containing 2% BSA. The wells were washed four times with 100 µl of PBS containing 0.05% Tween 20 and incubated for 2 h at room temperature with 25 µl of lysate or P-AKT standard. Wash wells four times with 100 µl of PBS containing 0.05% Tween 20, then incubate for 1 h at room temperature with 25 µl of phosphorylated Akt1 (S473) detection antibody (#843081) diluted 100 ng / ml in PBS containing 1% BSA. Wash wells four times with 100 µl of PBS containing 0.05% Tween 20, and incubate for 20 min at room temperature with 25 µl of streptavidin-HRP A (RnD# 890803) diluted 1:200 in PBS containing 1% BSA. Wash wells four times with 100 µl of PBS containing 0.05% Tween 20, and incubate for 10–20 min at room temperature with 25 µl of TMB substrate solution (Abcam TMBELISA high-sensitivity substrate (ab171523; batch number GR3427893-1)). Add 12.5 µl of stop solution (RnD# 895926 from Auxiliary Kit 2). Collect the standard curve and OD450 data for each sample using a Promega GloMAX Discover microplate reader. Interpolate the sample data against the standard curve, correct for dilution factors, plot the data, and draw the pAKT levels in Prism 10 (Graphpad).

[0082] Immunohistochemical (IHC) assay Muscle tissue was isolated from wild-type (WT) male C57BL / 6J (Jackson Labs, strain #000664) or C57BL / 10ScSn-Dmdmdx / J(mdx) (Jackson Labs, strain #001801) 4–72 hours after administration of 3–10 mg / kg CV1707-12. The tissue was fixed overnight in 4% paraformaldehyde, and then replaced with 30% sucrose after 24 hours. The muscle tissue was then embedded in OCT medium, cut into 10 μm sections, and mounted on glass slides. The tissue was then stained with rabbit anti-human IgG, followed by secondary antibody and DAB staining kit for hIgG. Briefly, sections were washed with PBS + 0.1% Tween-20 and permeabilized with PBS + 0.3% TrixonX-100. Next, the slide was blocked with hydrogen peroxide for 10 minutes, then incubated with rabbit anti-human IgG (Invitrogen #31143) at a dilution of 1:2000 for 24 hours. The slide was then washed three times with PBS + 0.1% Tween-20 and incubated with the secondary antibody, anti-rabbit IgG HRP polymer (Vector Labs). The slide was then washed three times with PBS + 0.1% Tween-20 and developed with DAB (3,3'-diaminobenzidine) substrate at room temperature for 3–5 minutes, followed by rinsing in deionized water for 2 minutes. The stained material was then counterstained with methyl green for 1 minute at room temperature, rinsed in deionized water, and dehydrated twice with 100% isopropanol for two minutes each time. Finally, the slide was covered with a coverslip and imaging was performed.

[0083] Western blot analysis and quantification of proteins Tissue lysates were generated from 4 mice at each treatment and time point. These samples were pooled and mixed 1:1 with 2x Laemmli sample buffer (Bio-Rad, #1610737) and β-mercaptoethanol according to the manufacturer's instructions, and boiled for 10 minutes. Kidney samples were further diluted 3-fold. 3.5 μL–9 μL were loaded onto 4%–15% SDS-PAGE gels (Bio-Rad, #4561086) and run at 200 V for approximately 45 minutes. The gels were transferred to PVDF transfer membranes (Millipore, #IPVH00010) using a standard Western blot procedure. The membranes were stained with Ponceau S to visualize the protein loading. Primary antibody incubation was performed overnight at 4°C, followed by secondary antibody incubation for 1 hour at room temperature. The antibodies used were: P-AKTS473 1:2,000 (Cell Signaling Technology, #4060), AKT 1:1,000 (Cell Signaling Technology, #9272), GAPDH 1:2,000 (Boster, 00227-1), and goat anti-rabbit IgG (H+L)-HRP 1:50,000 (Invitrogen, #31460). Signals were detected by incubation with a chemiluminescent substrate (ThermoScientific, #34580) and using autoradiography film (ECE Scientific, #E3018).

[0084] Generation of C2C12 Itga7 knockout cells By using the Alt-R CRISPR-Cas9 system (IDT), according to the manufacturer's protocol, targeted... Itga7 C2C12-ITGA7 KO cells were generated using gRNA oligonucleotides / AltR1 / rCrU rCrUrC rUrGrU rCrGrA rGrArC rUrCrA rUrArU rGrUrUrUrUrArGrArGrCrUrA rUrGrC rU / AltR2 / and Lipofectamine CRISPRMAX transfection reagent (Thermo Fisher Scientific, #CMAX00008). The absence of MATN3 was verified by immunofluorescence staining with anti-ITGA7 (Cavalry Biosciences, #CV1707-4).

[0085] Perform ELISA in 384-well plates according to the manufacturer's recommendations with slight modifications. Briefly, coat the wells of the 384-well plate with 25 μL of phosphorylated Akt1 (S473) capture antibody (RnD #841692) in PBS (6 μg / mL) (25 μL / 384 wells), seal, and incubate overnight at room temperature. Wash the wells three times with 100 μL of PBS containing 0.05% Tween 20, and block with 50 μL of PBS containing 2% BSA for 1–2 h at room temperature (RT). Wash the wells three times with 100 μL of PBS containing 0.05% Tween 20, and incubate for 2 h at room temperature with 25 μL of lysate or P-AKT standard (RnD #841694). Wash wells three times with 100 μL of PBS containing 0.05% Tween 20, then incubate for 1 h at room temperature with 25 μL of phosphorylated Akt1 (S473) detection antibody (RnD #843081) diluted 100 ng / mL in PBS containing 1% BSA. Wash wells three times with 100 μL of PBS containing 0.05% Tween 20, and incubate for 20 min at room temperature with 25 μL of streptavidin-HRP A (RnD #890803) diluted 1:200 in PBS containing 1% BSA. Wash wells three times with 100 μL of PBS containing 0.05% Tween 20, and incubate for 2–20 min at room temperature with 25 μL of TMB substrate solution (Abcam TMB ELISA highest sensitivity substrate, #ab171522) (time depends on the intensity of the colorimetric reaction). Add 12.5 μL of stop solution (RnD #DY994) and measure the OD450 of each well using a BMG Labtech CLARIOstar® Plus microplate reader. Calculate EC using Prism 10 software. 50 value.

[0086] Hind limb plaster cast fixation Mice were acclimatized for 14 days or longer upon arrival. Baseline muscle function was measured, and animals were assigned to balanced groups based on initial body weight and baseline muscle strength measurements. Mice were then subjected to hindlimb cast or sham cast immobilization on one limb, and administered the mediator or 10 mg / kg CAV-003 every 4 days via intraperitoneal injection. After 2 weeks of cast immobilization, the cast was removed, muscle function was measured again, and the mice were then euthanized and autopsied for further histological analysis.

[0087] For plaster cast application, mice were anesthetized by inhalation or approximately 5% isoflurane. Once sedated, the hind limbs were wiped with three alternating washes of povidone-iodine followed by 70% ethanol and allowed to dry. The hind limbs were then loosely wrapped in sterile surgical gauze and a custom-made plastic fixation device consisting of a small, tapered tube approximately the diameter of the mouse's leg. This device was then placed on the hind limb with the foot fully extended to ensure maximum in vivo unloading of the plantar flexor muscles. The cast extended proximally to the knee joint and distally to the ankle joint. The fixation device was then secured to the hind limb using Vetbond tissue glue. After sedation, the mice were removed from the anesthesia, warming pad, and surgical area and placed in a recovery cage. Once fully mobile, the mice were returned to their original habitat. Dietgel was administered to the animals during the study period. Animals were monitored daily for signs of inflammation around the cast to ensure animal welfare was not compromised. For sham cast application, the animals underwent all procedures, but the cast was not applied with Vetbond and was subsequently removed. The plastic tube used for plaster fixation is 7.9 mm in diameter and 25 mm in length, extending from directly above the mouse's knee joint to directly below the ankle joint. The limb is loosely wrapped in gauze and gently slid into the tube, with the paw fully extended. The mouse is able to walk relatively easily, simply dragging the plaster-fixed limb along the bottom of the cage. This plaster fixation method does not require specialized living or feeding conditions. If the plaster needs to be changed, it will be changed immediately.

[0088] hind limb muscle function Muscle performance was measured in vivo using a 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN). Mice were anesthetized by inhalation or approximately 5% isoflurane and placed on a temperature-controlled console, where anesthesia was maintained via a nasal cone (approximately 2%–3% isoflurane). The knee joint was dislocated by inserting a needle into the proximal tibia, and the foot was then securely fixed to a pedal on a motor shaft. For the plantar flexors, contraction was induced by transcutaneous electrical stimulation of the sciatic nerve. The optimal isometric twitching torque was determined by increasing the current, with a minimum interval of 30 seconds between each contraction to avoid fatigue. A series of stimulations was then performed at increasing stimulation frequencies (0.2 ms pulse, 500 ms pulse train duration): 1, 20, 40, 50, 60, 80, 100, 150 Hz, and the maximum peak isometric muscle force was collected and plotted.

[0089] Body mass and body composition Time-domain (TD)-NMR (LF90 Minispec, Bruker, Spring, TX, USA) was performed on mice to determine the body composition of fat, lean body mass, and body fluids in conscious mice under randomized feeding conditions. After body mass measurement, mice were placed in a Plexiglas sample holder (90 mm in diameter and 250 mm in length) with vents at both ends and around the circumference of the tube, and inserted into a 0.5 T magnet hole. All measurements were performed in triplicate, and the mean was reported. Mice were then returned to their cages after the measurements were completed.

[0090] FSHD mouse model Based on body weight, female FLExDUX4.CRE mice were assigned to one of two balanced groups. Mice were administered either the vector or 10 mg / kg CAV-003 via intraperitoneal injection every 4 days for 4 weeks. A group of littermate CRE WT mice served as a control and were also administered the vector via intraperitoneal injection every 4 days for 4 weeks. At the end of the study, hind limb muscle function was measured in vivo as described above, and the animals were euthanized for tissue collection.

[0091] Example 2. In vitro potency of IGF-1 targeting ITGA7 The in vitro potency of IGF-1 targeting ITGA7 (containing three polypeptide chains: SEQ ID NO: 2, 39, and 40) was tested in both non-myocellular (DU145) and differentiated myocellular (C2C12) cells using pAKT assays. The half-maximal effective concentration (EC50) of IGF-1 in DU145 cells was 4.6 nM, and the EC50 of IGF-1 targeting ITGA7 in DU145 cells was 30.5 nM. The half-maximal effective concentration (EC50) of IGF-1 in C2C12 cells was 7.8 nM, and the EC50 of IGF-1 targeting ITGA7 in C2C12 cells decreased to 7.1 nM. This indicates that IGF-1 targeting ITGA7 is more effective in differentiated myocellular cells compared to non-myocellular cells. Figure 1A and Figure 1B As shown.

[0092] Example 3: In vivo distribution of IGF-1 targeting ITGA7 Mice were injected with an antibody scaffold containing only the ITGA7 targeting arm or with IGF-1 targeting ITGA7 (containing SEQ ID NO: 2, 39, and 40). Blood was collected 0–150 hours post-injection to measure the pharmacokinetics of the antibodies in plasma via human IgG assay. Figure 2 As shown, the half-life (t1 / 2) of IGF-1 targeting ITGA7 in plasma 终末The half-life (64.7 hours) is shorter than that of antibody scaffolds with only an ITGA7 targeting arm. This suggests that IGF-1 targeting ITGA7 is either rapidly cleared from the system or translocated to other tissues or organisms.

[0093] Tissues from the lungs, liver, heart, diaphragm, forearm, and tibia of test mice were harvested 72 hours after injection. The amount of antibody in the tissues was measured by human IgG assay and normalized relative to the corresponding human IgG levels. The levels of IGF-1 targeting ITGA7 were significantly higher in muscle tissues (e.g., heart, diaphragm, forearm, and tibia) than in non-muscle tissues (e.g., lungs and liver). Figure 3A As shown. Conversely, plasma levels of IGF-1 targeting ITGA7 were lower than in the nonspecific human IgG control, such as... Figure 3B As shown in the figure, this indicates that IGF-1 targeting ITGA7 preferentially allocates to muscle tissue compared to nonspecific human IgG.

[0094] Muscle tissue from test mice was isolated 4–72 hours after injection and stained for human IgG. Figure 3C This indicates that, compared with nonspecific control IgG, more IGF-1 targeting ITGA7 is localized in muscle tissue.

[0095] Tissue samples were harvested from test mice 4–24 hours post-injection, and IGF-1 targeting ITGA7 was measured via pAKT assay and Western blot analysis. Muscle tissues (e.g., gastrocnemius, diaphragm) exhibited prolonged pAKT levels compared to non-muscle tissues (e.g., kidney, lung). Figure 3D and Figure 3E As shown. Tissues from test mice were harvested at 4 and 24 hours post-injection, and antibodies were measured via pAKT assay. The concentration of IGF-1 targeting ITGA7 at 24 hours post-injection was normalized relative to the concentration at 4 hours post-injection. The concentration of IGF-1 targeting ITGA7 at 24 hours post-injection was significantly higher in muscle tissues (e.g., heart, diaphragm, forearm, and gastrocnemius) compared to non-muscle tissues (e.g., liver, kidney, and lung). Figure 4 As shown in the figure, this indicates that IGF-1 targeting ITGA7 persists longer in muscle tissue than in non-muscle tissue.

[0096] Example 4: IGF-1 targeting ITGA7 increases body weight and muscle mass in young and old mice.

[0097] To evaluate the effects of IGF-1 targeting ITGA7 (containing three polypeptide chains: SEQ ID NO: 2, 39, and 40) on body weight and muscle mass, young mice (under 18 months old) were administered the IGF-1 fusion molecule targeting ITGA7 via intravenous administration at a dose of 10 mg / kg every 4 days. Figure 5A The study showed that the percentage of body weight gain from baseline during the 14-day treatment period was higher in the treated animals than in the untreated animals. Figure 5B The study showed a qualitative increase in forearm muscle mass in mice treated with IGF-1 targeting ITGA7 at the end of 14 days of treatment, compared with untreated mice. Figure 5C The study showed a significant increase in isolated tibialis anterior (TA) muscle mass in mice treated with IGF-1 targeting ITGA7 compared to untreated mice. These results indicate that IGF-1 targeting ITGA7 increases body weight and muscle mass in young mice.

[0098] To evaluate the effects of IGF-1 targeting ITGA7 on body weight and muscle mass, aged mice (18 months old) were administered the IGF-1 fusion molecule targeting ITGA7 via intravenous administration at a dose of 10 mg / kg every 4 days. Figure 6A The percentage of body weight gain from baseline during the 14-day treatment period is shown compared to untreated animals. Figure 6B The study showed a significant increase in isolated tibialis anterior (TA) muscle mass in mice treated with an IGF-1 fusion molecule targeting ITGA7 compared to untreated mice. These results indicate that IGF-1 targeting ITGA7 increases body weight and muscle mass in aged mice.

[0099] Example 5: IGF-1 targeting ITGA7 increases muscle weight in a hindlimb plaster fixation atrophy model.

[0100] Mice had their right leg immobilized in a plaster cast for 2 weeks. During this period, they were administered IGF-1 (containing three polypeptide chains: SEQ ID NO: 2, 39 and 40) targeting ITGA7 at 10 mg / kg every 4 days via the intraperitoneal route, followed by gastrocnemius muscle dissection and weight measurement. Figure 7 The study showed that, compared with the mediator group, mice treated with IGF-1 targeting ITGA7 had significantly increased muscle mass in both untreated (left leg) and treated (right leg) muscles.

[0101] Example 6: In facioscapulohumeral muscular dystrophy (FSHD) transgenic mice, IGF-1 targeting ITGA7 increased body muscle mass, thereby leading to increased muscle strength.

[0102] FSHD mice were administered IGF-1 (containing three polypeptide chains: SEQ ID NO: 2, 39 and 40) targeting ITGA7 at 10 mg / kg every 4 days via the intraperitoneal route. Figure 8A The study showed that during the 14-day treatment period, the percentage of weight gain from baseline in treated FSHD mice was higher than that in untreated FSHD mice and wild-type (WT) mice treated with the vector. Figure 8B The study showed that FSHD mice treated with IGF-1 targeting ITGA7 had a significant increase in isolated gastrocnemius / Gastroc muscle weight compared to untreated FSHD mice and wild-type (WT) mice treated with the vector. Figure 8C The study showed that FSHD mice treated with IGF-1 targeting ITGA7 exhibited a significant increase in maximal muscle strength compared to untreated FSHD mice and wild-type (WT) mice treated with the vector. Maximal muscle strength was measured using plantar flexor strength measurements.

[0103] Example 7: One month after treatment, IGF-1 targeting ITGA7 increased body weight and muscle mass and altered body composition.

[0104] Adult mice (6 months old) were administered an IGF-1 fusion molecule targeting ITGA7 (containing three polypeptide chains: SEQ ID NO: 2, 39 and 40) at 10 mg / kg every 4 days via the intraperitoneal route for 28 days. Figure 9A The study showed that mice had a significant increase in body weight compared to animals treated with a medium (saline). Figure 9B The isolated organs (liver, kidney, and heart) showed no weight difference between the treatment and vector groups. Isolated skeletal muscles (including extensor digitorum longus (EDL), tibialis anterior (TA), and gastrocnemius) showed a significant increase in mass compared to the vector-treated animals. Time-domain MRI was used to measure total lean body mass and fat content. Figure 9C The results showed that, compared with 2 weeks and 4 weeks after treatment, treated mice exhibited a significant increase in lean body mass percentage and a decrease in fat mass percentage from baseline to 2 weeks and 4 weeks after treatment.

[0105] Example 8: In vitro potency of IGF-1 targeting ITGA7 compared to variants with potency-reducing mutations in IGF-1.

[0106] The potency of IGF-1 targeting ITGA7 (containing three polypeptide chains: SEQ ID NO: 2, 39, and 40) and its variants with potency-reducing mutations (1790-3, 1790-4, and 1790-5) was determined in the DU145 potency assay. 1790-3 (containing three polypeptide chains: SEQ ID NO: 46, 51, and 52) contains an R37 deletion relative to IGF-1 targeting ITGA7. 1790-4 (containing three polypeptide chains: SEQ ID NO: 47, 53, and 54) contains a Y24L mutation relative to IGF-1 targeting ITGA7. 1790-5 (containing three polypeptide chains: SEQ ID NO: 48, 55, and 56) contains a Y31A mutation relative to IGF-1 targeting ITGA7. DU145 cells were stimulated with the specified molecules for 15 minutes, followed by measurement of phosphorylated AKT (P-AKT) levels via ELISA. Figure 10 The EC50 of IGF-1, IGF-1 targeting ITGA7, and its mutants, calculated in GraphPad Prism, is shown. 50 value.

[0107] Example 9: Compared with ITGA7 knockout (KO) C2C12 cells, IGF-1 targeting ITGA7 and variants with reduced potency mutations in IGF-1 are more effective in WT C2C12 cells.

[0108] IGF-1 targeting ITGA7 (containing three polypeptide chains: SEQ ID NO: 2, 39, and 40) and variants of IGF-1 with potency-reducing mutations (1790-3, 1790-4, 1790-5) or their non-ITGA7-targeting controls (1765-1, 1790-7, 1790-6) were measured in WT C2C12 cells or in ITGA7 KO C2C12 cells generated using the Alt-R CRISPR-Cas9 system. 1790-3 (containing three polypeptide chains: SEQ ID NO: 46, 51, and 52) contains an R37 deletion relative to IGF-1 targeting ITGA7. 1790-4 (containing three polypeptide chains: SEQ ID NO: 47, 53, and 54) contains a Y24L mutation relative to IGF-1 targeting ITGA7. 1790-5 (containing three polypeptide chains: SEQ ID NO: 48, 55, and 56) contains a Y31A mutation relative to IGF-1 targeting ITGA7. 1765-1 contains wild-type IGF-1. 1790-6 (containing three polypeptide chains: SEQ ID NO: 49, 57, and 58) contains an R37 deletion relative to IGF-1 targeting ITGA7. 1790-7 (containing three polypeptide chains: SEQ ID NO: 50, 59, and 60) contains a Y24L mutation relative to IGF-1 targeting ITGA7. Figure 11A Immunofluorescence detection of ITGA7 using 1707-4 (ITGA7 antibody) in WT C2C12 cells or ITGA7 KOC2C12 cells is shown. Figure 11B The levels of phosphorylated AKT (P-AKT) were shown by ELISA 15 minutes after stimulating WT C2C12 cells and ITGA7 KO C2C12 cells with IGF-1. Figure 11C The levels of phosphorylated AKT (P-AKT) are shown by ELISA, measured 15 minutes after stimulation of WT C2C12 cells and ITGA7 KOC2C12 cells with IGF-1 or an IGF-1 variant targeting ITGA7. EC50 values ​​were calculated in GraphPad Prism. Figures 11A-11C This indicates that, compared to ITGA7 knockout (KO) C2C12 cells, IGF-1 targeting ITGA7 and variants with reduced potency mutations in IGF-1 are more effective in WT C2C12 cells.

[0109] While preferred embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to embodiments of this disclosure may be employed in the practice of this disclosure. The following claims are intended to define the scope of this disclosure, thereby covering the methods and structures and their equivalents within the scope of these claims.

Claims

1. A medicament for selectively targeting muscle cells, the medicament comprising (a) an antigen-binding domain of mammalian ITGA7 and (b) a cargo coupled to the antigen-binding domain of said mammalian ITGA7.

2. The pharmaceutical agent according to claim 1, wherein the cargo includes a carrier.

3. The pharmaceutical agent according to claim 2, wherein the carrier is a non-viral carrier.

4. The agent according to claim 2, wherein the carrier is a viral carrier.

5. The agent according to claim 4, wherein the viral vector is adeno-associated virus (AAV).

6. The pharmaceutical agent according to any one of the preceding claims, wherein the cargo comprises lipid nanoparticles.

7. The pharmaceutical agent according to any one of the preceding claims, wherein the cargo comprises nucleic acid.

8. The pharmaceutical preparation according to claim 7, wherein the nucleic acid comprises ribonucleic acid (RNA).

9. The pharmaceutical preparation according to claim 8, wherein the RNA is small interfering RNA (siRNA).

10. The pharmaceutical preparation according to claim 8, wherein the RNA is short hairpin RNA (shRNA).

11. The pharmaceutical agent according to any one of the preceding claims, wherein the cargo comprises antisense oligonucleotides (ASO).

12. The pharmaceutical preparation according to claim 11, wherein the ASO is a morpholino oligonucleotide.

13. The pharmaceutical agent according to any one of the preceding claims, wherein the cargo includes a drug.

14. The pharmaceutical agent according to any one of the preceding claims, wherein the cargo comprises a toxin.

15. The pharmaceutical preparation according to any one of the preceding claims, wherein the preparation comprises an immunomodulator.

16. The pharmaceutical preparation according to claim 15, wherein the immunomodulator is an interleukin.

17. The pharmaceutical preparation according to claim 15 or 16, wherein the immunomodulator reduces the activity of the immune system.

18. The pharmaceutical preparation according to claim 16 or 17, wherein the interleukin is IL-10, IL-35, IL-4, IL-13, IL-27 or IL-37.

19. The pharmaceutical preparation according to any one of claims 1-18, wherein the antigen-binding domain binds to an epitope expressed in a mammalian cell, said mammalian cell being selected from muscle satellite cells, skeletal muscle cells, cardiomyocytes, and muscle fibers.

20. The pharmaceutical preparation according to any one of claims 1-19, wherein the antigen-binding domain comprises an antigen-binding fragment (Fab) domain or a single-stranded variable fragment (scFv).

21. The pharmaceutical preparation according to any one of claims 1-20, wherein the antigen-binding domain comprises any one of SEQ ID NO: 31-40.

22. The pharmaceutical agent according to any one of claims 1-21, wherein the antigen-binding domain comprises: VH CDR1, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 31; VH CDR2, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 32; and VH CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

33.

23. The pharmaceutical agent according to any one of claims 1-21, wherein the antigen-binding domain comprises: VL CDR1, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

34. VL CDR2, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 35, and VL CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

36.

24. The pharmaceutical agent according to any one of claims 1-21, wherein the antigen-binding domain comprises: A variable light (VL) chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 38, and Variable weight (VH) chain pestle comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:

37.

25. The pharmaceutical agent according to any one of claims 1-24, further comprising a connector, wherein the connector couples the cargo to the antigen-binding domain of the mammalian ITGA7 selectively binding.

26. The pharmaceutical agent according to any one of claims 1-25, further comprising a crystallizable fragment domain (Fc domain).

27. The pharmaceutical preparation according to claim 26, wherein the Fc domain is the human IgG1 Fc domain.

28. The agent according to claim 26 or 27, wherein the Fc structural domain comprises a pestle-and-mortar structure.

29. The pharmaceutical agent according to any one of the preceding claims, wherein the antigen-binding domain comprises (i) a human IgG1 heavy chain having a club-shaped mutation and an N297G mutation and (ii) a light chain.

30. The pharmaceutical preparation of claim 29, wherein the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO:

39.

31. The pharmaceutical preparation according to claim 29 or 30, wherein the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO:

40.

32. A pharmaceutical composition comprising (i) the pharmaceutical agent according to any one of claims 1-31 and (ii) one or more pharmaceutically acceptable excipients.

33. A method for regulating muscular dystrophy, the method comprising administering to a subject an agent according to any one of claims 1-32.

34. A method for selectively targeting muscle cells, the method comprising administering to a subject an agent according to any one of claims 1-32.

35. The method of claim 33 or 34, wherein the object is a mammal.

36. The method of claim 35, wherein the mammal is a human.

37. The method according to any one of claims 33-36, wherein the agent is delivered to a muscle cell or a region adjacent to a muscle cell.

38. The method according to any one of claims 33-37, wherein the application comprises injecting the object at a location near skeletal muscle, muscle satellite cells (e.g., skeletal muscle satellite cells), skeletal muscle, cardiac muscle, smooth muscle, or muscle fibers.

39. The method according to any one of claims 33 or 35-38, wherein the muscular dystrophy includes Duchenne muscular dystrophy (DMD), Benedictine muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD) and / or congenital muscular dystrophy.

40. An agent configured to selectively target a regulator to mammalian cells expressing integrin α7 (ITGA7), said agent comprising: (a) regulators; and (b) Selectively binds to the antigen-binding domain of mammalian ITGA7; The modulator is coupled to the antigen-binding domain of the mammalian ITGA7 that selectively binds.

41. A drug agent configured to selectively target a signal transduction pathway regulator to mammalian cells containing lysin α7 (ITGA7), said drug agent comprising: (a) Signal transduction pathway modulators; and (b) Selectively binds to the antigen-binding domain of mammalian ITGA7; The signal transduction pathway modulator is coupled to the antigen-binding domain of the mammalian ITGA7 that selectively binds to it.

42. The pharmaceutical preparation of claim 41, wherein the signal transduction pathway modulator regulates the insulin-like growth factor 1 (IGF-1) signal transduction pathway.

43. The pharmaceutical preparation according to claim 41 or 42, wherein the signal transduction pathway modulator comprises an IGF-1 peptide.

44. The pharmaceutical preparation according to claim 43, wherein the IGF-1 polypeptide is the wild-type form of IGF-1.

45. The pharmaceutical preparation according to any one of claims 41-43, wherein the IGF-1 polypeptide comprises a polypeptide having a reduced ability to activate the IGF-1 receptor (IGF-1R) relative to endogenous IGF-1.

46. ​​The pharmaceutical preparation according to any one of claims 41-43 and 45, wherein the ability of the polypeptide to activate the IGF-1R is reduced by at least about 5 times.

47. The pharmaceutical preparation according to any one of claims 41-43, 45 and 46, wherein the IGF-1 polypeptide comprises an IGF-1 variant.

48. The pharmaceutical preparation of claim 47, wherein the IGF-1 variant comprises an amino acid sequence of any one of SEQ ID NO: 1-11, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in any one of SEQ ID NO: 1-11.

49. The agent according to claim 41, wherein the signal transduction pathway modulator regulates the growth hormone (GH) signal transduction pathway.

50. The agent according to claim 41 or 49, wherein the signal transduction pathway modulator regulates the growth hormone receptor (GHR).

51. The pharmaceutical preparation according to any one of claims 41, 49 or 50, wherein the signal transduction pathway modulator comprises a GHR activator.

52. The pharmaceutical preparation according to any one of claims 41 or 49-51, wherein the signal transduction pathway modulator comprises a GH peptide.

53. The pharmaceutical preparation of claim 52, wherein the GH polypeptide comprises the amino acid sequence of SEQ ID NO: 12 or 13, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 12 or 13.

54. The pharmaceutical preparation of claim 41, wherein the signal transduction pathway modulator regulates the basic fibroblast growth factor (bFGF) signal transduction pathway.

55. The pharmaceutical preparation according to claim 41 or 54, wherein the signal transduction pathway modulator regulates the fibroblast growth factor receptor (FGFR).

56. The agent according to any one of claims 41, 54 or 55, wherein the signal transduction pathway modulator comprises an FGFR activator.

57. The pharmaceutical preparation according to any one of claims 41 or 54-56, wherein the signal transduction pathway modulator comprises a bFGF peptide.

58. The pharmaceutical preparation of claim 57, wherein the bFGF polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 14-17, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in any one of SEQ ID NO: 14-17.

59. The pharmaceutical preparation of claim 41, wherein the signal transduction pathway modulator regulates the interleukin-4 (IL4) signal transduction pathway.

60. The pharmaceutical preparation according to claim 41 or 59, wherein the signal transduction pathway modulator regulates the interleukin-4 receptor (IL4R).

61. The agent according to any one of claims 41, 59 or 60, wherein the signal transduction pathway modulator comprises an IL4R activator.

62. The pharmaceutical preparation according to any one of claims 41 or 59-61, wherein the signal transduction pathway modulator comprises an IL4 peptide.

63. The pharmaceutical preparation of claim 62, wherein the IL4 polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 18-21, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in any one of SEQ ID NO: 18-21.

64. The pharmaceutical preparation of claim 41, wherein the signal transduction pathway modulator regulates activin type II receptor (ActRII).

65. The pharmaceutical preparation according to claim 41 or 64, wherein the signal transduction pathway modulator comprises an ActRII inhibitor.

66. The pharmaceutical preparation according to any one of claims 41, 64 or 65, wherein the signal transduction pathway modulator comprises bimagglutinumab.

67. The pharmaceutical preparation according to any one of claims 41 or 64-66, wherein the signal transduction pathway modulator is an antibody or an antibody fragment.

68. The pharmaceutical preparation of claim 67, wherein the antibody or antibody fragment comprises: VH complementarity-determining region 1 (VH CDR1) contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

22. VH CDR2, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 23, and VH CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

24.

69. The pharmaceutical agent according to claim 67 or 68, wherein the antibody or antibody fragment comprises: VL CDR1 (VL CDR1) comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

25. VL CDR2, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 26, and VL CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

27.

70. The pharmaceutical preparation according to any one of claims 67-69, wherein the antibody or antibody fragment comprises a variable weight (VH) chain, the VH chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:

28.

71. The pharmaceutical preparation according to any one of claims 67-70, wherein the antibody or antibody fragment comprises a variable light (VL) chain, the VL chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:

29.

72. The pharmaceutical preparation according to any one of claims 67-71, wherein the antibody or antibody fragment comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 30 or 44.

73. The pharmaceutical preparation according to any one of claims 40-72, wherein the antigen-binding domain binds to an epitope expressed in a mammalian cell, said mammalian cell being selected from muscle satellite cells, skeletal muscle cells, cardiomyocytes, and muscle fibers.

74. The pharmaceutical preparation according to any one of claims 40-73, wherein the antigen-binding domain comprises an antigen-binding fragment (Fab) domain or a single-stranded variable fragment (scFv).

75. The pharmaceutical agent according to any one of claims 40-74, wherein the antigen-binding domain comprises: VH complementarity-determining region 1 (VH CDR1) contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

31. VH CDR2, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 32, and VH CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

33.

76. The pharmaceutical agent according to any one of claims 40-75, wherein the antigen-binding domain comprises: VL CDR1 (VL CDR1) comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

34. VL CDR2, comprising an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO: 35, and VL CDR3, which contains an amino acid sequence having at least about 70%, at least about 80%, at least about 90%, at least about 95%, or 100% sequence identity with the sequence shown in SEQ ID NO:

36.

77. The pharmaceutical agent according to any one of claims 40-76, wherein the antigen-binding domain comprises: A variable light (VL) chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 38, and Variable weight (VH) chain pestle comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:

37.

78. The pharmaceutical preparation according to any one of claims 41-77, wherein the signal transduction pathway modulator is coupled to the amino terminus of the antigen-binding domain.

79. The pharmaceutical preparation according to any one of claims 41-77, wherein the signal transduction pathway modulator is coupled to the carboxyl terminus of the antigen-binding domain.

80. The agent according to any one of claims 41-79, further comprising a connector, wherein the connector couples the signal transduction pathway modulator to the antigen-binding domain of the mammalian ITGA7 that selectively binds to the ITGA7.

81. The pharmaceutical agent according to any one of claims 40-80, further comprising a crystallizable fragment domain (Fc domain).

82. The pharmaceutical preparation according to claim 81, wherein the Fc domain is the human IgG1 Fc domain.

83. The agent according to claim 81 or 82, wherein the Fc structural domain comprises a pestle-and-mortar structure.

84. The pharmaceutical preparation according to claim 41, wherein: (a) The signal transduction pathway modulator comprises an amino acid sequence of any one of SEQ ID NO: 1-30, or an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with any one of the sequences shown in SEQ ID NO: 1-30, wherein the C-terminus or N-terminus of the signal transduction pathway modulator is coupled to a linker of GGGGSGGGGSGGGGS (SEQ ID NO: 41) or GGGGSGGGGS (SEQ ID NO: 42) and a human IgG1 Fc domain having the acetylene and N297G mutations; and (b) The antigen-binding domain comprises the heavy chain and light chain of human IgG1 with the club and N297G mutations.

85. The pharmaceutical preparation of claim 84, wherein the human IgG1 heavy chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO:

39.

86. The pharmaceutical preparation according to claim 84 or 85, wherein the light chain comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO:

40.

87. The pharmaceutical preparation according to any one of claims 41-86, wherein the pharmaceutical preparation comprises: a human IgG1 heavy chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39; and a light chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO:

40.

88. The pharmaceutical preparation according to any one of claims 41-87, wherein the pharmaceutical preparation comprises a human IgG1 heavy chain containing SEQ ID NO: 39 and a light chain containing SEQ ID NO:

40.

89. The pharmaceutical preparation according to any one of claims 41-88, wherein the pharmaceutical preparation comprises: a heavy chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 2; a human IgG1 heavy chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO: 39; and a light chain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity with the sequence shown in SEQ ID NO:

40.

90. The pharmaceutical preparation according to any one of claims 41-89, wherein the pharmaceutical preparation comprises a heavy chain containing SEQ ID NO: 2, a human IgG1 heavy chain containing SEQ ID NO: 39, and a light chain containing SEQ ID NO:

40.

91. A pharmaceutical composition comprising (i) the pharmaceutical agent according to any one of claims 40-90 and (ii) one or more pharmaceutically acceptable excipients.

92. A method for regulating muscular dystrophy, the method comprising administering to a subject an agent according to any one of claims 40-90.

93. The method of claim 92, wherein the object is a mammal.

94. The method of claim 93, wherein the mammal is a human.

95. The method according to any one of claims 92-94, wherein the agent is delivered to a muscle cell or a region adjacent to a muscle cell.

96. The method according to any one of claims 92-95, wherein the administration comprises injecting the object at a location near skeletal muscle, muscle satellite cells (e.g., skeletal muscle satellite cells), skeletal muscle, cardiac muscle, smooth muscle, or muscle fibers.

97. The method according to any one of claims 92-96, wherein the agent induces a reduction in signal transduction or inhibits ActRII in the IGF-1 signal transduction pathway, the GH signal transduction pathway, the bFGF signal transduction pathway, and the IL4 signal transduction pathway.

98. The method according to any one of claims 92-97, wherein the muscular dystrophy includes Duchenne muscular dystrophy (DMD), Benedictine muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD) and / or congenital muscular dystrophy.

Images